Reprogramming Pseudomonas aeruginosa: an in depth study on phage-host interaction
Abstract
Phages are bacterial viruses with high interest for science and technology due to their applicability in human health, biotechnology, agriculture, and veterinary, as well as in synthetic biology. As therapeutic agents, phages can be used to treat life-threatening bacterial infections, such as Pseudomonas aeruginosa infections, although its approval in human medicine is mainly limited by the lack of knowledge about phage mode of action under in vivo conditions and bacterial responses towards phage infection. In this work, RNA-seq of phage-infected bacteria grown under different media conditions and adhered to human lung epithelium was performed to capture a global view of the transcriptional events that occur under phage therapy. Thus, it was possible to prove that P. aeruginosa was significantly more virulent when cultured in media used to grow human lung epithelial cells, as well as when adhered to a monolayer of human lung epithelium, than when cultured in lysogeny broth. During phage infection, it was observed that global transcriptomes are substantially different at early infection, an important phase during which phage takeover of the bacterial cell occurs, with particular changes in metabolic pathways associated with energy acquisition. Curiously, in all growth conditions, there is a set of common mechanisms that are targeted by the phage, which influence the phage transcriptional landscape. These mechanisms include prophage induction, bacterial receptors shutdown, and motility inhibition. In addition, specific transcriptional events were captured for specific growth conditions, indicating that phage can adapt the bacterial cell to reallocate resources for infection and surpass the limitations imposed by bacterial defences. The results put into evidence the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, and the obviously phage versatility on bacterial cell invasion under different environments. Besides providing a better understanding on phage/host interactions, transcriptomics did not disclose the phage´s genes that are essential for phage replication. Therefore, a genomic approach based on the direct manipulation of phage´s genome was made to obtain phages with a reduced genome. This approach consists in exposing the phage to a mutagenic agent and heat treatments in the presence of a chelating agent. The surviving phages had large deletions (up to 3kb) in their early genes and revealed similar ability in the killing of the bacterial host. However, major changes occur in terms of the host range and in their long-term competitiveness, which might indicate that early genes are not essential for phage replication in lab conditions but confer competitive advantages to the phage. This approach enables the fast generation of mutants that can provide clues towards a better understanding of the role of unknown genes and potentially drive the discovery of novel phage sequences and phage core infection mechanisms at the molecular level.
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iii ACKNOWLEDGMENTS O limite do sonho somos nós mesmos, e por isso agradeço a todas as vozes que me mostraram isso e me deram carinho, apoio, partilha, e força ao longo deste percurso que foi o doutoramento! Em primeiro lugar gostaria de agradecer aos meus orientadores. À Professora Joana Azeredo, que me deu a oportunidade de seguir o doutoramento, acreditou em mim, sempre me desafiou com novas ideias e a superar os problemas. Obrigada pela orientação, pela paciência, e incentivo positivo constante! À Doutora Diana Priscila Pires, com a qual aprendi muito ao longo do Doutoramento, na resolução de problemas constante, na aprendizagem de novas técnicas, pela constante ajuda na escrita, pela orientação pertinente e objetiva, que foram essenciais ao longo do doutoramento. Obrigada pela constante preocupação, pela partilha de know-how, pela imensa disponibilidade, e pelo incentivo a fazer mais e melhor! To professor Rob Lavigne that kindly accepted me in his research group and introduced me to the world of transcriptomics, for its helpful insights that allowed me to grow as a researcher, and for supporting me in my work. Thank you, Rob, for all the challenges, all valuable opinions, and providing me all the means that allowed me to say today that Leuven was one of my research homes! Also, I would like to thank both Professor Jan Paeshuyse and Professor Abram Aertsen for allowing me to use their labs and giving me good research advice during my staying in KU Leuven. Of course, I also want to acknowledge the LogT members that embrace this “damaged Portuguese girl” and made my staying in Leuven one of the most amazing experiences of my life, even when things in the lab were wrong. Many thanks to my amazing co-workers that supported me with the transcriptomic data: Leena, Lucas, and Marleen. I also want to acknowledge to Laura for the instantaneous friendship, the kindness, and sharing so many good moments, helping me in so many aspects that make me feel everyday happy for finding such a singular friend! To Cédric for the friendship, help in the lab and all doubts during my adaptation in Belgium, and specially all patience. To Ana Lechuga, Marina, Rodrigo, Annegrette, Maggie, and Leena for the amazing moments that we spent together! Queria agradecer também a todos os meus colegas do Lphage que me ajudaram no dia-a-dia, que partilharam comigo bons momentos e muitas gargalhadas, que discutiram ciência comigo, que me deram motivação e ajudaram a encarar os desafios! Assim, um obrigado especial à Adriana, Alexandre, Alice, Carla, Cátia, Daniela, Diana R., Gracita, Luciana, Mary, Maria João, Marta M., Marta G, Rodrigo, Sílvio e Rute. Em particular à Mary pelo apoio psicológico, motivação, bons momentos e que façamos muitas mais viagens! À Dricas pelas caminhadas, por ser um doce de pessoa, pelos chocolatinhos, e teres essa visão bonita da vida! À minha parceira de lab, “JMC”, pelas gargalhadas na hora de desespero, pela boa companhia, ajuda e pela amizade carinhosa que a carateriza! Um obrigado ao Luís Melo por me ter dado uma oportunidade na ciência na altura que mais precisei, pelos sermões motivacionais (mesmo quando me põe a chorar) e bons insights científicos! Um obrigado ao Sílvio por todos os conselhos, pela paciência, e ajuda! Também agradeço aqueles elementos que já não estão no lab mas que nunca vou esquecer, Rita Costa e Catarina Duarte, estão para sempre no meu coração! Um obrigado também especial à Mariana, que tem sido desde então um grande apoio e parceira no crime. Um obrigado também à tua família por todo o carinho, à Filipa, à Juliana e ao Nelson pelos bons momentos e companheirismo. Queria ainda agradecer as pessoas que me ajudaram imenso antes de vir para doutoramento e incentivaram a não desistir da ciência: Dna. Adelaide, Joana, Madalena, Nela, e staff da Lusitana. Obrigada as minhas colegas de casa que me ajudaram a todos os níveis e as quais não esqueço a mão que me estenderam e grande amizade, Arlete e Xixio. Obrigada às minhas grandes parceiras de PhD, que se tornaram grandes amigas para a vida, que me acolheram como parte da vossa família, e que sei que posso contar para tudo, Andreia e Sílvia! Um grande obrigado aos meus mosqueteiros da Licenciatura, pela amizade que perdura independentemente do tempo e espaço que nos separem, Selénio, Fósforo, e Titânio. Obrigado, sobretudo ao Titânio por todo o companheirismo, amizade, por me chatear e irritar quando foi preciso, e estar sempre presente ao longo da minha vida, e porque o “phD começa a preto e branco, e termina a cores”. Ainda um especial agradecimento à minha Regina, que sempre foi um fiel ombro amigo para a tristeza e alegria, que deu a mão sempre que precisei e sempre esteve disponível para os meus desabafos na hora do cafezinho! Por fim um grande obrigado aos meus irmãos Samuel e Quininha, que me amam incondicionalmente! À Telma que me trata como ouro, e ao Afonso que é o orgulho desta tia babada! Por fim um obrigado a mim mesma! por ter ultrapassado muitos desafios, por ter aprendido ao longo destes anos que mesmo quando todos nos abandonam, os verdadeiros ficam para nos fazer sorrir! Hoje sei que o medo é o meu maior inimigo, mas com determinação nada é impossível! “The best things in life are on the other side of fear”
iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. University of Minho, December 30th, 2022
v ABSTRACT Phages are bacterial viruses with high interest for science and technology due to their applicability in human health, biotechnology, agriculture, and veterinary, as well as in synthetic biology. As therapeutic agents, phages can be used to treat life-threatening bacterial infections, such as Pseudomonas aeruginosa infections, although its approval in human medicine is mainly limited by the lack of knowledge about phage mode of action under in vivo conditions and bacterial responses towards phage infection. In this work, RNA-seq of phage-infected bacteria grown under different media conditions and adhered to human lung epithelium was performed to capture a global view of the transcriptional events that occur under phage therapy. Thus, it was possible to prove that P. aeruginosa was significantly more virulent when cultured in media used to grow human lung epithelial cells, as well as when adhered to a monolayer of human lung epithelium, than when cultured in lysogeny broth. During phage infection, it was observed that global transcriptomes are substantially different at early infection, an important phase during which phage takeover of the bacterial cell occurs, with particular changes in metabolic pathways associated with energy acquisition. Curiously, in all growth conditions, there is a set of common mechanisms that are targeted by the phage, which influence the phage transcriptional landscape. These mechanisms include prophage induction, bacterial receptors shutdown, and motility inhibition. In addition, specific transcriptional events were captured for specific growth conditions, indicating that phage can adapt the bacterial cell to reallocate resources for infection and surpass the limitations imposed by bacterial defences. The results put into evidence the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, and the obviously phage versatility on bacterial cell invasion under different environments. Besides providing a better understanding on phage/host interactions, transcriptomics did not disclose the phage´s genes that are essential for phage replication. Therefore, a genomic approach based on the direct manipulation of phage´s genome was made to obtain phages with a reduced genome. This approach consists in exposing the phage to a mutagenic agent and heat treatments in the presence of a chelating agent. The surviving phages had large deletions (up to 3kb) in their early genes and revealed similar ability in the killing of the bacterial host. However, major changes occur in terms of the host range and in their long-term competitiveness, which might indicate that early genes are not essential for phage replication in lab conditions but confer competitive advantages to the phage. This approach enables the fast generation of mutants that can provide clues towards a better understanding of the role of unknown genes and potentially drive the discovery of novel phage sequences and phage core infection mechanisms at the molecular level. Keywords: phage; RNA-seq; P. aeruginosa .
vi RESUMO Os fagos são vírus que infetam bactérias e que têm elevado interesse devido à sua aplicabilidade nas áreas da saúde humana, biotecnologia, agricultura, veterinária, e biologia sintética. Como agentes terapêuticos, os fagos podem ser usados para tratar infeções provocadas por bactérias resilientes, como P. aeruginosa . Assim, a sua aprovação está sobretudo limitada à falta de conhecimento no modo de ação dos fagos em condições in vivo e nas respostas bacterianas associadas. Neste trabalho foi realizado RNAseq de culturas bacterianas infetadas com fago que cresceram em dois meios distintos, e aderidas a epitélio de pulmão humano, para obter uma visão global dos eventos transcricionais que ocorrem durante a terapia fágica. Assim, foi possível provar que P. aeruginosa encontra-se significantemente mais virulenta quando cultivada em meio de crescimento de células epiteliais, assim como quando aderida a uma monocamada das mesmas, comparativamente em meio “Lysogeny broth”. Durante a infeção do fago, o transcriptoma global é muito diferente na fase inicial da infeção, etapa importante em que o fago faz uma reprogramação da célula bacteriana com alterações em metabolismos associados à produção de energia. Curiosamente, em todas as condições, há um conjunto de mecanismos comuns que são manipulados pelo fago e influenciam o transcriptoma bacteriano. Estes incluem a indução de profago, diminuição da expressão de recetores da superfície bacteriana, e inibição da motilidade. Ainda, eventos transcricionais específicos foram capturados em determinadas condições de crescimento, indicando que o fago adapta a célula bacteriana para realocar recursos essenciais à infeção e ultrapassar as limitações impostas pelas defesas bacterianas. Os resultados põem em evidência a relevância de usar condições complexas que mimetizem as condições in vivo para estudar a interação fago-bactéria, assim como a obvia versatilidade do fago em invadir a célula bacteriana em diferentes condições. Apesar de permitir conhecer a interação fagos/hospedeiro, os dados obtidos não permitiram inferir sobre os genes dos fagos essenciais para a sua replicação. Assim, recorreu-se a uma abordagem genómica para gerar fagos com genomes reduzidos. Esta abordagem consistiu em expor os fagos a um agente mutagénico e um tratamento térmico na presença de agente quelante. Os fagos sobreviventes possuem deleções consideráveis e são eficientes contra a bactéria hospedeira. Contudo, mudanças significativas ocorrem no espectro lítico do fago e na sua capacidade competitiva a longo termo, indicando que os genes iniciais não são essenciais para a replicação do fago em condições laboratoriais, mas conferem vantagens competitivas ao fago. O método permitiu gerar mutantes que providenciem conhecimento sobre o papel de genes de fago desconhecidos, e potencialmente descobrir novas sequências de fago e elucidar mecanismos de infeção do fago a nível molecular. Palavras-chave: fago; RNA-seq; P. aeruginosa .
vii TABLE OF CONTENTS Direitos de autor e condições de utilização do trabalho por terceiros ............................... ii Acknowledgments ........................................................................................................... iii Statement of integrity ....................................................................................................... iv Abstract ........................................................................................................................... v Resumo ............................................................................................................................ vi Table of Contents ............................................................................................................ vii List of Abbreviations ........................................................................................................ xii List of Figures ................................................................................................................ xvi List of Tables .................................................................................................................. xix Scientific Outputs ........................................................................................................... xxi Chapter I: Research goals and thesis outline .................................................................... 1 Chapter II: Literature review ............................................................................................ 3 2.1 Pseudomonas aeruginosa .................................................................................................. 4 2.1.1 A worldwide public health concern ............................................................................... 4 2.1.2 General traits and infection mechanisms ...................................................................... 5 2.1.3 Virulence, cell communication and biofilm formation .................................................... 5 2.1.4 Multidrug resistance mechanisms ................................................................................ 8 2.1.5 Strategies to control P. aeruginosa infections................................................................ 9 2.2 Phages: Viruses of bacteria .............................................................................................. 10 2.2.1 Life cycle and classification ........................................................................................ 10 2.2.2 A brief history of phage therapy .................................................................................. 12 2.2.3 Phage efficacy against biofilms: Lessons from in vitro assays ...................................... 12 2.2.4 In vivo studies, clinical cases and clinical trials using phages to fight P. aeruginosa ..... 13 2.2.5 Constraints of phage therapy ..................................................................................... 14 2.3 Phage genome modification ............................................................................................. 15 2.3.1 Creation of phage libraries through mutational techniques .......................................... 16 2.3.1.1 Selection of phage deletion mutants through heat treatment and chelating agents17 2.4 Phage-bacteria interactions .............................................................................................. 19
xiv PCR — Polymerase chain reaction PEG — Polyethylene glycol PFU — Plaque forming units poly(dT) — Poly (deoxyadenylate Deoxythymidylate PQS — Pseudomonas quinolone signal qPCR — Quantitative polymerase chain reaction QS — Quorum sensing Qst — Quorum sensing targeting protein RBPs — Receptor binding proteins RBS — Ribosome binding site RIN — RNA integrity number R-M — Restriction-modification systems RNA — Ribonucleic acid RNAP — RNA polymerase RNA-seq — RNA sequencing ROS — Reactive oxygen species rpm — Rotations per minute rRNA — Ribosomal RNA RT — reverse transcriptase RT-qPCR — Real-Time Quantitative Reverse Transcription PCR SNAMPs — synthetic mimics of antimicrobial peptides SNP —Single nucleotide polymorphisms ss — Single-stranded T1SS — Type I secretion system T2SS — Type II secretion system T3SS — Type III secretion system T4SS — Type IV secretion system T5SS — Type V secretion system T6SS — Type VI secretion system TCA — Tricarboxylic acid cycle tRNA — Transfer RNA TSS — Transcription start sites
xv US — United States UTIs — Urinary tract infections VBNC — Viable but non-culturable cells vRNAP — Virion RNAP WHO — World Health Organization
xvi LIST OF FIGURES Chapter II Figure 2.1Main steps of bacterial biofilm formation……………………………………………………………………8 Figure 2.2Phage life cycles (lytic, lysogenic, peudolysogenic, chronic infection)……………………………..11 Figure 2.3Overview of bacteria-phage interactions…………………………………………………………………..20 Figure S2.1Pseudomonas LUZ19 phage genome……………………………………………………………………58 Chapter III Figure 3.1Percentage of reads that map to the genomes of phage LUZ19 and P. aeruginosa PAO1 for individual replicate samples during synchronized infections in LB or MCCM medium……………………….65 Figure 3.2Distribution of cDNA reads over phage genome at early (t=5min), middle (t=10min) and late (t=15min) infection when LB or MCCM media were used……………………………………………………………66 Figure 3.3Number of up and downregulated genes per COG category from non-infected bacterial cultures. DE are relative to MCCM versus LB……………………………………………………………………………68 Figure 3.4Principal Component Analysis of transcriptome obtained from samples acquired during different time points of synchronized assays when LB or MCCM media were used……………………………69 Figure 3.5Up and downregulated P. aeruginosa PAO1 genes observed in both LB and MCCM media at early infection stage……………………………………………………………………………………………………………70 Figure S3.1One-step growth curves of LUZ19 phage on LB or MCCM medium……………………………….86 Figure S3.2P. aeruginosa PAO1 genes differentially expressed at early infection (t=5min). Venn diagram shows the interception of LB and MCCM conditions, where 56 genes are commonly significantly regulated………………………………………………………………………………………………………………………….87 Figure S3.3Heat map of P. aeruginosa PAO1 genes expression in different time points of synchronized assays performed in LB and MCCM medium……………………………………………………………………………87 Chapter IV Figure 4.1Overview of the methodology used for RNA samples acquisition from the complex mixture of Nuli-1 epithelial cells-bacteria-phage, library preparation and Illumina RNA sequencing…………………..103
xvii Figure 4.2Pseudomonas phage LUZ19 transcriptional landscape on P. aeruginosa PAO1 adhered to Nuli-1 epithelial cells. 5min samples, 10min samples and 15min samples represent the phage transcripts at the early middle, and late infection stage, respectively ………………………………………………………107 Figure 4.3Principal Component Analysis of total transcriptomes from samples acquired at early (5min), middle (10min), and late infection (15min) in the presence of LB media, MCCM media, and Nuli-1 epithelial cells).………………………………………………………………………………………………………………..108 Figure 4.4Volcano plots representing P. aeruginosa PAO1 DEGs after 5min (A), 10min (B), and 15min (C) of infection with LUZ19 phage (-1.5≤Log2Fold change≤1.5, and padj≤0.05)……………………………110 Figure 4.5Venn diagram showing genes that are exclusively differentially transcribed in each media and the genes that are commonly expressed in each condition.……………………………………………………….111 Figure 4.6Heat maps of genes Log2Fold values for each categorical group of genes at different stages of infection (5-, 10-, and 15-min post-infection) in bacteria adhered to Nuli-1 epithelial cells monolayer, grown on MCCM media, and grown on LB media..…………………………………………………………………112 Figure 4.7 - Heat maps with Log2Fold values for each group of genes that are specifically targeted at Nuli1 at different stages of infection (5-, 10-, and 15-min post-infection) in bacteria adhered to Nuli-1 epithelial cells monolayer, grown on MCCM media, and grown on LB media. …………………………………………….113 Figure 4.8Main overview of transcriptional changes induced on bacteria by phage during infection in the presence of human lung epithelial cells………...………………………………………………………………………115 Figure S4.1Percentage of reads mapping to P. aeruginosa PAO1 adhered to Nuli-1 epithelial cells and Pseudomonas LUZ19 phage in each sample acquired at 0 (control-uninfected bacteria), 5-, 10-, and 15min post-infection………...………………………………………………………………………………………………….127 Figure S4.2Phage LUZ19 transcriptional landscape over the time of infection of P. aeruginosa PAO1 grown in LB medium, MCCM, and Nuli-1 epithelial cells……………………………………………………………128 Figure S4.3Pseudomonas LUZ19 one-step growth curve performed on P. aeruginosa PAO1 adhered to Nuli-1 epithelial cells monolayer …………...…………………………………………………………………………….128 Figure S4.4Heat map representing the expression of iron-related genes (Log2Fold values relative to uninfected bacteria) during each stage of phage infection, under the presence of different growth conditions.………………………………………………………………………………………………………………….….129
xviii Chapter V Figure 5.1P. aeruginosa PAO1 bacterial lawns with diluted lysate from wildtype LUZ19 phage and with lysate obtained after 10 consecutive cycles of heat treatment in the presence of 0.1M EDTA and enrichment in bacterial cultures grown in LB medium………………………………………………………………144 Figure 5.2Visualization of deletion region for each phage mutant. Reads from sequencing of each phage mutant were mapped to the LUZ19 genome using Genius software ………………………………..145 Figure 5.3OSGC for each phage mutants (LUZ19:KO1, LUZ19:KO2, LUZ19:KO3, LUZ19:KO4, and LUZ19:KO5) and wildtype LUZ19 phage……………………………………………………………..….…………….146 Figure 5.4Efficiency of LUZ19, LUZ19:KO1, LUZ19:KO2, LUZ19:KO3, LUZ19:KO4, and LUZ19:KO5 mutants on reducing optical density (at 600nm) of P. aeruginosa PAO1 exponential cultures (≈0.55) when infected at a final MOI of 1..…………………………………………………………..….………………………………..147 Figure 5.5Surviving biofilm bacterial cells from a 24hold P. aeruginosa PAO1 biofilm after being exposed to phage LUZ19 and mutant KO2 at 0.1MOI, after different time points………………….…………………….148 Figure 5.6Overview of the relative abundance of LUZ19 phage and KO2 phage during several cycles of competition assays which begin with the same amount of each phage to infect P. aeruginosa PAO1 exponential-phase cultures…….………………………….…………………………………..….……………………….149 Figure S5.11% Agarose gel containing PCR products of phage plaque mutants and wildtype phage LUZ19, using primers targeting gp0.1 to gp10..…………………………………………………………..…………158 Figure S5.2Agarose gel containing genomic DNA from mutant phages KO1, KO2, KO3, KO4, and KO5.……………………………………………………..….…………………………………………………………………..159 Figure S5.3Optical density and cell count for P. aeruginosa PAO1 cultures grown in LB medium at 37°C, with agitation, for 24 to 54h. ……………………….…………………………………..….……………………..………163
xix LIST OF TABLES Chapter II Table S2.1Detailed Genome annotation of Pseudomonas LUZ19 phage. Location, name, and size of each feature product are detailed. ..................................................................................................... 56 Chapter III Table 3.1Common differentially expressed P. aeruginosa PAO1 genes at 5min post-infection in both LB and MCCM media.. .......................................................................................................................... 71 Table 3.2Differentially expressed P. aeruginosa PAO1 genes at 5min post-infection that have opposite expression according to the medium. ................................................................................................ 75 Table S3.1Upregulated genes in MCCM medium relative to LB medium. These genes are involved in iron acquisition and their expression at early infection in both medium is represented………………………………………………………………………………………………………………………88 Table S3.2Upregulated genes in MCCM medium comparatively to LB medium. These genes have various functions related with bacterial pathogenicity and virulence. .............................................................. 93 Chapter IV Table S4.1Absolute values of reads mapping for each sample of P. aeruginosa infected with LUZ19 phage under the presence of Nuli-1 epithelial cells.. ........................................................................ 126 Table S4.2DEG of P. aeruginosa PAO1 adhered to Nuli-1 epithelial cells monolayer during phage LUZ19 infection at different stages of infection using as control uninfected bacteria. ................................... 129 Table S4.3P. aeruginosa PAO1 genes that are differentially expressed in all bacterial growth conditions (LB medium, MCCM medium, and Nuli-1). ...................................................................................... 132 Table S4.4P. aeruginosa PAO1 genes that are differentially expressed only in Nuli-1 condition, being absent the genes that codifies for hypothetical proteins. .................................................................. 133 Chapter V Table 5.1Titer reduction of wildtype LUZ19 phage lysates with 0.1M of EDTA when heat treated to different temperatures and periods. ................................................................................................ 143 Table 5.2Log reduction of phage titer lysates obtained after production in bacterial cultures grown either in LB or MCCM media after each cycle of heat treatment. ............................................................... 143
xx Table S5.1Genomic DNA extraction from phage mutants using Quick-DNA Viral Kit (Zymo Research)……………………………………………………………………………………………………………………….159 Table S5.2Short repeat nucleotides that are before and after each deletion site.................................159 Table S5.3List of polymorphisms found in each mutant retrieved from Geneious software. ............. 160 Table S5.4Common polymorphisms found in the different mutants in relation to wildtype phage .... 162 Table S5.5Phage host range of the different phage mutants (KO1, KO2, KO3, KO4, KO5) and wildtype LUZ19 phage in different P. aeruginosa clinical isolates ................................................................... 163 Table S5.6EOP of the different phage mutants (KO1, KO2, KO3, KO4, KO5) and wildtype LUZ19 phage in different P. aeruginosa clinical isolates. ....................................................................................... 166 Table S5.7Efficiency of each phage to form plaques on P. aeruginosa PAO1 lawns incubated at different temperatures. ................................................................................................................................. 167
xxi SCIENTIFIC OUTPUTS Papers in peer reviewed journals: ● Brandão, A., Putzeys, L., Pires, D.P., Voet, M., Paeshuyse, J., Lavigne, R., and Azeredo, J. (2022) Impact of phage predation on P. aeruginosa adhered to human airway epithelium: major transcriptomic changes in metabolism and virulence-associated genes. Under review in RNA Biology. ● Brandão, A., Pires, D.P., Coppens, L., Voet, M., Lavigne, R., and Azeredo, J. (2021) Differential transcription profiling of the phage LUZ19 infection process in different growth media. RNA Biol 00: 1–13. ● Pires, D.P., Meneses, L., Brandão, A.., and Azeredo, J. (2022) An overview of the current state of phage therapy for the treatment of biofilm-related infections. Curr Opin Virol 53: 1–7. Selected talks in conferences: ● Brandão, Ana; Pires, Diana; Voet, Marleen; Coppens, Lucas; Lavigne, Rob; Azeredo, Joana. “Differential Gene Expression Analysis of LUZ19 Phage and Pseudomonas Growing in Epithelial Cell Culture Medium vs Luria Broth Medium”. Oral presentation in Section IV: Phage-Host Interactions at 23rd Biennial Evergreen International Phage Meeting, 2019. ● Brandão, Ana; Pires, Diana; Voet, Marleen; Coppens, Lucas; Lavigne, Rob; Azeredo, Joana. “Differential transcription profiling of the phage LUZ19 infection process in different growth media”. Oral presentation in Health Biotechnology @ CEB session of Center of Biological Engineering Biennial Scientific Meeting, Braga, 16th May, 2022. Posters in conferences: ● Brandão, A.; Putzeys, L.; Pires, D. P.; Azeredo, J.; Lavigne, R., “Impact of phage predation on bacterial transcriptome under simulated human airway conditions”. VoM 2022 - Viruses of Microbes - The Latest Conquests (Program and Abstract Book). Guimarães, Portugal, June 1722, 365, 2022. ● Brandão, A.; Putzeys, L.; Pires, D.; Lavigne, R.; Azeredo, J., “ Pseudomonas aeruginosa transcriptome during phage predation under simulated human airway epithelium conditions”. Poster presentation at 32nd ECCMID, 2022. Award among the best 100 abstracts. ● Brandão, A.; Pires, D. P.; Voet, M.; Coppens, L.; Lavigne, R.; Azeredo, J., “Analysis of phage infection on P. aeruginosa lacking G3P encoding gene under different growth media”. i-poster presentation at World Microbe Forum, 2021.
CHAPTER I: RESEARCH GOALS AND THESIS OUTLINE
Chapter I: Research goals and thesis Outline __________________________________________________________________________________ Brandão, A.|2022 2 Bacteriophages (hereinafter designated by phages) are intracellular parasites that usurp cell machinery for their own propagation. The mechanisms behind bacterial cell reprogramming during phage infection are poorly known, as well as the function of many early genes annotated as hypothetical proteins. Transcriptomics is a powerful tool to study phage-host interactions and has been widely applied to discover regulatory sequences and the role of phage genes. However, the phage biology knowledge has been limited to studies of host-phage interactions under well-controlled laboratory conditions that fail to mimic the complexity of microbial communities in real environments. From a phage therapy perspective, it is important to understand how phages interact with their bacterial hosts in human physiological conditions. Thus, the main goal of the work described in this thesis was to elucidate the interaction between a potential therapeutic phage, the Pseudomonas LUZ19 phage, and its host P. aeruginosa PAO1, a pathogen responsible for many difficult-to-treat infections, such as lung infections. This thesis is divided in 5 chapters, each addressing specific aims of the work. Chapter II: aims at providing an overview of the current knowledge about phage-host interaction in general and specifically the interplay between P. aeruginosa and its infecting phages in a context of phage therapy. Chapter III: aims at disclosing the influence of bacterial growth media in capturing phage-bacteria interactions at the transcriptional level, and how the media affects both phage and bacteria transcriptomes. It also aims at understanding phage-host interaction in more complex conditions, such as in the presence of a medium that supports the growth of human airway epithelial cells (Mammalian Cell Culture Medium, MCCM medium). To assess that, RNA-seq of synchronized phage LUZ19 infected cultures of P. aeruginosa PAO1 in LB medium and MCCM medium was performed. Chapter IV: aims at understanding phage-bacteria interactions under conditions that mimics P. aeruginosa -associated infections in the human lung. To achieve that, transcriptome analysis of a complex mixture containing human lung epithelial cells-bacteriaphage was performed. Chapter V aims at identifying in phage’s genome, genes that are not essential for phage particle viability. To achieve that, a simple, non-cost, and fast method was used, which was based on the combination of a chelating agent with heat treatment to select thermostable phages with large deletions in the genome. Together with transcriptomic data, the results described in chapter V contributed to a better elucidation of phage-bacteria interactions, and give clues towards phage genomes manipulation to achieve safer and efficient phages, for instance for phage therapy purposes.
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 9 antibiotics (such as β-lactams and quinolone that pass slowly through porin channels). Additionally, bacteria have multidrug efflux pumps that actively pump antibiotics as they pass through, and have periplasmatic enzymes (like β-lactamases) that degrade or inactivate antibiotics [34]. In turn, acquired resistance is a result of spontaneous genetic mutations, or it can arise through the acquisition of resistance determinants by horizontal transfer of mobile genetic elements (e.g., plasmids, transposons, interposons, and integrons) via transduction, transformation, and/or conjugation [27,34,35]. Additionally, bacterial resistance can be a transient mechanism that is induced by variation of growth conditions (e.g., nutrients available, biofilm formation and swarming motility, environmental stimulation, continuous and inappropriate exposure to sub-inhibitory antibiotic concentrations, stress caused by pH and heat shock variations, DNA stress, and anaerobiosis) being usually reported as an adaptive resistance [36,37]. The small fraction of cells surviving antibiotic treatment due to non-mutational mechanisms are called “persister cells”, and these constitute a phenotypic variation where the cells are metabolically inactive and present less transcription, translation, and replication of the genome. These characteristics allow them to be highly tolerant to antibiotic treatment but once the antibiotic therapy is stopped, “persister cells” become metabolically active again [38]. “Persister cells” are highly prevalent in P. aeruginosa biofilms and are responsible for the high tolerance of biofilms to antibiotics together with the diffusional limitations imposed by the matrix and high population heterogeneity, as discussed above. Thus, eradication of P. aeruginosa biofilms is difficult to achieve with antibiotics since the bacterium that escape from these treatments become fully prepared to resist subsequent treatments and the small fraction of survived-population quickly leads to the beginning of a well-established disease and therefore, antibiotic treatment leads to an active selection for MDR P. aeruginosa isolates. Thus, there is an urgent need to develop new strategies to combat P. aeruginosa infectious diseases, particularly those associated with biofilm formation [39,40]. 2.1.5 Strategies to control P. aeruginosa infections A common therapeutic approach consists of combined antibiotic therapy because it extends the effective life of each used antibiotic, it has a synergistic effect and reduces the development of antibiotic resistance. However, synergy is rarely tested in the biofilm state and its understanding requires deeper knowledge, particularly extensive work is necessary to optimize the combination and concentration of each antibiotic. Besides the apparent success of antibiotics conjugation therapy, biofilms have a resilient nature and multi-drug resistance, being still difficult to eradicate [41].
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 10 Several approaches have been developed to minimize the infections caused by P. aeruginosa , including the use of gene editing (e.g., CRISPR/Cas9), antimicrobial peptides, bacteriocins, probiotics, vaccines, synthetic mimics of antimicrobial peptides (SNAMPs), innate defence regulatory (IDR) peptides, anti-virulence products (e.g., QS inhibitors), and nanoparticles [42,43]. In addition, there is a naturally occurring alternative, the use of phages or phage-derived products. Phage research gained a renewed interest once these have been pointed out as one of the best solutions to fight MDR bacteria and work as antibiotic substitutes. The next section describes in detail phage basic biology as a promising alternative to fight P. aeruginosa -associated infections. 2.2 Phages: Viruses of bacteria Phages are small viruses that specifically prey prokaryotes for their replication, being the most abundant entities in nature that can be found in several habitats [44,45]. Phages are known to be harmless to humans, plants, and animals, and are even considered as important ecosystem shapers in the sense that, phages modulate bacterial abundance and evolution in microbial communities at a global and local scale [46]. Their discovery was made independently by Frederic Twort (1915) and Félix d’Hérelle (1917) [44], where D’Hérelle was the first one to suggest that phages are bacterial viruses. Since that time, phage research has evolved over more than one century, laying the foundation for molecular biology and becoming one of the first approaches to combat human infections caused by bacterial pathogens. 2.2.1 Life cycle and classification Virulent phages with a strictly lytic cycle begin their infection by adsorbing to bacterial cell surface receptors, performing an attachment that allows them to inject their genetic material into the host cytoplasm. Here, host replication, transcription, and translation machinery are redirected to produce new phage particles that are released at the end of the infection cycle through the action of phage enzymes that led to cell lysis. Temperate phages, on the other hand, can integrate their genomic material into the bacterial genome, remaining as a prophage (without production of progeny and consequently bacterial lysis) and replicate together with the bacterial genome for several generations, until the lytic cycle is induced by an external, stress-related stimulus [46,47]. In addition, phages can also adopt other life cycles described as pseudolysogeny (phage doesn’t integrate into the bacterial chromosome and is transmitted to daughter cells), carrier state (large production of phages and accumulation into the cell without release), and chronic infection (phage release without cell lysis) [47,48] (Figure 2.2).
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 11 Phage diversities are also extended to its genomic traits and morphology, based on a classification established by the International Committee on Taxonomy of Viruses (ICTV), which comprises their morphology (size and shape), genetic material, life cycle, host, proteomic and genomic analysis [49]. The most abundant phages in nature are tailed phages with double-stranded DNA (dsDNA) genome, which belong to Caudoviricetes class [50]. Tailed phages have a similar morphology in terms of possessing an icosahedral capsid containing genomic material (together they constitute the “head”), a head-to-tail connector, and a tail, being the last trait that differentiates these phage families. In terms of morphology, phages are usually divided into three morphotypes: a long-rigid-contractile tail with a sheath around the tail tube (myovirus), a long-flexible-non-contractile tail (siphovirus), and a short tail (podovirus) [51]. Figure 2.2Phage life cycles (lytic, lysogenic, pseudolysogenic, chronic infection). a) In lytic cycle phages infect bacteria and produce new virions that are released in the end-of-life cycle as a result of cell lysis; on the other side, in lysogenic life cycle phage genome can be integrated in bacterial chromosome and passed to bacterial daughter cells until a signal stimulates phage release and lytic life cycle progress. Related to lysogenic cycle, pseudo-lysogenic life cycle is referred to phages that are maintained and transmitted to next bacterial generations as plasmids or episomes. Differently from other life cycles, in the chronic life cycle the phage virions are released without bacteria lysis, which is the opposite of carrier state (not represented), where virions are produced but these are accumulated into the bacterial cell. b) different life cycles can be distinguished by means of persistence, replication and
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 12 virions release, being highlighted these traits for the different life cycles. The image was adapted from [48]. 2.2.2 A brief history of phage therapy Natural phage therapy consists of using strictly lytic phages to fight bacterial infections. Phage's first application as a therapeutic agent began in 1919 to treat dysentery, and after that, they were mostly applied in Eastern Europe (Poland and the Soviet Union). In the West, phage therapy was largely abandoned due to the rise of the “antibiotics-era” and due to the lack of knowledge about phages basic biology [44,52]. With the emergence of MDR isolates and the increase of outbreaks caused by resistant bacteria, a renewed interest for phage therapy has emerged in the last two decades as a promising adjunct to antibiotics. A basic rationale to revisit phage therapy was because of the multiple advantages of phages over antibiotics, such as rapid, easy, and inexpensive phage production; high level of specificity, which implies a reduced impact in human microbiota; harmlessness to eukaryotic cells [52]; and minimal side effects and self-replication [44,53]. As a consequence, the number of studies demonstrating phages’ safety and efficacy against human diseases has increased. A special relevance is made to the studies that take into account the infections driven by biofilms. 2.2.3 Phage efficacy against biofilms: Lessons from in vitro assays Most of the studies begin by using in vitro evaluation of phage efficacy against biofilm-related infections, which usually consists of studying mono-species biofilms formed on polystyrene microtiter plates and testing single phage preparations, phage cocktails, or phages combined with antibiotics to assess its efficacy by cell viability, biomass quantification, metabolic activity, or microscopic visualization [54]. In some cases, an attempt to simulate real infections is made by using other types of materials inside the microtiter plates including, stainless steel coupons [55], catheter sections [56,57], collagen [9] or human cells monolayers [59]. However, the in vivo complexity of biofilms in human infections is not well mimicked in these assays, since clinical biofilms harbour a complex and heterogeneous community with multiple bacterial species and sometimes even fungi [60]. Even by performing mixed biofilms (typically dual-species biofilms), in vitro models are not entirely capable of reproducing real infection conditions. Therefore, the translation of in vitro results into clinical outcomes is not straightforward. Indeed, in addition to harbouring a heterogenous polymicrobial community (including viable but non-culturable cells-VBNC), other factors might influence phage action on real biofilms. For instance, in vivo stressors/inhibitory compounds, bacteria under different metabolic states, fluids
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 13 dynamics, and host immune system are factors that are not reproduced by in vitro methods. Nonetheless, all possible knowledge acquired from the in vitro studies can be a major advantage to investigate the variables that rule biofilm control by phages and understanding the potential of phage therapy. Thus, in vitro biofilm-forming methods are useful to assess the biofilm-killing efficacy of phages alone or combined with other antimicrobial agents; to test phage formulations in vitro (to understand its release, dose, and stability) before doing in vivo tests; administration strategy (simultaneously vs. sequential) [61,62]; to predict the emergence of phage-resistant variants (related with bacterial modifications on phage receptors [11,12], defective growth, reduced virulence [15], re-sensitization to antibiotics [65,66] and the immune system [67]); its long-term implication in the therapeutic context (for instance, increase biofilm thickness due to the release of extracellular DNA under lower levels of phage predation [68]); and to study phageantibiotics synergy [62,69–73]. However, extrapolations towards the outcomes for phage-antibiotic combinations should be carefully addressed since many factors may influence the phage-antibiotic synergy such as the antibiotic class, the in vivo biological environment (for example, the presence of human serum), phage species, as well as phage and antibiotic concentrations [24]. It is also important to consider the long-term effects of the phage-antibiotics combination, since there are reports suggesting that the accumulation of extracellular DNA ultimately leads to thicker biofilms and that antibiotics can function as communication molecules to modulate microbial communities [75]. This draws attention to the need for improved in vitro methods that better assess phage formulations and mimic long-term infections by using dynamic conditions or surfaces that simulate human tissue/device surfaces. Considering the limitations of using in vitro methods to guarantee phage efficacy and safety, it has been observed an increased number of in vivo studies performed in animal models, as well as an increasing number of clinical trials and clinical cases in humans. 2.2.4 In vivo studies, clinical cases and clinical trials using phages to fight P. aeruginosa Positive results obtained from laboratory studies allowed the expansion of phage therapy in western Europe. Recently, case studies focused on the use of phages to treat P. aeruginosa infections in humans have been published and, in general, most of them report infection resolution or a clinical improvement [76–90]. An increasing number of studies have also demonstrated the phage's ability to kill P. aeruginosa in vivo using different animal models, as reviewed by Melo and collaborators [91]. To date, the application of phages against P. aeruginosa infections were previously studied in the following animal models: mouse model of respiratory infection [92,93], mice models of bacteraemia, lung and wound
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 14 infections [94–98], keratitis model in rabbits [99], sheep sinusitis model [100], porcine model [101] and Galleria mellonella infection models [102]. These studies highlight that phage researchers are now addressing the importance to test and develop phage products in vivo before jumping to precipitated conclusions given by in vitro methods and testing on humans. This has prompted the emergence of clinical trials that would be useful for validating phages as therapeutic agents. The first clinical trial used to treat P. aeruginosa infections with phages was done by Wright et al. in 2009 and consisted in applying a six-phage cocktail to treat chronic P. aeruginosa otitis. In this trial, the results demonstrated a significant reduction of bacterial counts in the phage-treated group, while in the placebo group there was no significant bacterial reduction. In general, there were no significant side effects in both groups, which sounded a promise of phage therapy success and safety [103]. However, recently it was published the results from a randomized phase I/II trial where a cocktail of lytic P. aeruginosa phages was used to treat wound infections, which was the first trial made under good manufacturing practices (GMP) and good clinical practices (GCP). This trial was stopped due to insufficient efficacy of the cocktail since during the follow-up period, isolated bacteria revealed to be resistant to phage. Another limitation was associated with adverse effects that were observed (23%) and with the shelf-life of the phage cocktail that was very reduced [104]. At the moment, there are several phase I/II trials going on and planned to start this year, which will evaluate different phage products against P. aeruginosa: the APPA02 cocktail to treat serious respiratory infections (NCT04596319), YPT01 cocktail to treat chronic P. aeruginosa infections in CF (NCT04684641), phage cocktail TP102 to treat multispecies infections on diabetic foot ulcers (NCT04803708), BX004-A (NCT05010577), PL03BM (NCT04815798) and PGX0100 (NCT04323475) [105]. This evolution in the development of phage-based products has been driven by the development of dedicated protocols for phage therapy in humans. For instance, in Belgium, a national regulation for magistral preparation was implemented for tailor-made phage medicine to treat individual patients [106]. Similarly, in France, phages can be used under the nominative Temporary Authorization for Use (ATUn), and in the United States through FDA emergency investigational new drug (eIND) pathway [107]. 2.2.5 Constraints of phage therapy Although, considering all the advantages of using phages that were already mentioned above, there are also some limitations associated to phage therapy: less accessibility of intracellular bacterial pathogens to phages; narrow host range; possible phage clearance by the immune system, especially when applied using higher phage titers; release of intracellular toxic components like bacterial endotoxins
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 15 after cell lysis; emergence of phage-resistant bacteria during treatment; diffusional limitations of phages through the biofilm matrix and low metabolic activity of biofilm cells; inhibition of phage infection by QS; and the need for phage production according to GMP guidelines and in large amounts [44,45] (with exception of magistral preparations in Belgium). In addition, there are still regulatory constraints to phage application, because most of the genes encoded in phage genomes are still of unknown function [52,108], and many phages can carry in their genomes genes that encode for virulent or toxic products, antibiotic resistance, and lysogeny. Besides these disadvantages there is the potential chance to isolate new phages with novel features, such as encoding proteins that grants phage higher efficiency against biofilms, or for instance that infect a high panel of bacterial isolates, but also novel phages that might have compact genomes free of genes that codify for toxins or genes associated to genetic material exchange, being thus safer for therapeutic purposes. Besides, many phage therapy strategies had emerged to better overcome the barriers posed by biofilms, such as the use of mechanical debridement; dispersing enzymes; antimicrobial phage-derived enzymes; the use of phage cocktails to increase the spectrum of activity; and exploiting phage synergy with other antimicrobials [45,52]. In an ultimate approach, phages can be genetically engineered towards acquiring new functionalities, including the expansion of their host range [109], delivering antimicrobials [110,111], changing phage life cycle from temperate to virulent phages [112], removal of virulence/toxic genes, to have smaller genomes without unknown ORFs [102], to encode CRISPR/Cas systems that target the bacteria [113,114] or even encoding genes that inhibit bacterial resistance to phages (e.g., anti-CRISPR proteins) [92], among others. Phage engineering opens a near future in which phages can be validated as a therapeutic agent and substitute the current antibiotic therapy. For that, biological properties of phages need to be fully elucidated, so that it can be understood the consequences of its applications and better predict the patients’ outcomes. 2.3 Phage genome modification The genetic modification of phages is an innovative strategy to improve several phage traits that limit its generalized application (reviewed in [115]). Hence, these genetically manipulated phages can be rebooted to fulfil the demands of numerous research fields, which has prompted the development itself of the available genome engineering and synthetic biology tools. To date, several phage-engineering techniques have emerged, including techniques based in Homologous Recombination [116–122],
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 16 Bacteriophage Recombineering of Electroporated DNA (BRED) [123–128], Rebuilding/Refactoring phage genomes in vitro [129], Whole Genome Synthesis [130–132], Yeast-based platform [118,133–135], Cellfree transcription-translation systems [136], CRISPR-Cas based techniques [137–144], and L-form bacteria [145]. Nevertheless, most of referred techniques still have several limitations, namely in the difficulty of screening recombinant phages [125], low recombination rates [120–122], low transformation efficiency [118,129,133–135], lack of available tools for phages that infect non-model bacteria, limitations in terms of genome size [129,130–132], location of protospacer adjacent motif (PAM) sequences that restrict the genetic modifications in the case of CRISPR-Cas systems [141,142,146], and in some cases modification of more than one sequence can only be performed in a sequential steps [147–150]. Phage genome modifications began with small edits of phage genomes such as point mutations using mutagenic agents. Later, after the discovery of specific phenomena such as recombination and transposon, other new methods have been developed. In the next subsections, the ancient techniques used to create phage mutants without using synthetic biology tools will be addressed. 2.3.1 Creation of phage libraries through mutational techniques Currently, there is an urgent demand for techniques that can generate phage libraries in a high throughput manner. In addition to the above-mentioned techniques where a rational design is performed to achieve one or more modifications in phage genomes, introducing small changes in phage genomes is also possible through other standardized techniques. The most often used approach is to perform phage genome mutagenesis randomly. This usually leads to point mutations that are spread across the entire genome and that, can or not, lead to new phage variants/impair phage particle viability. Because of this, they are often used to study phage evolutionary processes [151]. This can be easily performed through chemical mutation using mutagenic agents such as ethyl methanesulfonate (EMS) [151,152] and hydroxylamine (HA) [153,154], or it can be used, for instance, ultraviolet (UV) radiation [155,156]. The advantage of this technique is that it leads to semibiased mutations, not depending on host replication. These mutations are influenced primarily by the mutagenic agent that might favour specific transitions or transversion mutations [157]. This approach is not suitable to insert or swap genes, nor for well-rationalized phage engineering aims. In addition to chemicals and UVs, mutations in the phage genome can also be achieved thought several phage propagations in a host that harbours a vector that encodes proteins that damage DNA or reduce DNA replication fidelity, thus leading to phage genomic mutations after several passages [152]. Nevertheless, this last method limits phage engineering to a specific host, as well as generated phage libraries. In
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 17 general, the random mutagenesis techniques lead to high diversity in the generated libraries, which are not programmed, and if the frequency of the mutations is aimed to be studied, it will demand depth sequencing. Phage-targeted mutagenesis is also possible and is usually restricted to a specific locus. This can be used to discover a specific gene function when its role is already predicted, being analysed by the generated variants in comparison to the unchanged sequence. This targeted mutagenesis is often achieved by error-prone PCR (epPCR) [158], DNA shuffling [159], nicking mutagenesis [160,161], and PCR amplification using degenerate or random primers [161,162]. This targeted mutagenesis is a wellprogrammed approach, and there are several approaches to obtain it (including some of the abovementioned techniques): homologous recombination [163], lambda Red recombineering [164], using sitespecific recombinases [165], CRISPR systems [162,166], and bacterial retroelements [144]. 2.3.1.1 Selection of phage deletion mutants through heat treatment and chelating agents As described above, random mutagenesis generates several phage mutants and, among them, many are undesirable. From this pool, phages with the desired phenotype can be enriched by using a selective pressure, such as heat treatment [151,167–169]. This technique has been used to select naturally occurring phage mutants that have gene deletions thought to occur due to recombineering phenomena or incorrect replication across direct repeat sequences of 7-10 nucleotides [168,170,171]. The selection of genome-reduced phages using heat treatment resulted from early phage stability studies under high temperatures, being noticed that these heat-resistant phages usually have deletions in their sequences [154,167,168,172,173]. These deleted genes (0.5-3.5kb) are commonly referred to as nonessential genes for lytic development [169,171,173]. This phenomenon is observed to occur in a linear relationship between the heat inactivation curve of the mutant and the DNA content of that mutant [172,174]. Similar to temperature, chelating agents induce the same selection of a small proportion of the phage population (1-10% [171]) that are treatment-resistant and that contain gene deletions [172]. In the case of a chelating agent, it is proposed that chelating cations present in the phage surface destabilize the virion conformation [171]. The destabilization of the viral particle under high temperatures and chelating agent presence is a consequence of DNA lose from phage heads, being more pronounced when the two effects (ionic strength and temperature) are combined [169,171]. Initially, it was proposed to be related to smaller genomes exerting less pressure on the head structure [173,174]. Later, studies on viral metastability
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 18 have elucidated the balance between instability required for phage genome release upon cell infection and the stability of packaged genome in the capsid to survive under environmental conditions. In phages, this balance is ensured by a portal complex that forms a thin channel through the capsid wall and allows DNA release from phage capsid only after irreversible binding to the cell surface. Still, it should be noted that phage DNA is packed under high pressure (tens of atmospheres) provoked by DNA bending stress and forces provoked by electrostatic repulsion and hydration between neighbouring DNA helices [175]. Even though, in most environmental conditions, the viral capsid is highly resilient in maintaining packed DNA without premature injection. Nevertheless, thermostability is limited, and at high temperatures, the internal DNA pressure increases lead to DNA leakage from the capsid [175]. Several studies have proven that densely packed viruses also have higher internal pressures and that the balance between capsid stability and internal capsid pressure governs DNA injection. This DNA injection (which is spontaneous and irreversible) usually occurs at lower temperatures (<70°C) than the major capsid proteins disassemble temperature and than viral DNA melting temperature [175]. Studies using DSC analysis (Differential scanning calorimetry) have shown that both enthalpy and temperature associated with DNA injection are shifted under chelating conditions (such as EDTA presence) [175]. These conditions reduce the temperature necessary for viral DNA injection and increase the injection enthalpy. By contrast, the presence of cations, such as Mg2+, increases phage thermostability since when these ions permeate into phage capsid it stabilizes DNA and reduces repulsive forces, thereby reducing capsid internal pressure [174]. Curiously, it was also proven that genomes with smaller density (smaller length) have lower enthalpy (therefore smaller DNA pressure), resulting in a higher ability to resist to high temperatures [174,175]. Altogether, the elucidation of these molecular processes allows to explain the mechanism through which several authors have isolated phage mutants with deletions in their genomes after temperature and chelating agents’ challenging. The advantage of this method is that it is easy to perform, fast and inexpensive, however, it is not suitable for targeted phage modifications. As such, the generation and selection of phage mutants is primarily used as a support to study the characterization of phage-host interactions. For instance, these mutants can help in the elucidation of essential phage genes, elucidation of phage receptor-receptor binding proteins for different bacterial hosts by the discovery of crucial residues that play a role in proteins functionality, validate genes annotation (usually based in homology with proteins present in databases), phage immunogenicity, phage efficiency, phage stability, creating detailed libraries that provide information for machine learning -based programs, reveal genes involved in phage lysogeny, insertion of reporter genes, understanding phage genes essentiality under different contexts, understanding phage evolutionary processes, and identification of phage regulation
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 25 were related with amino acid metabolism, and a downregulation in genes related with bacterial surface components was also observed ( wbp , type IV pilus genes, and arn operons) [204]. According to highly conserved DNA replication genes, the Phikmvvirus members follow similar patterns of T7 coliphage replication. In short, DNA polymerase binds to a short RNA fragment synthetized by the host RNAP upon promotor recognition, allowing host DNA polymerase to manufacture the leading strand. The lagging strand synthesis are facilitated through the combined action of helicase/primase/single-strand binding protein that allows the replication in the opposite direction [236]. This bidirectional replication is hypothesized to start in the ori, which is thought to occur in the DNA polymerase gene [236]. Parallel with phage genome replication it was observed the breakdown of the host genome, most likely performed by 5’ to 3’ exonuclease (gene 22) and the type VII endonuclease (gene 23) to provide nucleotides essential for viral genome assembly [204]. For LUZ19 phage, the genome replication only occurs at 14 min post-infection, being this moment coincident with the start of host DNA degradation [204]. After DNA replication, the next phase is the production of structural proteins that constitute phage procapsids (major capsid proteins, scaffolding proteins, and head-tail connector proteins) and their assembly [237]. Although during infection the most abundant proteins seem to be gp10 (hypothetical protein), gp13, gp21, gp32 (major capsid protein), and gp41 (putative tail fiber) [204]. Next, phage DNA is packaged by the action of two terminase proteins (gp42-gp43) that identify the viral DNA concatemer, cut it into individual genomes and push it into the procapsid. Filled capsids are subsequently attached to phage tails to obtain mature viral particles, which are released upon cell lysis due to the combined activity of holin and an endolysin [238]. The former is responsible for causing small holes in the bacterial membrane, that allow endolysin access to the periplasm to exert its function of peptidoglycan degradation [239]. The fusion between inner and outer membranes is probably performed by the double action of Rz/Rz-like proteins (gp46-gp46.1), which are thought to encode spanins [238,240]. In the next sections interesting interactions captured during LUZ19 infection are highlighted as well as for other Pseudomonas phages, being these interactions powerful examples that drives the discovery of new mechanisms both from bacteria and phage sides. 2.4.4 Temporal gene expression of Pseudomonas phages Besides the diversity of strategies employed by phages to use transcriptional machinery, the temporal expression schemes of P. aeruginosa phages genes appear to follow a consistent pattern: early (hypothetical proteins that are involved in host machinery reprogramming), middle (genes related to
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 26 nucleic acid metabolism), and late (phage structural proteins) genes expression. For example, phages, phages ɸKZ [208], PAK_PA3 [209], PaP3 [211], and LUZ19 [204] are some of the Pseudomonas phages that seem to transcribe its genes in this order. A common feature observed during Pseudomonas phages infection is the tight control of host genes transcription, which is usually marked by the decrease in bacterial transcripts over time during infection. This was observed for phages PaP1 [214], PAK_P3 [209], PA5oct [213], LUZ19 [204], and ɸKZ. Although, an exception was observed for the dsRNA phiYY phage that has a lesser proportion of phage reads over time compared to host reads, probably due to the small size of its genome, indicating a high dependency on host genes for its reproduction [212]. Apart from this last phage example, in general, phage diminishes the transcription of host “non-essential genes” by encoding proteins that bind to host RNAP and inhibit when it is necessary (e.g., gp25.1 from LUZ19, gp36 from LKA1, and other proteins from Phikmvvirus phages) [205,228]. In addition to the generalized shutdown of host gene expression, the selective degradation of host transcripts is also a target process, where RNA degradosome (multicomplex that includes endoribonuclease RNase E) can be modulated. For example, Dip protein from ɸKZ phage inhibits the degradation of phage transcripts by interaction with bacterial degradome, balancing therefore the host and phage transcripts turnover [241]. 2.4.5 Manipulation of host transcription and replication machinery for new viral particles production The mechanisms of how P. aeruginosa phages modulate the host transcriptional machinery are not yet fully understood and major efforts are yet to be done to deeply characterize the phages’ mode of action. In addition to characterizing the unknown ORFs from phage genomes, advances in the discovery of phage regulatory sequences also need to be done. Currently, efforts have been made to better annotate and predict transcription start sites (TSS), promotor sequences, antisense regulation mechanisms, and function of non-coding small RNAs, among others [207,208,213,215]. In addition to the lack of knowledge on underlying mechanisms behind phage transcriptional landscapes, there is also a big gap in understanding phage manipulation of the bacterial host towards a successful infection. While phages shutdown “non-essential” bacterial functions, specific molecular mechanisms are recruited for the phage's benefit. According to phage replication needs, some metabolic pathways can be targeted including the upregulation of genes involved in transcription, translation, and post-translational modifications as verified for dsRNA phage phiYY, PAK_P3, PaP3, and LUZ19 [205,209,211,212]. According to the phage infection stage, the expression levels of those genes is tightly regulated, for instance, for phage PaP3, the downregulation of genes that encode ribosomal RNA and ribosomal
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 27 proteins is specifically achieved during late infection [211]. This orchestration of specific bacterial pathways at specific points of infection has been reported to be attained mainly through targeting bacterial global transcriptional regulators. An important target for phage PaP3 seems to be RpoS, a bacterial global response regulator that governs bacterial responses towards starvation, heat shock, osmolarity, antibiotic, and oxidative stress [242], since expression of phage gp70.1 leads to RpoS downregulation. In addition, other bacterial regulators like dadA and fabA are also downregulated [211]. A way to control regulators is to inhibit their expression, such as occurs during phiYY phage infection, where a cluster of transcriptional regulators is downregulated [212]. The manipulation of host regulators goes beyond the transcriptional level since these interactions also occur with functionally expressed host proteins. An example of this is the small protein SrpA encoded by P. aeruginosa , which is targeted by several phage proteins from phage K5. This host protein governs central cellular processes such as T3SS, chemotaxis, cell motility, cell shape, T6SS system, pyocyanin synthesis, biofilm formation, and it is an important regulator for phage DNA replication and phage transcription [243]. These examples prove the relevance of controlling bacterial global regulators to modulate bacterial metabolism. Recently, the protein product of an early gene from Pseudomonas LUZ24 (gp9, called “Igy”) was demonstrated to inhibit DNA gyrase subunit B of P. aeruginosa , impeding cell DNA replication, but the ultimate biological role during phage infection is unknown [244]. In some phages, modulation of host metabolism can be achieved by the expression of auxiliary metabolic genes (AMGs). These genes facilitate the acquisition of energy and metabolic products used by phages during infection. It’s hypothesized that these genes have been acquired from the bacteria and improve phage infection. Phages that encode AMGs are PaP1 (thymidylate synthase, ORF 110) [214] and PAK_P3 (gp67, gp69, gp57, gp155 related to host mRNA and genome degradation; and gp151 encodes a cell wall hydrolase similar to the host) [209]. Thereby, the use of global bacterial regulators, phage AMGs, and phage regulatory proteins/factors seem to efficiently coordinate bacterial metabolism according to phage demands. For some phages, it is possible to correlate the phage-targeted pathways with the stage of phage infection. For example, during the “middle infection stage”, a phase where the viral genome is replicated, it is common to see an upregulation of host genes associated with replication and nucleic acid metabolism, as well as phage genes necessary for viral DNA replication. This has been described for Pap3 infection where it is seen the co-expression of ORFs (ORF32, 39, and 40) involved in DNA replication with host genes dnaN, gyrB, recF related to DNA replication, recombination, modification, and repair [211]. Indeed, for phage genome replication the abundance of nucleotides is critical, which explains their
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 28 high abundance and the associated metabolites (e.g., pyrimidine) during the infection, as seen for P. aeruginosa infection with PAK_P3 [209]. Besides replication and transcription metabolisms, many other pathways seem to be significantly changed during phage predation, for example energy-associated metabolisms a common target which will be detailed in subsection 2.4.6. 2.4.6 Recruitment of viral building blocks by P. aeruginosa phages during infection Bacterial metabolism control is crucial during viral infection since the phage needs to tightly manage energy consumption and ‘phage building blocks’ to successfully assemble new virions. Regarding this, the exploitation of amino acid metabolisms appears to be vital during the phage life cycle in order to obtain sufficient amino acids for phage proteins production. Indeed, this has been observed for several P. aeruginosa phages: ɸKZ upregulates amino acid biosynthesis pathways (including arginine, leucine, glutathione, and phenylalanine biosynthesis pathways) [208] although, looking to a distinct phage, for instance PaP3, this pathway is regulated in a opposite manner [211]. Similarly, differences are found among the different phages, namely in the degradation of amino acids, where for dsRNA Phage phiYY [212] and PaP1 [214] genes related to the metabolism of amino acids are downregulated, but high coverage metabolism on P. aeruginosa infection with PAK_P3 verified that at late stage the metabolites from amino acid-related pathways were significantly reduced [209]. This possibly indicates that all recruited amino acids are being used for phage production. The recruitment/shutdown of amino acids metabolism is a common trait during phage infection, but so far it isn’t understood the differences among the different phages, which might be related to specific strategies that vary according to the phase of infection, media conditions/bacterial fitness, or the phage itself. Since many of the produced proteins during infection function as enzymes, the acquisition of associated co-factors is very important. One of the most important co-factors in P. aeruginosa is iron and during phage infection, it appears to be essential for phage replication. This is because iron is an essential cofactor of enzymes during new virions production (such as for DNA synthesis enzymes during viral DNA replication and ATP generation as energy for phage production) and even as an incorporated iron present in virions (in receptor binding proteins of tail fibers). Therefore, the upregulation of iron recruitment pathways is a must during infection. The interference with iron metabolisms is common in several P. aeruginosa phages: For example, ɸKZ increases the expression of heme-binding bacterioferritin bfrB [204], as well as PAK_PA4 upregulates three operons related to the transport and production of siderophores (operons homologous to PAO1 pchEFG, pchABCD, fpt system, pvdIJD, phuSTUVW operon,
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 29 and phuR receptor gene, among others) [217]. In addition to iron, vitamins, such as vitamin B6 degradation can also be inhibited during phage infection [211]. 2.4.7 Mechanisms of P. aeruginosa phages to obtain energy Throughout the lytic cycle, phage impose a high energy burden on cells. As such, one of the main targets of bacterial metabolism is the pathways associated with energy production, even when it expresses phage encoded AMGs to facilitate this process. Intriguingly, it seems that phages tend to downregulate energy-associated pathways: downregulation of oxidative phosphorylation and nitrogen metabolism PaP1 [214]; in PaP3 there is a downregulation of NADH dehydrogenase encoding gene, F-type ATPase, succinate dehydrogenase, cytochrome c reductase, and cytochrome c oxidase [211]; downregulation of heme-copper oxidase family (F-type ATPase of complex V and the cytochrome bo3 ubiquinol oxidase) during ɸKZ infection [208]; downregulation of pyruvate metabolism in early infected P. aeruginosa -PaP3 cells [211]; and downregulation of energy metabolism and carbon metabolism in phage phiYY [212]. On the other hand, small molecule transport involved genes narK1 , narH , narJ seem to be upregulated in PaP1 [214]. In the case of PaP3 the secondary metabolite production seems to be unimportant for phage infection, as well as degradation of naphthalene [211]. Nevertheless, conclusions from these data should be carefully considered because in the majority of the transcriptomic/metabolic/proteomic studies, significant changes are obtained through the comparison of late-infected cultures to non-infected cultures, and consequently, these differences might be specific for the infection stage that is being evaluated. Besides the acquisition of required concentrations of phage components, the recruitment of enzymes and metabolic pathways to produce new virions, phage needs also to control other bacterial aspects that might contribute to phage infection progress and if possible, increase the chances to spread and propagate in the neighbouring environments. 2.4.8 Phage mechanisms that influence lytic cycle success and viral progeny propagation In the past decade, more attention has been given to the bacterial side during phage infection, since multiple underlying mechanisms of bacteria respond to phage infection and that some can lead to phage resistance. Functions related to bacterial phenotypes have then been of special interest such as changes in the expression of bacterial chemotaxis and motility. This was noted in PaP3-infected P. aeruginosa due to the downregulation of motility-related genes pilI and fliC genes [242]. Also, it was observed the downregulation of motility-associated transcriptional regulators in ɸKZ [208], and
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 30 chemotaxis repression in phages phiYY [212], PaP1 [214], and PaP3 [211]. This phenomenon has been described as a mechanism of phage to repress the dispersion of host cells to maintain cell proximity, and thus increase the success of infection of bacterial cells by the newly produced phages. This last explanation is consistent with the upregulation of putative adhesin CdrA involved in biofilm formation or cell aggregation seen in ɸKZ, as a mechanism of enhancement of infection rate [208]. Similarly, during P. aeruginosa infection with phage PA5oct, the napABCDEF operon was upregulated (periplasmic nitrate reductase). This operon is involved in biofilm/cell aggregation [213]. Likewise, PA5oct infection leads to a strong upregulation of operon psl E-J that is involved in the synthesis of Psl exopolysaccharide, associated with the formation of matrix. Once again, the induction of cell aggregation can be seen as a mechanism to increase phage spread through the neighbouring cells, but authors suggest that it can also be a mechanism of avoiding secondary infections by masking cell LPS [213]. Curiously, PB1-like Pseudomonas phages seem to encode peptides that interact with diguanylate cyclase YfiN, increasing cdi-GMP levels that affect bacterial morphology and induce small colony variants. These peptides ultimately lead to reduced cellular motility and increased biofilm formation [245]. Conversely, other studies reported the opposite, where they verified the inhibition of biofilm formation: during the expression of gp70.1 from phage PaP3 on P. aeruginosa biofilm was inhibited by the interaction of gp70.1 with biofilm regulator gene [242]. In terms of bacterial surface, different responses have been captured during phage infection. In the case of ɸKZ-infected P. aeruginosa PAO1, there is a downregulation of LPS-associated genes ( wzx and wzy ) [191,208], which was hypothesized to be a defence mechanism to avoid cell infection with other phages. The last hypothesis is also suggested to explain dsRNA phage phiYY infection, which downregulates ADP-L-glycerol-D-mannoheptose epimerase ( rfaD , PA3337), which is involved in LPS biosynthesis, limiting phage adsorption [212]. By contrast an upregulation of wall/LPS/capsule-related genes is observed in PaP3-infected bacteria [211]. In another study, precursor metabolites of the cell wall (UDP-N-acetyl-D-glucosamine or UDP-N-acetyl-D-galactosaminuronic acid) were founded to be increased after phage infection [209]. The contradiction among different studies is not yet understood and requires further studies to elucidate these tendencies. 2.4.9 Phage-encoded proteins to bypass host defences Not of less importance are the strategies that phage employs to overcome bacterial phageresistance mechanisms. To bypass bacterial surface modifications, it has been established that phages can evolve by changing their own receptor binding proteins (RBPs) [246]. After the adsorption process,
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 31 the phage injects its genome and rapidly needs to counter-interact the bacterial defences that recognize and degrade phage DNA (e.g., RM defence system and the CRISPR/Cas system). One way to resist to CRISPR immunity is by acquiring point mutations on the targeted DNA region [247], or the phage can encode anti-defence proteins that are expressed immediately after phage DNA injection such as antiCRISPR/Cas proteins usually found in prophages and temperate phages [248–250]. In lytic phages, there are some examples of identified proteins that can avoid phage DNA degradation by interaction with host defences, such as LUZ24 gp4 (Mip, the MvaT inhibiting protein) that is highly expressed at an early stage and interacts with host MvaT by inhibiting its binding to AT-rich DNA (present on phage DNA) and consequent degradation [251]. MvaT/MvaU is also implicated in the Pseudomonas Pf4 prophage superinfection mechanism, being responsible for the inhibition of Pf4. Upon MvaT/MvaU loss, Pf4 can replicate and kill bacterial cells [252]. During infection, phage ɸKZ also employs a mechanism that downregulates a type I restriction enzyme (PA2732) to avoid its DNA restriction, as well as upregulates antitoxin ParD to protect viral DNA from intracellular ParE toxins [208]. Similarly, dsRNA phage phiYY upregulates anti-oxidative stress genes ( ahpB and katB ) probably to protect against genomic RNA degradation which is more sensitive to degradation rather than DNA genomes [212]. 2.4.10 Other bacterial responses toward phage infection Curiously, another mechanism often induced during P. aeruginosa phages infection (PA5oct LUZ19, PEV2, PAK_P3, and PAK_P4) is the transcription of prophage-associated genes contained within the host genome [204,209,213,217]. Their transcription is a direct consequence of a global bacterial stress that is induced by phage infection. This increase in bacterial stress functions as a signal for prophage survival which is induced under these conditions. The relevance of prophage induction during lytic Pseudomonas phages infection remains to be well elucidated. This phenomenon should be carefully addressed from a phage therapy context, since the release of temperate phages can have a negative impact. For example, Pseudomonas prophages have been described to positively impact biofilm formation [253], virulence [254,255], interspecies competition [253,256], and type IV pili inhibition [191]. Also, “morons” (prophage sequences) can increase bacterial lysogen fitness against further lytic phage infections [257]. A recent study revealed that wounds from mice, pigs, and humans colonized with P. aeruginosa harbouring pf prophage have impaired healing comparing with wounds colonized with bacteria that lack Pf prophage [258].
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 32 QS is also one way for bacteria to defend the population from phage attack [259]. Nevertheless, phage-bacteria interactions can shape PQS-mediated virulence of P. aeruginosa [259]. For some lytic P. aeruginosa phages, PQS metabolites were found to be abundant in infected cultures [225] and were proposed to be a host-mediated stress response towards phage infection. But for some phages (PEV2, ɸKZ, and LUZ19) this phenomenon isn’t valid, indicating that these phages might evolve mechanisms to block PQS synthesis [217,225,260]. This might be the case for ɸKZ that during its infection downregulates PQS transcriptional regulators to decrease bacterial virulence [208]. For LUZ19, the role of a phage-encoded protein (Qst, ‘quorum sensing targeting protein) was described, and induces decreased PQS levels by direct interaction of Qst with PqsD (a host enzyme from quinolone signal biosynthesis pathway), and curiously phage infection is limited in the absence of this enzyme. Also, the lysogenic phage DMS3 encodes in its genome a QS anti-activator protein (Aqs1) that inhibits the master regulator of QS LasR and inhibits type IV pilus assembly (blocking superinfection) [261]. During the later stages of infection, bacterial stress responses have been captured, such as the ones associated with heat shock response [262,263], oxidative stress, and proteolysis [264,265]. The upregulation of SOS and DNA repair systems during phage infection was proposed to be a result of phage targeting by host CRISPR immunity [266]. However, on the other hand, it was reported that during ɸYY infection, genes that encode antioxidant products are upregulated, which was attributed to a phage mechanism that induces genes that minimize host stress responses during infection [212]. Bacterial responses to phage infection are highly dependent on the phage-host pair and vary according to the viral cycle phase. 2.4.11 P. aeruginosa virulence manipulation by lytic phages In several transcriptome studies the impact of phage infection on bacterial virulence has been addressed to guarantee the safety of phage therapy. This is a relevant question from a phage therapy context because it is known that phage-resistant bacteria have reduced virulence [267], which aids in the efficacy of antimicrobials and patients’ immune systems [268]. From a molecular point of view, it was observed that host-associated virulence genes are downregulated upon phage infection. For example, T3SS system, needle protein ( pscF ), ATPase (PA1697), the virulence regulator VqsR, ExsA the transcriptional activator of the P. aeruginosa T3SS were all downregulated in P. aeruginosa infection with ɸKZ phage [208]. In PaP1-infected P. aeruginosa , authors also reported the repression of a twocomponent system, as well as, the downregulation of operon BetAB involved in glycine betaine production, resulting in the inhibition of choline–glycine betaine pathway, which is highly toxic for P. aeruginosa [214].
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 33 In addition, PaP3 phage also encodes a protein that globally represses bacterial virulence by interacting with transcriptional regulators argR and vqsR related to QS system and virulence respectively. This reduction in virulence mediated by PaP3 ORF was demonstrated in mice survival assays [242]. Likewise, a small protein SrpA from P. aeruginosa was identified as essential for phage K5 infection. SrpA is a modulator of several bacterial mechanisms such as chemotaxis, motility, biofilm, pyocyanin syntheses, protein secretion, and virulence in the infection model of Caenorhabditis Elegans [243]. Many mechanisms of phage-bacteria interactions have yet to be discovered and with the application of integrative omics technologies, the knowledge gap in the phage field could be resolved. As a first step towards the implementation of these global omics approaches in phage research, obtaining high-resolution transcription maps is crucial to fully elucidate the gene expression profile phage-host pair and elucidate regulatory processes at a molecular level [194]. 2.5 References 1. Royer S, Morais AP, da Fonseca Batistão DW: Phage therapy as strategy to face postantibiotic era: a guide to beginners and experts. Arch Microbiol 2021, 203:1271–1279. 2. WHO: WHO priority pathogens list for R&D of new antibiotics. 2017, 3. CDC: Antibiotic Resistance Threats in United States, 2019 . 2019. 4. ECDC: Antimicrobial resistance in the EU/EEA . 2022. 5. ECDC: Healthcare-associated infections acquired in intensive care units . 2019. 6. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FSL, Hufnagle WO, Kowallk DJ, Lagrou M, et al.: Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000, 406:959–964. 7. Wagner VE, Bushnell D, Passador L, Brooks AI, Iglewski BH: Microarray Analysis of Pseudomonas aeruginosa Quorum-Sensing Regulons : Effects of Growth Phase and Environment. J Bacteriol 2003, 185:2080–2095. 8. Pereira SG, Rosa AC, Ferreira AS, Moreira LM, Proença DN, Morais P V., Cardoso O, Proença DN, Morais P V., Cardoso O: Virulence factors and infection ability of Pseudomonas aeruginosa isolates from a hydropathic facility and respiratory infections. J Appl Microbiol 2014, 116:1359–1368. 9. Neves PR, McCulloch JA, Mamizuka EM, Lincopan N: Pseudomonas: Pseudomonas aeruginosa . Elsevier; 2014.
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 34 10. Wu W, Jin Y, Bai F, Jin S: Pseudomonas aeruginosa . Elsevier Ltd; 2014. 11. Burrowes E, Baysse C, Adams C, Gara FO, O’Gara F: Influence of the regulatory protein RsmA on cellular functions in Pseudomonas aeruginosa PAO1, as revealed by transcriptome analysis. Microbiology 2006, 152:405–418. 12. Wagner VE, Gillis RJ, Iglewski BH: Transcriptome analysis of quorum-sensing regulation and virulence factor expression in Pseudomonas aeruginosa . Vaccine 2004, 22. 13. Goodman AL, Lory S: Analysis of regulatory networks in Pseudomonas aeruginosa by genomewide transcriptional profiling. Curr Opin Microbiol 2004, 7:39–44. 14. Strateva T, Mitov I: Contribution of an arsenal of virulence factors to pathogenesis of Pseudomonas aeruginosa infections. Ann Microbiol 2011, 61:717–732. 15. Waite RD, Papakonstantinopoulou A, Littler E, Curtis M a: Transcriptome Analysis of Pseudomonas aeruginosa growth: Comparison of Gene Expression in Planktonic Cultures and Developing and Mature Biofilm. J Bacteriol 2005, 187:6571–6576. 16. Elborn JS: Cystic fibrosis. Lancet 2017, 388:2519–2531. 17. Park JE, Yung R, Stefanowicz D, Shumansky K, Akhabir L, Durie PR, Corey M, Zielenski J, Dorfman R, Daley D, et al.: Cystic fibrosis modifier genes related to Pseudomonas aeruginosa infection. Genes Immun 2011, 12:370–377. 18. Reynolds D, Kollef M: The Epidemiology and Pathogenesis and Treatment of Pseudomonas aeruginosa Infections: An Update. Drugs 2021, 81:2117–2131. 19. Kung VL, Ozer EA, Hauser AR: The Accessory Genome of Pseudomonas aeruginosa . Microbiol Mol Biol Rev 2010, 74:621–641. 20. Dötsch A, Schniederjans M, Khaledi A, Hornischer K, Schulz S, Bielecka A, Eckweiler D, Pohl S, Häussler S: The Pseudomonas aeruginosa transcriptional landscape is shaped by environmental heterogeneity and genetic variation. MBio 2015, 6. 21. El-Shaer S, Shaaban M, Barwa R, Hassan R: Control of quorum sensing and virulence factors of Pseudomonas aeruginosa using phenylalanine arginyl β-naphthylamide. J Med Microbiol 2016, 65:1194–1204. 22. Qin S, Xiao W, Zhou C, Pu Q, Deng X, Lan L, Liang H, Song X, Wu M: Pseudomonas aeruginosa : pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduct Target Ther 2022, 7:1–27. 23. Harrison JJ, Turner RJ, Ceri H: Persister cells, the biofilm matrix and tolerance to metal
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 41 Bacteriophages Containing Anti-CRISPR Suppress Infection of Antibiotic-Resistant P. aeruginosa . Microbiol Spectr 2022, 0:e0160222. 93. Lin Y, Quan D, Chang RYK, Chow MYT, Wang Y, Li M, Morales S, Britton WJ, Kutter E, Li J, et al.: Synergistic activity of phage PEV20-ciprofloxacin combination powder formulation-A proofof-principle study in a P. aeruginosa lung infection model. Eur J Pharm Biopharm 2021, 158:166–171. 94. Peng H, Rossetto D, Mansy SS, Jordan MC, Roos KP, Chen IA: Treatment of Wound Infections in a Mouse Model Using Zn(2+)-Releasing Phage Bound to Gold Nanorods. ACS Nano 2022, 16:4756–4774. 95. Arumugam SN, Manohar P, Sukumaran S, Sadagopan S, Loh B, Leptihn S, Nachimuthu R: Antibacterial efficacy of lytic phages against multidrug-resistant Pseudomonas aeruginosa infections in bacteraemia mice models. BMC Microbiol 2022, 22:187. 96. Chen F, Cheng X, Li J, Yuan X, Huang X, Lian M, Li W, Huang T, Xie Y, Liu J, et al.: Novel Lytic Phages Protect Cells and Mice against Pseudomonas aeruginosa Infection. J Virol 2021, 95. 97. Alvi IA, Asif M, Tabassum R, Aslam R, Abbas Z, Rehman SU: RLP, a bacteriophage of the family Podoviridae , rescues mice from bacteremia caused by multi-drug-resistant Pseudomonas aeruginosa . Arch Virol 2020, 165:1289–1297. 98. Chow MYT, Chang RYK, Li M, Wang Y, Lin Y, Morales S, McLachlan AJ, Kutter E, Li J, Chan H-K: Pharmacokinetics and Time-Kill Study of Inhaled Antipseudomonal Bacteriophage Therapy in Mice. Antimicrob Agents Chemother 2020, 65. 99. Rahimzadeh G, Saeedi M, Nokhodchi A, Moosazadeh M, Ghasemi M, Rostamkalaei SS, Mortazavi P, Eghbali M, Pourbakhshian R, Rezai MS, et al.: Evaluation of in-situ gel-forming eye drop containing bacteriophage against Pseudomonas aeruginosa keratoconjunctivitis in vivo. Bioimpacts 2021, 11:281–287. 100. Łusiak-Szelachowska M, Weber-Dąbrowska B, Żaczek M, Górski A: Anti-biofilm activity of bacteriophages and lysins in chronic rhinosinusitis. Acta Virol 2021, 65:127–140. 101. Guillon A, Pardessus J, L’Hostis G, Fevre C, Barc C, Dalloneau E, Jouan Y, Bodier-Montagutelli E, Perez Y, Thorey C, et al.: Inhaled bacteriophage therapy in a porcine model of pneumonia caused by Pseudomonas aeruginosa during mechanical ventilation. Br J Pharmacol 2021, 178:3829–3842. 102. Pires DP, Monteiro R, Homens DM, Fialho A, Lu TK, Azeredo J, Mil-Homens D, Fialho A, Lu TK,
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 42 Azeredo J: Designing P. aeruginosa synthetic phages with reduced genomes. Sci Rep 2021, 11:1–10. 103. Wright A, Hawkins CH, Änggard EE, Harper DR: A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa ; a preliminary report of efficacy. Clin Otolaryngol 2009, 34:349–357. 104. Jault P, Leclerc T, Jennes S, Pirnay JP, Que YA, Resch G, Rousseau AF, Ravat F, Carsin H, Le Floch R, et al.: Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial. Lancet Infect Dis 2019, 19:35–45. 105. Reig S, Le Gouellec A, Bleves S: What’s new in the antiPseudomonas aeruginosa clinical development pipeline since the 2017 WHO alert? Front Cell Infect Microbiol 2022, 12. 106. Pirnay JP, Verbeken G, Ceyssens P-J, Huys I, De Vos D, Amelotte C, Fauconnier A: The Magistral Phage. Viruses 2018, 10. 107. Pires DP, Costa AR, Pinto G, Meneses L, Azeredo J: Current challenges and future opportunities of phage therapy. FEMS Microbiol Rev 2020, 44:684–700. 108. Pelfrene E, Willebrand E, Cavaleiro Sanches A, Sebris Z, Cavaleri M: Bacteriophage therapy: a regulatory perspective. J Antimicrob Chemother 2016, 71:2071–2074. 109. Chen Y, Yang H, Luo S, Wang L, Lu S, Fu Z: Engineering Phage Tail Fiber Protein as a Wide-Spectrum Probe for Acinetobacter baumannii Strains with a Recognition Rate of 100. Anal Chem 2022, 94:9610–9617. 110. Lu TK, Collins JJ: Dispersing biofilms with engineered enzymatic bacteriophage. Proc Natl Acad Sci 2007, 104:11197–11202. 111. Chen X, Liu M, Zhang P, Xu M, Yuan W, Bian L, Liu Y, Xia J, Leung SSY: Phage-Derived Depolymerase as an Antibiotic Adjuvant Against Multidrug-Resistant Acinetobacter baumannii . Front Microbiol 2022, 13:845500. 112. Monteiro R, Pires DP, Costa AR, Azeredo J: Phage Therapy: Going Temperate? Trends Microbiol 2019, 27:368–378. 113. Park JY, Moon BY, Park JW, Thornton JA, Park YH, Seo KS: Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus . Sci Rep 2017, 7. 114. Nath A, Bhattacharjee R, Nandi A, Sinha A, Kar S, Manoharan N, Mitra S, Mojumdar A, Kumar
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 43 P, Patro S, et al.: Biomedicine & Pharmacotherapy Phage delivered CRISPR-Cas system to combat multidrug-resistant pathogens in gut microbiome. Biomed Pharmacother 2022, 151:113122. 115. Pires DP, Cleto S, Sillankorva S, Azeredo J, Lu TK: Genetically Engineered Phages: a Review of Advances over the Last Decade. Microbiol Mol Biol Rev 2016, 80:523–543. 116. Court DL, Sawitzke JA, Thomason LC: Genetic engineering using homologous recombination. Annu Rev Genet 2002, 36. 117. Friedberg EC: Damage and Repair. Nature 2003, 421. 118. Ando H, Lemire S, Pires DP, Lu TK: Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell Syst 2015, 1:187–196. 119. Guttman B: Phage Crosses. In Brenner’s Encyclopedia of Genetics . Edited by Maloy S, Hughes KBT-BE of G (Second E. Academic Press; 2013:270–271. 120. Loessner MJ, Rees CE, Stewart GS, Scherer S, Loessner MJ, Rees CED, Stewart GSAB: Construction of luciferase reporter bacteriophage A511 :: luxAB for rapid and sensitive detection of viable Listeria cells.. Appl Environ Microbiol 1996, 62:1133–1140. 121. Mahichi F, Synnott AJ, Yamamichi K, Osada T, Tanji Y: Site-specific recombination of T2 phage using IP008 long tail fiber genes provides a targeted method for expanding host range while retaining lytic activity. FEMS Microbiol Lett 2009, 295:211–217. 122. Le S, He X, Tan Y, Huang G, Zhang L, Lux R, Shi W, Hu F: Mapping the Tail Fiber as the Receptor Binding Protein Responsible for Differential Host Specificity of Pseudomonas aeruginosa Bacteriophages PaP1 and JG004. PLoS One 2013, 8:1–8. 123. Nobrega FL, Costa AR, Santos JF, Siliakus MF, Van Lent JWM, Kengen SWM, Azeredo J, Kluskens LD: Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine. Sci Rep 2016, 6:1–12. 124. Fehér T, Karcagi I, Blattner FR, Pósfai G: Bacteriophage recombineering in the lytic state using the lambda red recombinases. Microb Biotechnol 2012, 5:466–476. 125. Shin H, Lee JH, Yoon H, Kang DH, Ryu S: Genomic investigation of lysogen formation and host lysis systems of the salmonella temperate bacteriophage SPN9CC. Appl Environ Microbiol 2014, 80:374–384. 126. Marinelli LJ, Hatfull GF, Piuri M: Recombineering: A powerful tool for modification of bacteriophage genomes. Bacteriophage 2012, 2:5–14.
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 44 127. Sharan SK, Thomason LC, Kuznetsov SG, Court DL: Recombineering: A Homologous Recombination-Based Method of Genetic Engineering. Nat Methods 2009, 4:206–223. 128. Marinelli LJ, Piuri M, Swigonová Z, Balachandran A, Oldfield LM, van Kessel JC, Hatfull GF, Swigoňová Z, Balachandran A, Oldfield LM, et al.: BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS One 2008, 3. 129. Chan LY, Kosuri S, Endy D: Refactoring bacteriophage T7. Mol Syst Biol 2005, 1:E1–E10. 130. Sanger F, Coulson AR, Friedmann T, Air GM, Barrell BG, Brown NL, Fiddes JC, Hutchison CA, Slocombe PM, Smith M: The nucleotide sequence of bacteriophage φX174. J Mol Biol 1978, 125:225–246. 131. Smith HO, Hutchison CA, Pfannkoch C, Venter JC: Generating a synthetic genome by whole genome assembly: ΦX174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci 2003, 100:15440–15445. 132. Yang R, Han Y, Ye Y, Liu Y, Jiang Z, Gui Y, Cai Z: Chemical synthesis of bacteriophage G4. PLoS One 2011, 6:2–7. 133. Lu TK, Koeris MS, Chevalier BS, Holder JW, McKenzie GJ, Brownell DR: Recombinant phage and methods. 2013, 134. Sagona AP, Grigonyte AM, MacDonald PR, Jaramillo A: Genetically modified bacteriophages. Integr Biol (United Kingdom) 2016, 8:465–474. 135. Jaschke PR, Lieberman EK, Rodriguez J, Sierra A, Endy D: A fully decompressed synthetic bacteriophage øX174 genome assembled and archived in yeast. Virology 2012, 434:278–284. 136. Shin J, Jardine P, Noireaux V: Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-Free reaction. ACS Synth Biol 2012, 1:408–413. 137. Makarova KS, Brouns SJJ, Horvath P, Sas DF, Wolf YI: Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 2012, 9:467–477. 138. Deveau H, Garneau JE, Moineau S: CRISPR/Cas System and Its Role in Phage-Bacteria Interactions. Annu Rev Microbiol 2010, 64:475–493. 139. Horvath P, Barrangou R: CRISPR/Cas, the Immune System of Bacteria and Archaea. Science (80- ) 2010, 327:167–170. 140. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero D a, Horvath P: CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science (80- ) 2007, 315:1709–1712.
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 45 141. Yaung SJ, Esvelt KM, Church GM: CRISPR/Cas9-mediated phage resistance is not impeded by the DNA modifications of phage T4. PLoS One 2014, 9:3–9. 142. Tao P, Wu X, Tang W-C, Zhu J, Rao V: Engineering of Bacteriophage T4 Genome Using CRISPR-Cas9. ACS Synth Biol 2017, 20:1952–1961. 143. Lemay ML, Tremblay DM, Moineau S: Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9. ACS Synth Biol 2017, 6:1351–1358. 144. Ramirez-chamorro L, Boulanger P, Rossier O: Strategies for Bacteriophage T5 Mutagenesis : Expanding the Toolbox for Phage Genome Engineering. Front Microbiol 2021, 12:1–12. 145. Kilcher S, Studer P, Muessner C, Klumpp J, Loessner MJ: Cross-genus rebooting of custommade, synthetic bacteriophage genomes in L-form bacteria. Proc Natl Acad Sci 2018, 115:201714658. 146. Martel B, Moineau S: CRISPR-Cas: An efficient tool for genome engineering of virulent bacteriophages. Nucleic Acids Res 2014, 42:9504–9513. 147. Oda M, Morita M, Unno H, Tanji Y: Rapid detection of Escherichia coli O157 : H7 by using green fluorescent protein-labeled PP01 bacteriophage. Appl Environ Microbiol 2004, 70:527–534. 148. Bremer E, Silhavy TJ, Weinstock GM: Transposable λ placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli . J Bacteriol 1985, 162:1092–1099. 149. Tanji Y, Furukawa C, Na SH, Hijikata T, Miyanaga K, Unno H: Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage. J Biotechnol 2004, 114:11–20. 150. Sarkis GJ, Jacobs WR, Hatfuii GF: L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria. Mol Med Microbiol 1995, 15:1055–1067. 151. Favor AH, Llanos CD, Youngblut MD, Bardales JA: Optimizing bacteriophage engineering through an accelerated evolution platform. Sci Rep 2020, 10:1–10. 152. Yosef I, Goren MG, Globus R, Molshanski-mor S, Qimron U: Extending the Host Range of Bacteriophage Particles for DNA Transduction Technology Extending the Host Range of Bacteriophage Particles for DNA Transduction. Mol Cell Technol 2017, 66:721–728. 153. Tessman I: Mutagenic treatment of doubleand single-stranded DNA phages T4 and
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 46 S13 with hydroxylamine. Virology 1968, 35:330–333. 154. Villafane R: Construction of Phage Mutants. In Bacteriophages: Methods in Molecular BiologyMethods in Molecular Biology . Edited by Clokie MR, Kropinski A. Humana Press; 2009:223–237. 155. Arimoto-kobayashi S, Anma N, Yoshinaga Y, Douki T, Cadet J, Hayatsu H: Oxidative damage and induced mutations in M13mp2 phage DNA exposed to Nnitrosopyrrolidine with UVA radiation. Mutagenesis 2000, 15:473–477. 156. Drake JW: Ultraviolet Mutagenesis in Bacteriophage T4 I. Irradiation of Extracellular Phage Particles. J Bacteriol 1966, 157. Shibai A, Takahashi Y, Ishizawa Y, Motooka D, Nakamura S: Mutation accumulation under UV radiation in Escherichia coli . Sci Rep 2017, 7. 158. Dunne M, Rupf B, Tala M, Qabrati X, Ernst P, Shen Y, Sumrall E, Heeb L, Loessner MJ, Kilcher S: Reprogramming Bacteriophage Host Range through Structure-Guided Design of Chimeric Receptor Binding Proteins. Cell Rep 2019, 29:1336–1350. 159. Stemmer WP: Rapid evolution of a protein in vitro by DNA shuffling. Nature 1994, 370:389–391. 160. Wrenbeck EE, Klesmith JR, Stapleton JA, Adeniran A, Tyo KEJ, Whitehead TA: Plasmid-based one-pot saturation mutagenesis. Nat Methods 2016, 13:928–930. 161. Faber MS, Leuven JT Van, Ederer MM, Sapozhnikov Y, Wilson ZL, Wichman HA, Whitehead TA, Miller CR: Saturation Mutagenesis Genome Engineering of Infective Φ X174 Bacteriophage via Unamplified Oligo Pools and Golden Gate Assembly. ACS Synth Biol 2020, 9:125–131. 162. Yehl K, Lemire S, Yang AC, Ando H, Mimee M, Torres MDT, Fuente-nunez CD la, Lu TK: Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis. Cell 2019, 179:459–469. 163. Marinelli LJ, Piuri M, Hatfull GF: Chapter 6 Genetic Manipulation of Lytic Bacteriophages with BRED: Bacteriophage Recombineering of Electroporated DNA. In Bacteriophages: Methods and Protocols . . 2019:69–80. 164. Jensen JD, Parks AR, Adhya S, Rattray AJ, Court DL: λ Recombineering Used to Engineer the Genome of Phage T7. Antibiotics 2020, 9. 165. Huss P, Meger A, Leander M, Nishikawa K, Raman S: Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning. Elife 2021, 10.
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 47 166. Kantor A, Mcclements ME, MacLaren R: CRISPR-Cas9 DNA Base-Editing and PrimeEditing. Int J Mol Sci 2020, 21:1–21. 167. Ritchie DA, Malcolm FE: Heat-stable and density mutants of phages T1, T3 and T7. J Gen Virol 1970, 9:35–43. 168. Cole AW, Tran SD, Ellington AD: Heat adaptation of phage T7 under an extended genetic code. Virus Evol 2021, 7:1–9. 169. Studier FW: Genetic analysis of non-essential bacteriophage T7 genes. J Mol Biol 1973, 79:227–236. 170. Cunningham CW, Jeng K, Husti J, Badgett M, Molineux IJ, Hillis DM, Bull JJ: Parallel Molecular Evolution of Deletions and Nonsense Mutations in Bacteriophage T7. Mol Biol Evol 1997, 9:113–116. 171. Gutiérrez D, Fernández L, Rodríguez A, García P: Practical Method for Isolation of Phage Deletion Mutants. methods Protoc 2018, 1. 172. Davis RW, Parkinson JS: Deletion mutants of bacteriophage lambda. J Mol Biol 1971, 56:403–423. 173. Studier FW: Bacteriophage T7. Science (80- ) 1972, 176:367–376. 174. Bauer DW, Evilevitch A: Influence of Internal DNA Pressure on Stability and Infectivity of Phage λ. J Mol Biol 2015, 427:3189–3200. 175. Bauer DW, Li D, Huffman J, Homa FL, Wilson K, Leavitt JC, Casjens SR, Baines J, Evilevitch A: Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages. J Virol 2015, 89:9288–9298. 176. Tock MR, Dryden DTF: The biology of restriction and anti-restriction. Curr Opin Microbiol 2005, 8:466–472. 177. Kronheim S, Daniel-Ivad M, Duan Z, Hwang S, Wong IM, Nodwell JR, Maxwell KL: A chemical defence against phage infection. Nature 2018, 564:283–286. 178. Cohen D, Melamed S, Millman A, Shulman G, Oppenheimer-Shaanan Y, Kacen A, Doron S, Amitai G, Sorek R: Cyclic GMP-AMP signalling protects bacteria against viral infection. Nature 2019, 574:691–695. 179. Millman A, Bernheim A, Stokar-Avihail A, Fedorenko T, Voichek M, Leavitt A, OppenheimerShaanan Y, Sorek R: Bacterial Retrons Function In Anti-Phage Defense. Cell 2020, 183:15511561.e12.
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 48 180. Tal N, Morehouse BR, Millman A, Stokar-Avihail A, Avraham C, Fedorenko T, Yirmiya E, Herbst E, Brandis A, Mehlman T, et al.: Cyclic CMP and cyclic UMP mediate bacterial immunity against phages. Cell 2021, 184:5728-5739.e16. 181. Maxwell KL: The Anti-CRISPR Story: A Battle for Survival. Mol Cell 2017, 68:8–14. 182. Maxwell KL: Phages Tune in to Host Cell Quorum Sensing. Cell 2019, 176:7–8. 183. Chen Q, Dharmaraj T, Cai PC, Burgener EB, Haddock NL, Spakowitz AJ, Bollyky P: Bacteriophage and Bacterial Susceptibility, Resistance, and Tolerance to Antibiotics. Pharmaceutics 2022, 14. 184. Sweere JM, Van Belleghem JD, Ishak H, Bach MS, Popescu M, Sunkari V, Kaber G, Manasherob R, Suh GA, Cao X, et al.: Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection. Science 2019, 363. 185. Veldkamp KE, Heezius HC, Verhoef J, van Strijp JA, van Kessel KP: Modulation of neutrophil chemokine receptors by Staphylococcus aureus supernatent. Infect Immun 2000, 68:5908– 5913. 186. Waldor MK, Mekalanos JJ: Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 1996, 272:1910–1914. 187. Figueroa-Bossi N, Bossi L: Inducible prophages contribute to Salmonella virulence in mice. Mol Microbiol 1999, 33:167–176. 188. Secor PR, Michaels LA, Smigiel KS, Rohani MG, Jennings LK, Hisert KB, Arrigoni A, Braun KR, Birkland TP, Lai Y, et al.: Filamentous Bacteriophage Produced by Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo . Infect Immun 2017, 85. 189. Mazaheri Nezhad Fard R, Barton MD, Heuzenroeder MW: Bacteriophage-mediated transduction of antibiotic resistance in enterococci. Lett Appl Microbiol 2011, 52:559–564. 190. Secor PR, Sweere JM, Michaels LA, Malkovskiy A V, Lazzareschi D, Katznelson E, Rajadas J, Birnbaum ME, Arrigoni A, Braun KR, et al.: Filamentous Bacteriophage Promote Biofilm Assembly and Function. Cell Host Microbe 2015, 18:549–559. 191. Schmidt AK, Fitzpatrick AD, Schwartzkopf CM, Faith DR, Jennings LK, Coluccio A, Hunt DJ, Michaels LA, Hargil A, Chen Q, et al.: A Filamentous Bacteriophage Protein Inhibits Type IV Pili To Prevent Superinfection of Pseudomonas aeruginosa . MBio 2022, 13:e02441-21. 192. Shan J, Korbsrisate S, Withatanung P, Adler NL, Clokie MRJ, Galyov EE: Temperature dependent bacteriophages of a tropical bacterial pathogen. Front Microbiol 2014, 5:599.
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 49 193. Fister S, Robben C, Witte AK, Schoder D, Wagner M, Rossmanith P: Influence of Environmental Factors on Phage-Bacteria Interaction and on the Efficacy and Infectivity of Phage P100. Front Microbiol 2016, 7:1152. 194. Parmar KM, Gaikwad SL, Dhakephalkar PK, Kothari R, Singh RP: Intriguing interaction of bacteriophage-host association: An understanding in the era of omics. Front Microbiol 2017, 8:559. 195. Carninci P: Is sequencing enlightenment ending the dark age of the transcriptome? Nat Methods 2009, 6:711–713. 196. Garalde DR, Snell EA, Jachimowicz D, Sipos B, Lloyd JH, Bruce M, Pantic N, Admassu T, James P, Warland A, et al.: Highly parallel direct RNA sequencing on an array of nanopores. Nat Methods 2018, 197. Bankevich A, Pevzner PA: TruSPAdes: Barcode assembly of TruSeq synthetic long reads. Nat Methods 2016, 13:248–250. 198. Byrne A, Beaudin AE, Olsen HE, Jain M, Cole C, Palmer T, DuBois RM, Forsberg EC, Akeson M, Vollmers C: Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells. Nat Commun 2017, 8:1–11. 199. Blasdel B, Ceyssens P, Lavigne R: Preparing cDNA libraries from lytic phage infected cells for whole transcriptome analysis by RNAseq. In Bacteriophages: Methods in Molecular Biology . Edited by Clokie M, Kropinski A, Lavigne R. Humana Press; 2017:Methods in Molecular Biology. 200. Gliddon HD, Herberg JA, Levin M, Kaforou M: Genome-wide host RNA signatures of infectious diseases: discovery and clinical translation. Immunology 2017, 201. Doron S, Fedida A, Hernández-Prieto MA, Sabehi G, Karunker I, Stazic D, Feingersch R, Steglich C, Futschik M, Lindell D, et al.: Transcriptome dynamics of a broad host-range cyanophage and its hosts. ISME J 2015, 10:1437–1455. 202. Yang H, Ma Y, Wang Y, Yang H, Shen W, Chen X: Transcription regulation mechanisms of bacteriophages. Bioengineered 2014, 5:300–304. 203. Guzina J, Djordjevic M: Inferring bacteriophage infection strategies from genome sequence: Analysis of bacteriophage 7-11 and related phages. BMC Evol Biol 2015, 15:1–8. 204. Lavigne R, Lecoutere E, Wagemans J, Cenens W, Aertsen A, Schoofs L, Landuyt B, Paeshuyse J, Scheer M, Schobert M, et al.: A Multifaceted Study of Pseudomonas aeruginosa Shutdown by Virulent Podovirus LUZ19. MBio 2013, 4:1–13.
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 50 205. Ceyssens PJ, de Smet J, Wagemans J, Akulenko N, Klimuk E, Hedge S, Voet M, Hendrix H, Paeshuyse J, Landuyt B, et al.: The Phage-Encoded N-Acetyltransferase Rac Mediates Inactivation of Pseudomonas aeruginosa Transcription by Cleavage of the RNA Polymerase Alpha Subunit. Viruses 2020, 12. 206. Ceyssens P-J, Minakhin L, Van den Bossche A, Yakunina M, Klimuk E, Blasdel B, De Smet J, Noben J-P, Blasi U, Severinov K, et al.: Development of Giant Bacteriophage ΦKZ Is Independent of the Host Transcription Apparatus. J Virol 2014, 88:10501–10510. 207. Gerovac M, Wicke L, Chihara K, Schneider C, Lavigne R, Vogel J: A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions. MBio 2021, 12. 208. Wicke L, Ponath F, Coppens L, Gerovac M, Lavigne R, Vogel J: Introducing differential RNAseq mapping to track the early infection phase for Pseudomonas phage ɸKZ. RNA Biol 2020, doi:10.1080/15476286.2020.1827785. 209. Chevallereau A, Blasdel BG, De Smet J, Monot M, Zimmermann M, Kogadeeva M, Sauer U, Jorth P, Whiteley M, Debarbieux L, et al.: Next-Generation “-omics” Approaches Reveal a Massive Alteration of Host RNA Metabolism during Bacteriophage Infection of Pseudomonas aeruginosa . PLoS Genet 2016, 12:1–20. 210. Cui X, You J, Sun L, Yang X, Zhang T, Huang K, Pan X, Zhang F, He Y, Yang H: Characterization of Pseudomonas aeruginosa Phage C11 and Identification of Host Genes Required for Virion Maturation. Sci Rep 2016, 6:1–14. 211. Zhao X, Chen C, Shen W, Huang G, Le S, Lu S, Li M, Zhao Y, Wang J, Rao X, et al.: Global Transcriptomic Analysis of Interactions between Pseudomonas aeruginosa and Bacteriophage PaP3. Sci Rep 2016, 6:1–12. 212. Zhong Q, Yang L, Li L, Shen W, Li Y, Xu H, Zhong Z, Chen M, Le S: Transcriptomic Analysis Reveals the Dependency of Pseudomonas aeruginosa Genes for Double-Stranded RNA Bacteriophage phiYY Infection Cycle. iScience 2020, 23:101437. 213. Lood C, Danis-Wlodarczyk K, Blasdel BG, Jang H Bin, Vandenheuvel D, Briers Y, Noben JP, van Noort V, Drulis-kawa Z, Lavigne R, et al.: Integrative omics analysis of Pseudomonas aeruginosa virus PA5oct highlights the molecular complexity of jumbo phages. Environ Microbiol 2020, 22:2165–2181. 214. Zhao X, Shen M, Jiang X, Shen W, Zhong Q, Yang Y, Tan Y, Agnello M, He X, Hu F, et al.:
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 57 16,447 16,887 gene gp23 endonuclease type 7 441 16,884 17,930 gene gp24 hypothetical protein 1,047 17,940 18,311 gene gp25 hypothetical protein 372 18,304 18,654 gene gp25.1 hypothetical protein 351 18,663 21,110 gene gp26 phage-specific RNA polymerase 2,448 21,283 21,534 gene gp27 hypothetical protein 252 21,534 22,007 gene gp28 GNAT family Nacetyltransferase 474 21,973 22,248 gene gp29 hypothetical protein 276 22,260 23,792 gene gp30 head-tail connector protein 1,533 23,796 24,764 gene gp31 scaffolding protein 969 24,765 24,784 regulatory promoter phage-specific promoter sequence 20 24,817 25,824 gene gp32 major capsid protein 1,008 25,921 26,475 gene gp33 tail tubular protein A 555 26,478 28,958 gene gp34 tail tubular protein B 2,481 28,958 29,503 gene gp35 internal virion protein 546 29,503 32,199 gene gp36 internal virion protein 2,697 32,203 36,216 gene gp37 internal virion protein 4,014 36,218 36,973 gene gp38 phage particle protein 756 36,973 37,431 gene gp39 tail fibre protein 459 37,424 38,329 gene gp40 tail fibre protein 906 38,333 38,938 gene gp41 tail fibre protein 606 38,938 39,243 gene gp42 terminase small subunit 306 39,253 41,058 gene gp43 terminase large subunit 1,806 41,055 41,255 gene gp44 phage holin 201 41,252 41,734 gene gp45 lysozyme 483 41,692 42,021 gene gp46 Rz protein 330 41,909 42,109 gene gp46.1 Rz1 protein 201 42,111 42,425 gene gp47 phage particle protein 315 42,473 42,718 gene gp48 phage particle protein 246 42,728 42,943 gene gp49 hypothetical protein 216 43,077 43,548 repeat region - - 472
Chapter II: Literature review __________________________________________________________________________________ Brandão, A.|2022 58 Figure S2.1Pseudomonas LUZ19 phage genome. Coding sequences are identified in yellow, regulatory sequences in green, and repeat regions in orange. Image was performed in Geneious Prime.
CHAPTER III: TRANSCRIPTOMICS OF PSEUDOMONAS LUZ19 PHAGE INFECTED BACTERIA UNDER IN VITRO CONDITIONS This chapter is based on following scientific article: ⎯ Brandão, A., Pires, D.P., Coppens, L., Voet, M., Lavigne, R., and Azeredo, J. (2021) Differential transcription profiling of the phage LUZ19 infection process in different growth media. RNA Biol 00: 1–13. Brandão, A. kindly acknowledges to Coppens, L. for the bioinformatic analysis of obtained sequencing data, to Voet, M. for aiding in the construction of cDNA libraries and running the sequencing in a in house MiniSeq sequencer. Many thanks also for supervision and writing assistance of above-mentioned article to supervisors: Pires, D.P., Lavigne, R., and Azeredo, J. A special thanks to Pires, D.P. who helped with all RNA extraction methodology troubleshooting and for teaching how to work with P. aeruginosa .
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 60 Abstract RNA-seq of phage-infected bacterial cultures offers a snapshot of transcriptional events occurring during the infection process, providing insights into the phage transcriptional organization as well as the bacterial response. To better mimic real environmental contexts, it was performed RNA-seq of P. aeruginosa PAO1 cultures infected with phage LUZ19 in MCCM medium to better simulate a phage therapy event and the data were compared to LB medium. Regardless of the media, phage LUZ19 induces significant transcriptional changes in the bacterial host over time, particularly during early infection (t=5min) and gradually shutdown bacterial transcription. In a common response in both media, 56 P. aeruginosa PAO1 genes are differentially transcribed and clustered into several functional categories such as metabolism, translation and transcription. The data allowed to tease apart a medium-specific response during infection from the identified infection-associated responses. This reinforces the concept that phages overtake bacterial transcriptome in a strict manner to gain control of the bacterial machinery and reallocate resources for infection, in this case overcoming the nutritional limitations imposed by MCCM medium. From a phage therapy perspective, this study contributes towards a better understanding of phage-host interaction in human physiological conditions and demonstrates the versatility of phage LUZ19 to adapt to different environments.
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 61 3.1. Introduction Phages are highly diverse viruses that specifically infect bacteria and are ubiquitous in nature. These viruses have been intensively studied and have highly contributed to the progress of various research fields [1]. Besides the major advances in phage research, such as high-throughput genome sequencing that contributed to better understand the enormous molecular diversity of phages, these viruses still represent the “dark matter” of biological world [2]. The principal knowledge gaps in phage biology are linked to the identification and comprehension of the interactions between phage and bacteria during the viral infection cycle, at a systems biology level [3,4]. Understanding these interactions can reveal how phage modulate the bacterial metabolism and inhibit bacterial defence mechanisms to establish an effective infection [5]. Several omics approaches have contributed to the discovery of new phage-host interactions and mechanisms. Among these, transcriptomics analysis using RNA-seq is emerging as a valuable tool to study phage and bacteria gene expression during specific steps of infection, enabling the discovery of new genes and their functions, and the identification of regulatory RNAs [6]. The first studies have applied RNA-seq to unveil interactions between phage-bacteria pairs [6–15]. These works are focused on the study of phage and bacterial transcriptome in standard growth media at different stages of infection (usually early, middle, and late infection). Despite providing a deeper knowledge on phage-host interactions, these studies do not provide insights about the molecular mechanisms of interaction that occur when bacteria are exposed to non-optimal growth conditions. Therefore, increasing the complexity of these assays by introducing conditions that better mimic the real environments where phage and bacteria interact, might clarify if there are novel phage-host interactions to be discovered. This is particularly important in a context of phage therapy, where phages interact with bacteria in human physiological conditions, which are far from the ideal growth environments provided by standard optimal growth media. Here it was studied the transcriptional profiles of P. aeruginosa PAO1 cultures infected with phage LUZ19 under a standard optimal growth medium versus a MCCM medium. LUZ19 phage is a wellcharacterized virulent podovirus that targets P. aeruginosa [16,17], and a candidate phage for therapeutic purposes. Previous studies of LUZ19 transcriptome revealed a clear temporal expression pattern within the phage, as well as a major impact on transcription of host metabolic genes and on type IV pilus expression regulation [16]. In this chapter, it is investigated the effect of different media in both phage and bacteria transcriptome at different stages of infection. Using RNA-seq, it is aimed to understand the gene
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 62 expression patterns associated with the infection process in standard LB medium versus MCCM medium. This work constitutes a first step towards a better understanding of phage-host interactions under conditions that better mimic the real P. aeruginosa infections in human patients. 3.2. Materials and Methods 3.2.1. Bacterial strains, phage propagation and growth conditions The reference strain P. aeruginosa PAO1 (DSM22644) from The German Collection of Microorganisms and Cell Cultures was used in all experiments and was grown at 37°C on Lysogeny Broth (LB) or LB agar (LB with 1.2% (w/v) of agar). Specific experiments were performed using Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM:F12) supplemented with 2% (v/v) of Ultroser-G Serum Substitute Medium. This medium supports the growth of human airway epithelial cells and was chosen to mimic human bronchial physiological conditions, referred as MCCM [18]. Pseudomonas phage LUZ19 [17] was propagated on P. aeruginosa PAO1 cells by standard soft agar overlay, followed by PEG8000 precipitation and stored in phage buffer pH 7.5 (10mM Tris-HCl, 10mM MgSO4, 150mM NaCl) at 4°C [19]. Phage titration was performed using the double‐agar layer [20]. One-step Growth Curves (OSGC) were established on either LB or MCCM media. Briefly, cultures grown until OD600nm=0.3 were infected with phage at a multiplicity of infection (MOI) of 0.01 and incubated at 37ºC with 120rpm for 5min. The mixture was centrifuged (7,000× g at 4°C, 5min) to remove free phages and then the pellet was resuspended in fresh medium. Phage titer was quantified at 5min intervals over a period of 30min. The PFU/cell was accessed through the ratio between the PFU/mL in each time point and the initial PFU/mL (t=0min). 3.2.2. Synchronized infection assays P. aeruginosa PAO1 overnight cultures were diluted 1:100 in 25mL of either fresh LB or MCCM medium and grown to the early-exponential phase (OD600nm=0.3, corresponding to approximately 2×108 CFU/mL). At this point, the number of colony forming units of the culture (CFU/mL) were counted by plating serial dilutions in LB plates [21] and a sample of 4.5mL of culture was taken simultaneously (t=0min). This sample was immediately mixed with 1:10 vol of an ice-cold stop solution (1:10 buffered phenol, 9:10 absolute EtOH) and kept on ice to stabilize RNA. The remaining culture (≈20mL) was transferred to a pre-warmed Erlenmeyer flask and infected with phage LUZ19 at a MOI = 75 (to ensure synchronous infection of the culture) and incubated at 37°C and 120rpm.
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 63 At t=5 (early infection), t=10 (middle infection) and t=15 (late infection) minutes post-infection, 4.5mL of culture was mixed with chilled stop solution and placed on ice. Immediately after, all collected samples were frozen at -80°C to block RNA transcription and degradation until sample processing. Biologically independent infection experiments, showing a CFU reduction of at least 95% within 5min post-infection (only 5% bacterial culture survival to LUZ19 infection) were considered as synchronized infection. Triplicate samples from synchronized infections were used for total RNA extraction. 3.2.3. Total RNA extraction, quantification, quality assessment and rRNA depletion Samples from synchronized assays were thawed on ice and centrifuged (3,345× g , 20min, 4°C). Total RNA was extracted from cell pellets using the Purelink RNA Mini Kit (Invitrogen), according to the manufacturer’s instructions. Subsequently, each sample was treated with TURBO DNAse (Invitrogen) at 37°C to remove genomic DNA (gDNA) and then co-precipitated overnight at -20°C with 1:10 (v/v) NaOAc and 2.5 vol of absolute EtOH. Precipitated RNA samples were centrifuged (16,000× g , 4°C, 1h) and pellets were washed twice with 70% (v/v) EtOH. The air-dried RNA pellets were dissolved in 60µl of DNAse and RNAse free water (Invitrogen). Total RNA concentration and purity were measured on a Nanodrop 2000 Spectrophotometer (Thermo Scientific, MA, USA) and on a Qubit 4 Fluorometer (ThermoFisher Scientific). In addition, the absence of gDNA contamination was confirmed by PCR using specific primers for both phage LUZ19 (Forward: 5’-ccgacaagatcgtccgcatgc-3’, Reverse: 5’-ggctgaagctacgcttggcc-3’) and its host (Forward: 5’- cggtcggaatctctacgcacc-3’, Reverse: 5’-cgacgccaacaacgccacg-3’). To assess RNA integrity, samples were analysed on a Bioanalyzer (Agilent, Santa Clara, California, US) with a RNA 6000 Nano Kit (Agilent) according to manufacturer’s instructions. Samples with good RNA integrity (RNA integrity number (RIN) > 8) were treated with RiboMinus™ Transcriptome Isolation Kit (bacteria) (Invitrogen) to deplete ribosomal RNA (rRNA), according to the manufacturer’s instructions. 3.2.4. cDNA libraries preparation and sequencing Depleted rRNA samples were precipitated and washed as mentioned previously and resuspended in 18-19µL of Fragment Prime Finish mix from TruSeq Stranded mRNA Library Prep (Illumina). Successful rRNA depletion was verified on a Bioanalyzer and the final concentrations were determined using the Qubit 4 Fluorometer (Invitrogen).
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 64 rRNA-depleted samples were processed immediately, according to TruSeq Stranded mRNA Library Prep recommendations. In the end, cDNA libraries were evaluated on a Bioanalyzer (Agilent) to confirm average fragments between the range of 200-300bp and final concentrations were measured on a Qubit. The libraries’ normalization and pooling were performed according to MiniSeq System Denature and Dilute Libraries Guide: protocol A from Illumina. For each run, a library of three samples with equimolar amounts was combined with PhiX control (spike-in of 0.5-2%) and paired end sequenced (2×75bp) on an in-house Illumina Miniseq sequencer. 3.2.5. Data analysis The quality of cDNA reads was analysed using FastQC (version 0.11.2) [22]. Paired-end reads were aligned to the LUZ19 and P. aeruginosa PAO1 reference genomes (NC_010326.1 and NC_002516.2) using Bowtie 2 [23]. Next, the aligned reads were assigned to gene features using featureCounts [24]. Reads that mapped to rRNA (60-80%) were removed in silico . Subsequently, counts were first normalized by gene length and then by the number of non-ribosomal reads in each sample to obtain RPKM values. Samples variance was analysed using Principal Component Analysis (PCA) in R and outliers were excluded from transcriptional analysis. Therefore, samples from assay MCCM_3 were discarded while samples from two independent assays in MCCM and samples from three independent assays for LB medium were considered for further analysis. Heat map of P. aeruginosa PAO1 genes was also built (Figure S3.3). Differential gene expression analysis was performed using negative binomial distribution test from DESeq Bioconductor package in R [25]. ANOVA test was performed to identify the most significantly up or downregulated genes. The p-values were adjusted using the “BH” method [26], which is the standard method built into the DEseq package and are referred to as “padj”. Raw data and processed data are available under the accession GSE162278. 3.3. Results 3.3.1. Phage transcripts gradually take-over the total transcriptome in both LB and MCCM media Unveiling phage-host interactions depends on the knowledge acquired during infection process in a one-step infection cycle, as previously optimized [16]. To get a global overview of phage infection at the transcriptional level, it was tracked the non-rRNA transcripts as a function of time during the infection, both for P. aeruginosa PAO1 grown in LB and in MCCM medium, as illustrated in Figure 3.1. Before
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 65 infection (t=0min), all transcripts logically map to the host genome. Five minutes post infection, this percentage varies between 60% and 85%. However, at the middle (t=10min) and late (t=15min) time points, the total number of host reads are reduced to 20-25% and 3-8%, respectively. At this late stage of infection, the reads that map to phage genome dominate the sample in both LB and MCCM cultures. Figure 3.1Percentage of reads that map to the genomes of phage LUZ19 and P. aeruginosa PAO1 for individual replicate samples during synchronized infections in LB or MCCM medium. Comparing the phage transcriptional maps for time points 5, 10 and 15min in both media, expression patterns of the individual time points appear to correlate well to each other (Figure 3.2). Furthermore, these transcriptional maps correlate well with previous data and imply a logical progression from early (host adaptation), to middle (DNA replication) and late (structural and lysis proteins) expression for this phage [16]. However, it should be noted that a slight delay in phage transcription was observed for the MCCM-grown, infected cultures. Nevertheless, this minor discrepancy does not impact the results and interpretations outlined in the next sections, in fact OSGC demonstrated that the infection occurs equally in both medium in terms of latent period (Figure S3.1), but there was a visible change in the burst size when it is used different media. Zooming in on the expression levels of individual phage genes allows to identify differentially expressed genes (p-value<0.05) at early, middle and late stages of infection (comparing with uninfected cultures) for both growth media conditions. Overall, phage gene expression levels do not differ between the LB and MCCM grown bacterial cultures.
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 66 Figure 3.2Distribution of cDNA reads over phage genome at early (t=5min), middle (t=10min) and late (t=15min) infection when LB or MCCM media were used. 3.3.2. P. aeruginosa PAO1 transcription is markedly impacted by the growth media used To better understand the influence of growth media on phage infection, the medium impact on the bacterial transcriptome (without phage infection) was analysed as a first step. The indication that
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 73 structure and biogenesis PA4740 pnp [J] Translation, ribosomal structure and biogenesis 0.8 2.90E -02 1.4 1.22E -04 0.53 0.17 1.88 0.00 0.07 0.95 1.10 0.01 PA4936 Probable rRNA methylase (23 S) [J] Translation, ribosomal structure and biogenesis 0.9 7.00E -03 1 2.10E -02 -0.24 0.66 0.85 0.15 -0.73 0.46 -0.62 0.49 PA1097 fleQ [K] Transcription - 1.1 5.50E -06 -1.2 1.00E -03 -1.00 4.67E -04 -1.06 0.02 -0.81 0.09 -1.39 1.66E03 PA4462 rpoN [K] Transcription - 0.8 2.10E -02 -1.8 3.10E -04 -0.95 3.52E -03 -1.54 0.02 -1.07 0.01 -1.77 3.81E03 PA4581 rtcR [K] Transcription - 0.8 2.10E -02 -1.6 4.00E -03 -0.55 0.30 -1.31 0.22 -0.03 0.99 -1.36 0.33 PA4745 nusA [K] Transcription 0.9 1.20E -02 2.2 1.68E -10 0.65 0.04 1.61 1.88E -04 0.49 0.45 1.68 5.65E05 PA5255 algQ [K] Transcription - 0.7 1.70E -02 -1.7 8.25E -08 -1.14 1.41E -03 -1.97 4.41E -07 -1.36 0.00 -2.10 6.25E07 PA3159 wbpA [M] Cell wall/membrane/ envelope biogenesis Cellular processes and signalling 0.8 8.00E -03 1 4.60E -02 0.27 0.62 0.06 0.97 -0.26 0.73 -0.31 0.78 PA0623 Probable bacteriophage tail tube protein [S] Function unknown Poorly characterized 0.9 1.40E -02 0.9 4.80E -02 2.55 2.28E -12 2.50 7.20E -10 2.03 5.84E -07 2.58 4.46E09 PA3234 Probable sodium: solute symporter [S] Function unknown 1.3 2.00E -03 0.9 2.50E -02 1.60 0.01 -0.41 0.66 0.35 0.85 -0.58 0.58 PA4611 Hypothetical protein [S] Function unknown - 2.5 3.40E -02 -1.7 3.97E -04 -1.96 0.14 -1.17 0.10 -3.40 0.02 -1.28 0.12 PA4746 Hypothetical protein [S] Function unknown 0.8 2.80E -02 2.5 5.81E -06 1.17 5.76E -04 2.16 8.62E -04 0.39 0.57 2.15 1.06E03 PA3496 Hypothetical protein [S] Function unknown - 0.9 6.00E -03 -2.2 2.59E -06 -0.75 0.09 -0.48 0.77 0.16 0.88 0.09 0.97 PA3233 Hypothetical protein [T] Signal transduction mechanisms Cellular processes and signalling 1.3 1.40E -02 1.5 2.00E -03 1.82 2.60E -05 -1.29 0.09 0.48 0.78 -1.17 0.23
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 74 3.3.4.2. Impact of phage infection on the host’s two-component regulatory systems Among the 56 PAO1 genes targeted at 5min post-infection, many are involved in two-component regulatory systems ( wspAchemotaxis family, C4-dicarboxilates transport and RpoN –Ntrc family, FdnHNarL family, braDE -OmpR family, arcB -OmpR family), and have similar expression profiles in both media. It is worth noting that the observed genes are all upregulated, with the exception of transcriptional regulator RpoN, which is not consistent with dctPQM genes expression. Apart from the wspA gene, which encodes a probable chemotaxis transducer, the majority of these genes are involved in metabolism ( braDE : transport of amino acids, arcB : L-arginine metabolism, dctPQM genes: transport of c4-dicarboxilates, fdnH : nitrate respiration, rpoN: transcriptional repressor dctPQM genes). According to KEGG pathways, all mentioned genes are also part of two-component systems, which are responsible for bacterial recognition and transduction of certain signals. 3.3.4.3. Impact of phage infection on replication-, transcriptionand translationassociated genes Expression levels of bacterial genes involved in DNA replication, transcription (transcriptional regulators, RNA repair systems, rRNA methylation) and translation (mRNA degradation and processing) represent a second category of genes influenced by phage infection. These include elongation factor G, ribonuclease E, fleQ , pnp , rRNA methylase, rpoN , rtcR , nusA , and algQ genes. These genes are differentially transcribed after phage infection in different media, leading to the same response (except elongation factor G and ribonuclease HII), yet a little more attenuated in MCCM medium. 3.3.5. Growth medium influences gene expression levels at early infection Our initial experiments show that the growth media has only a minor influence on the phage transcriptome but greatly affect bacterial transcripts. In this section it is highlight the specific host responses during early infection that are driven by the growth medium. Table 3.2 presents PAO1 genes that were significantly, yet differentially expressed at early infection between both media. A first example is glpD gene, that is upregulated in MCCM medium and downregulated in LB medium, indicating a higher necessity of the expression of this gene in MCCM. Another bacterial gene that was upregulated in MCCM yet downregulated in LB medium after phage infection is thiI gene. Additionally, a clearly differentiated response between LB and MCCM during phage infection was also observed for dadX and dadA , where are upregulated in MCCM medium, but in LB medium, are downregulated.
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 75 Beyond leading to a different transcription of metabolic genes during phage infection, the growth medium also influenced genes with functions related with transcription and translation. Namely, the gene that encodes ribonuclease HII (degrades RNA from RNA-DNA hybrids) [27] is targeted after phage infection, being downregulated in LB but upregulated in MCCM medium. Similarly, elongation factor G ( fusA2 ), that is responsible for the catalysation of GTP-dependent ribosomal translocation step during translation elongation, an important step during translation [28], is upregulated in MCCM but the same is not observed for LB media. Another interesting observation is the differential transcription for the transcriptional regulator betI . This gene is upregulated in MCCM medium and downregulated in LB medium. When transcribed, betI represses betT , that is involved in choline transport and represses betAB genes expression, which in turn are involved in choline oxidation to generate glycine betaine [29]. Table 3.2Differentially expressed P. aeruginosa PAO1 genes at 5 min post-infection that have opposite expression according to the medium. Green cells indicate upregulation, red cells indicate downregulation, yellow cells indicate padj<0.05. Gene Annotation COG category Functional Category LB t=5min MCCM t=5min LB t=10min MCCM t=10min LB t=15min MCCM t=15min Log2 Fold padj Log2 Fold padj Log2 Fold padj Log2 Fold padj Log2 Fold padj Log2 Fold padj PA3584 glpD [C] Energy production and conversion Metabolism -1.3 1.00E03 2.5 1.32E06 -1.03 2.28E02 1.51 9.88E02 -0.27 8.06E01 0.41 8.81E01 PA2204 Probable binding protein component of ABC transporter [E] Amino acid transport and metabolism -2 9.32E05 2.9 3.70E15 -1.06 9.15E02 1.49 6.39E02 -0.37 8.06E01 0.78 6.26E01 PA5304 dadA [E] Amino acid transport and metabolism -0.9 2.20E02 2.8 7.28E10 0.10 8.54E01 3.82 4.45E20 0.67 2.29E01 2.69 1.21E08 PA5429 aspA [E] Amino acid transport and metabolism -1.2 4.68E06 1.9 5.00E03 -0.67 2.88E02 3.02 2.91E06 -0.53 3.63E01 2.75 5.32E05 PA0975 Hypothetical protein [H] Coenzyme transport and metabolism -0.8 4.00E03 1.1 1.60E02 -0.43 2.36E01 0.23 8.77E01 -0.16 9.08E01 0.52 6.60E01 PA5118 thiI [H] Coenzyme transport and metabolism -0.9 1.00E03 1.1 3.30E02 -1.05 5.70E05 1.32 3.06E02 -0.88 5.46E02 1.28 4.40E02 PA2557 Probable AMP-binding enzyme [I] Lipid transport and metabolism 1.2 3.50E02 -1 1.40E02 1.48 7.41E03 -2.14 1.67E04 1.27 2.83E01 -3.29 4.52E08 PA2071 elongation factor G [J] Translation, ribosomal structure and biogenesis 1.1 1.00E03 -1.2 3.00E03 0.74 1.25E01 -1.15 4.50E02 0.41 7.65E01 -1.61 3.12E02
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 76 PA0483 Probable acetyltransferase [K] Transcription Information storage and processing 1.1 4.10E02 -1.6 2.00E03 0.37 6.07E01 -2.20 2.25E03 0.36 8.39E01 -3.65 2.14E03 PA0528 Transcriptional repressor [K] Transcription -1.2 6.00E03 1.8 4.70E02 -1.08 1.98E02 1.48 4.27E01 -0.74 4.47E01 -0.45 9.50E01 PA5374 betI [K] Transcription -1.3 1.00E03 4.5 2.09E16 -1.63 5.78E05 4.55 8.04E16 -1.34 2.25E02 4.35 3.08E07 PA3642 ribonuclease HII, rnhB [L] Replication, recombination and repair -0.8 3.20E02 1 2.70E02 -1.64 1.64E05 0.45 6.90E01 -1.30 1.09E02 0.14 9.48E01 PA5302 dadX [M] Cell wall/membrane/envelope biogenesis Cellular processes and signaling -1 9.00E03 2.9 4.67E07 -0.18 7.59E01 3.65 6.07E16 0.51 5.36E01 2.88 3.62E08 PA1181 Hypothetical protein, sensor protein [T] Signal transduction mechanisms Cellular processes and signaling -0.9 2.00E03 1.1 9.00E03 -0.71 5.00E02 1.21 2.72E02 0.02 9.89E01 0.62 5.73E01 3.4. Discussion 3.4.1. Phage transcripts are not impacted by the growth media but bacterial transcripts are markedly affected A global tracking of bacterial and phage transcripts over time indicated that the host machinery is required to a high degree for phage reproduction as observed previously [16], and it appears that this pattern is independent of the used media. Independent of growth conditions, it is also the phage transcriptional map, which suggests a logical progression from early (host adaptation), to middle (DNA replication) and late (structural and lysis proteins) genes expression for this phage [16]. A slight delay in phage transcription was observed for the MCCM-grown infected cultures as a probable result of reduced bacterial fitness in this media, since that starvation and metabolic state have been already shown to impact phage infection parameters [30]. This phenomenon is also observed in the phage burst size, that variates according to bacterial growth rate, as demonstrated by Nabergoj and collaborators that showed that the burst size of T4 phage increases linearly with the increase of E. coli k-12 growth rate [31]. Nevertheless, this minor discrepancy does not impact the results obtained for phage transcriptome in both media, since that overall, phage gene expression levels do not differ between the LB and MCCM grown bacterial cultures. Similarly, the impact of the media on bacterial transcriptome during phage infection was intended to be analysed, considering that media has a significant impact on bacterial behaviour, as mentioned in a study by Wagner and collaborators, which highlighted that the expression levels of QS genes is
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 77 influenced by growth medium [32]. Here it was founded a high number of differentially expressed genes specially with categories related with metabolism, transcription, and cell wall/membrane/envelope biogenesis. In particular, genes regulated by iron availability were induced in MCCM media comparing with LB media, indicating iron starvation in MCCM medium. This is consistent with in vivo host conditions, where iron availability is low due to the presence of iron binding proteins [33], and with the fact that mechanisms of iron uptake are related to P. aeruginosa virulence and pathogenicity in lung infections [34]. Consistently, genes involved in bacterial pathogenicity and virulence were also induced under MCCM relative to LB medium, including genes involved in secretion systems (type I, II, III and IV), two-component system, biofilm formation, QS, multidrug resistance and virulence factors. Thus, all together these difference between MCCM and LB media proves that the composition of the media drastically influences the P. aerugino sa pathogenicity and virulence in addition to the bacterial metabolism. Within the focus of this research, these differences among the media serve as a control, enabling teasing out the phageinfection induced transcriptional responses between both cultures. 3.4.2. Elucidating the bacterial response to phage infection When the total transcriptome is examined during infection, the major bacterial responses to phage infection occur during the early infection period, as observed previously [5]. Therefore, this early phase is critical to reveal potential bacterial defence mechanisms, as well as processes triggered by phage for its infection cycle, since that there is a transcriptional convergence observed for the middle and late samples for both growth media. This indicates that during the course of infection, the impact of the growth medium is nullified, and that the transcriptional differences between samples are impacted by the high expression of phage genes and by bacterial genes targeted by phage. Thus, during middle and late infection, bacterial responses that are associated with growth medium conditions decrease relative to responses triggered by phage infection. 3.4.2.1. Phage infection impact in energy metabolism regardless of bacterial growth conditions Considering the importance of early infection stage, differentially expressed genes in both growth conditions were tracked, indicating a strong manipulation of metabolic pathways, particularly related with energy production through TCA cycle. An example, is the induction of pathways related with C4dicarboxilates acquisition, being these known to be the P. aeruginosa preferential carbon and energy source [35,36]. This suggests that during early infection, the transport of these substrates is crucial to obtain high levels of energy. This hijacking of molecular resources is consistent with the coliphage T4
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 78 infection process, and it is associated with energy requirement for phage particle formation [37]. The changes in metabolism observed in LUZ19-infected P. aeruginosa cultures at early stage are driven by phage at the very beginning of infection to assure the progression of infection. Another example is the induction of riboflavin and its biological active forms, which are widely used as cofactors by bacterial enzymes during redox reactions [38], but when its biosynthesis is inhibited, it leads to nutrient deficiency and oxidative stress [39]. The differentially expressed genes associated with vitamins and cofactors biosynthesis suggests that phages require their presence during infection. Correspondingly, an additional clue towards energy metabolism is provided by the upregulation fdnH gene, that is involved in the conduction of electrons from formate to nitrate. This enzyme has particular relevance during nitrate respiration that often occurs under anaerobic conditions, being the gene expression induced in the presence of nitrate [37]. In this dataset, the expression of the β-subunit of this protein is upregulated in early infection, compared with uninfected cultures and regardless of the media. This indicates that under phage infection, the expression of formate dehydrogenase-N might be required since this enzyme is responsible for proton motive force generation that ultimately results in energy production. Metabolic pathways that are also triggered after phage infection involve the conversion of putrescine to produce succinate, a TCA intermediate, and ultimately leads to the production of energy. However, related with this pathway, the transport of putrescine and spermidine is also induced, which hints at a structural relevance for spermidine transport, beyond the purpose of energy acquisition [40]. Indeed, it was previously suggested that spermidine compacts and stabilizes viral DNA in the capsid [41,42], and it is essential for N4-like Pseudomonas phages infection cycle [39]. This is a good example of multipurpose genes that are probably activated by phage infection to participate, in this specific case, in two mechanisms such as energy generation and phage DNA stabilization. 3.4.2.2. Impact of phage infection on the host’s two-component regulatory systems The two-component regulatory systems consist of mechanisms that enable P. aeruginosa PAO1 to sense and respond to environmental stimuli and potentially to phage infection. The results demonstrated several genes related with two-component system being altered. Among them was rpoN that is reported to positively regulate dctPQM genes [36], although in this data, expression of this transcriptional regulator is not consistent with dctPQM genes expression. Still, C4-dicarboxilates abundance stimulates the expression of its own transporters, such as dctPQM gene products. Another example is gene fdnH, that is triggered upon nitrate presence that works as a signal for promotion of nitrogen metabolism.
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 79 Other two-component system involved gene that was impacted by phage infection according to the obtained results were genes involved in the signal transduction of anaerobic respiration inhibition by quinone electron carriers ( arcB gene). Other signal mechanisms targeted during early infection were: the signal mechanism that promotes bacitracin efflux when it is abundant ( braDE genes); and signal obtained from surface cell contact that induces extracellular polysaccharide production ( wspA ). Also, RpoN participates in several signal-response mechanisms that results in a myriad of activities: the induction of amino acids uptake and metabolism; attaching and effacing (AE) lesions; pilA expression; regulation of biofilm formation, motility and virulence; flagellar assembly. In future, it would be of interest to understand whether the upregulation of two-component regulatory system genes is a bacterial answer to phage infection or if these genes are induced by phage for successful infection. Presumably, the higher expression levels of genes involved in two-component regulatory systems represent a bacterial response to phage infection, as suggested in a previous work, in which the upregulation of pqsABCDE operon was proposed to function as a signal to shutdown bacterial metabolism of non-infected cells [7]. While expression of pqs operon was higher during late infection in LB, this difference wasn’t significant compared to uninfected cultures. By contrast, the operon was significantly downregulated in MCCM, highlighting the importance of growth conditions during infection assays that also contribute to changes in gene expression. 3.4.2.3. Impact of phage infection on replication-, transcriptionand translationassociated genes Several changes were observed concerning the transcription process at early infection, including changes in the expression of genes involved in mRNA degradation ( pnp gene-polyribonucleotide nucleotidyltransferase, and ribonuclease E) [43,44], indicating that, at early infection, mRNA turnover is tightly controlled by the phage. Another major change was related with transcription termination/antitermination protein ( nusA ), indicating the necessity of a strict regulation of transcriptional mechanisms during phage infection. The upregulation of this gene might be part of an underlying mechanism that phage deploys to tightly control host machinery. It would be interesting to investigate this mechanism further in future. In addition, a tight regulation of RNA turnover early in infection is also confirmed by the differential expression of RNA repair system regulators rpoN (RNA polymerase sigma-54 factor) and rtcR (transcriptional regulator of rctAB genes) which are downregulated independent of the growth medium. Besides rpoN transcriptional regulator expression was not consistent with dctPQM genes, its transcription was consistent with rctAB operon system expression, indicating that the upregulation of this
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 80 transcriptional regulator has as purpose the influence of RNA turnover mechanisms. Additionally, RtcR is known to work through RpoN to positively activate the expression of rctAB operon that encodes for two constituents of tRNA repair system [45]. The downregulation of these two regulators indicates that the RNA repair system is repressed, which would be distinct from the P. aeruginosa strain PAK infected by phage PAK_P3, where the expression of rtcAB was significantly upregulated [5]. This indicates that a broad transcriptional regulator, such as RpoN, functions in distinct ways in bacterial responses and other mechanisms are probably associated to ensure expression regulation. Two additional transcriptional regulators are significantly targeted at this early phase: transcriptional regulator fleQ and betI. FleQ, together with FleR, is involved in a regulatory cascade that controls motility and adhesion in P. aeruginosa [46]. Considering the crucial role of fleQ in the control of motility and adhesion, its downregulation can lead to a non-control of this important bacterial aspect, which might impact bacterial infection establishment. Similarly, the downregulation of global transcriptional P. aeruginosa regulator and repressor of lasR and rhl, the algQ gene, might suggest that bacterial control is no longer available, leading to a fail in the regulation of QS systems [47]. With this data it was not possible to determine if the shutdown of bacterial functions such as QS, motility and adhesion would be beneficial for phage infection, being this a probable mechanism of phage to manipulate its host. 3.4.3. Phage targets differently specific metabolisms to assure successful infection Under different media, at early infection, phage manipulates glycerol metabolism. In MCCM medium glpD is induced, but not in LB. This gene encodes a glycerol-3-phosphate dehydrogenase (G3P), which acts as a key player in energy metabolism (involved in glycerol metabolism). It is known that the complete abolishment of this gene leads to alterations in amino acids metabolism and in the production of TCA intermediates, ending in a general stress response [48]. The tight control of this gene expression seems to be imperative for proper metabolism functionalization. As such, it appears to be an essential gene to target to achieve ideal infection conditions. A similar behaviour is observed for gene thiI that codes for a protein involved in the biosynthesis of thiazole (precursor of thiamine -vitamin 1). In bacteria, thiamine is an important cofactor with a fundamental role in energy metabolism [48]. The induction of this gene exclusively in MCCM media might be associated with the low concentration of this vitamin 1 precursor in this medium, which in the infection context might be highly required. Alanine metabolism-associated genes ( dadAX genes) seem to be important under MCCM medium presence. The presence of L-alanine (prevenient from intrinsic metabolism) is reported to be the
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 81 main inducer of dadAX genes [49], as well as nitrogen starvation. The obtained results demonstrate that possibly, the MCCM medium represents an energy starvation condition for PAO1, contrary to LB, which would activate these genes to produce nitrate and in pyruvate fermentation. Indeed, it was previously reported that nitrate can serve as alternative of electrons acceptor under anaerobic conditions in PAO1, and that pyruvate fermentation plays an important role as a general endogenous survival metabolism in response to energy starvation [50]. As such, the use of MCCM medium might better mimic anaerobic conditions, typical of biofilm and cystic fibrosis infection conditions, as reflected by the shift in amino acids metabolism. Finally, in MCCM medium, an induction of betI reveals a potential increase in glycine betaine production. This product is reported to function as an osmo-protector, promoting P. aeruginosa virulence and survival in mouse lung [29]. Inhibition of choline-betaine pathway is highly toxic to P. aeruginosa but upon phage infection in MCCM medium, the upregulation of betI gene leads to the repression of betAB genes, which was also observed in PaP1-infected P. aeruginosa [51]. Overall, this work was able to show differences in bacterial gene expression patters following phage infection in LB compared to MCCM, and it is tempting to speculate that these changes are triggered by the phage, although a response of the bacterium in this medium, in the presence of phage remains equally plausible. Regardless, these data suggest optimal conditions for phage propagation are attained, in view of the expression profile convergence later in infection. 3.5. Conclusions RNA-seq of LUZ19 - infected P. aeruginosa PAO1 cultures demonstrates that major transcriptional changes occur at the early infection stage, during bacterial takeover. This early expression stage is marked by specific changes in expression levels, targeting energy metabolism, translation and transcription. A medium-specific response to phage infection was also observed, targeting a handful or regulatory genes, that share a common response in terms of adapting to the limitations imposed MCCM medium compared to lysogeny broth. It is interesting that this response visualized at early infection doesn’t influence LUZ19 infection that proceeds largely uninterrupted and efficiently, despite these limitations. From this chapter, it is tempting to speculate that this small, yet densely coded phage has the genomic versatility to readily adapt the host machinery towards its needs. This would cast a different light on the types of analyses needed to understand some of the ‘viral dark matter’ genes, and their role under non-standard growth conditions.
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 82 From a phage therapy perspective, a first step towards assessing the efficiency and impact of this phage in MCCM medium was taken, paving the way to uncover the phage infection process in conditions comprising the three partners involved: phage-bacteria-human cell. Within this context, the versatility shown by LUZ19 appear promising, as this phage is well adapted to the imposed different environmental conditions. 3.6. References 1. Salmond GPCC, Fineran PC: A century of the phage: Past, present and future. Nat Rev Microbiol 2015, 13:777–786. 2. Hatfull GF: Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. J Virol 2015, 89:8107–8110. 3. Zhao X, Chen C, Shen W, Huang G, Le S, Lu S, Li M, Zhao Y, Wang J, Rao X, et al.: Global Transcriptomic Analysis of Interactions between Pseudomonas aeruginosa and Bacteriophage PaP3. Sci Rep 2016, 6:1–12. 4. De Smet J, Hendrix H, Blasdel BG, Danis-Wlodarczyk K, Lavigne R: Pseudomonas predators: understanding and exploiting phage–host interactions. Nat Rev Microbiol 2017, 15:517–530. 5. Chevallereau A, Blasdel BG, De Smet J, Monot M, Zimmermann M, Kogadeeva M, Sauer U, Jorth P, Whiteley M, Debarbieux L, et al.: Next-Generation “-omics” Approaches Reveal a Massive Alteration of Host RNA Metabolism during Bacteriophage Infection of Pseudomonas aeruginosa . PLOS Genet 2016, 12:e1006134. 6. Sacher JC, Flint A, Butcher J, Blasdel B, Reynolds HM, Lavigne R, Stintzi A, Szymanski CM: Transcriptomic analysis of the Campylobacter Jejuni response to T4-like phage NCTC 12673 infection. Viruses 2018, 10. 7. Blasdel BG, Ceyssens P-JJ, Chevallereau A, Debarbieux L, Lavigne R: Comparative transcriptomics reveals a conserved Bacterial Adaptive Phage Response (BAPR) to viral predation. bioRxiv 2018, doi:10.1101/248849. 8. Yang Z, Yin S, Li G, Wang J, Huang G, Jiang B, You B, Gong Y, Zhang C, Luo X, et al.: Global Transcriptomic Analysis of the Interactions between Phage ɸAbp1 and Extensively DrugResistant Acinetobacter baumannii . Am Soc Microbiol 2019, 4:1–12. 9. Mojardín L, Salas M: Global Transcriptional Analysis of Virus-Host Interactions between Phage ϕ29 and Bacillus subtilis . J Virol 2016, 90:9293–9304.
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 89 Siderophores transport PA2466 2.59 1.92E-11 Ferrioxamine receptor, FoxA: siderophore transport -0.26 8.79E-01 -0.65 3.85E-01 PA2467 3.67 1.13E-21 Anti-sigma factor Foxr 0.12 9.63E-01 -4.24 9.19E-15 PA2468 4.50 2.30E-36 ECF sigma factor FoxI 0.09 9.67E-01 -4.18 8.80E-23 PA4156 3.23 1.67E-10 FvbA, siderophore transport 1.10 2.62E-01 -0.24 8.63E-01 PA4675 2.88 1.03E-12 ChtA: siderophore transport -0.63 2.84E-01 -0.53 4.61E-01 PA1365 2.40 3.91E-13 Probable siderophore receptor: outer membrane receptor for ferrienterochelin and colicins -0.10 9.63E-01 0.29 7.67E-01 PA1910 2.42 8.76E-09 Ferric-mycobactin receptor, FemA -0.32 8.78E-01 -0.57 4.81E-01 PA1911 4.77 1.44E-16 Sigma factor regulator, FemR 0.43 9.06E-01 -3.09 2.23E-05 PA1912 5.19 4.27E-23 ECF sigma factor, FemI 1.63 1.48E-01 -2.74 6.31E-07 PA2033 7.52 1.02E-90 Hypothetical protein: fad-binding domain, ferredoxin reductase-type 0.39 8.32E-01 -2.08 1.16E-06 PA0931 3.67 3.11E-32 Ferric enterobactin receptor PirA: outer membrane receptor for ferrienterochelin and colicins -0.44 6.81E-01 -1.08 1.23E-02 PA4160 3.36 9.09E-05 Ferric enterobactin transport protein FepD 1.27 6.34E-01 -2.65 3.48E-02 Ferripyoverdine PA2398 5.21 3.53E-67 Ferripyoverdine receptor, FpvA -0.54 2.62E-01 0.91 1.01E-01 PA2409 2.61 3.12E-13 FpvE -0.20 8.90E-01 0.87 5.67E-02
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 90 PA2403 4.04 7.03E-29 FpvG: pyoverdine biosynthetic process -0.70 4.57E-01 0.30 6.66E-01 PA4168 2.63 4.44E-11 Second ferric pyoverdine receptor FpvB 0.15 9.06E-01 -0.34 7.18E-01 Phenazine biosynthesis PA1902 4.05 2.23E-03 Phenazine biosynthesis protein Phzd, PhzD2 0.12 9.89E-01 0.34 9.68E-01 PA1905 2.15 7.96E-03 Probable pyridoxamine 5'-phosphate oxidase, PhzG2 1.20 3.82E-01 1.52 2.65E-01 PA4210 6.43 8.41E-03 Probable phenazine biosynthesis protein, Phza1 2.19 3.04E-01 -0.40 9.77E-01 PA4211 5.52 4.13E-03 Probable phenazine biosynthesis protein, PhzB1 0.88 6.77E-01 -0.24 9.84E-01 PA4213 3.31 1.08E-02 Phenazine biosynthesis protein PhzD, PhzD1 -0.12 9.88E-01 1.36 8.37E-01 PA4215 2.11 2.42E-02 Probable phenazine biosynthesis protein, Phzf1 -0.01 9.98E-01 0.53 8.39E-01 PA4216 2.12 8.68E-03 Probable pyridoxamine 5'-phosphate oxidase, Phzg1 0.07 9.87E-01 1.21 4.21E-01 PA4217 3.81 2.20E-17 Flavin-containing monooxygenase, PhzS 0.57 5.39E-01 0.98 2.59E-01 Iron obtaining by transport and degradation of heme PA0122 2.69 2.62E-02 RahU: haemolysis by symbiont of host erythrocytes 0.07 9.63E-01 -0.40 9.49E-01 PA0672 5.72 2.38E-54 Heme oxygenase, HemO -0.54 7.78E-01 -0.93 2.45E-02 PA0471 3.68 5.68E-22 FiuR: periplasmic ferric-dicitrate binding protein FerR, regulates iron transport through sigma-19 -0.81 6.45E-01 -3.80 4.40E-14 PA0472 4.40 1.76E-25 FiuI: DNA-directed RNA polymerase specialized sigma subunit, sigma24 family -0.10 9.75E-01 -4.31 7.87E-19
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 91 PA1302 2.24 2.44E-09 Probable heme utilization protein precursor: outer membrane receptor proteins, mostly Fe transport -0.44 6.38E-01 0.21 8.23E-01 Heme uptake by Has system PA3405 6.24 1.85E-07 Metalloprotease secretion protein, HasE 0.14 9.86E-01 1.23 6.59E-01 PA3406 4.63 1.84E-13 Transport protein HasD 0.18 9.67E-01 1.13 2.47E-01 PA3407 5.98 1.78E-48 Heme acquisition protein HasAP 0.04 9.87E-01 0.74 7.68E-01 PA3408 4.71 1.14E-35 Heme uptake outer membrane receptor HasR precursor -0.42 7.61E-01 -1.77 8.43E-03 PA3409 6.02 1.94E-43 HasS -0.41 8.96E-01 -3.34 1.50E-15 PA3410 5.15 3.27E-38 HasI 0.13 9.54E-01 -3.87 4.45E-24 Heme uptake by Phu system PA4705 2.54 3.35E-19 PhuW -0.45 5.09E-01 0.47 4.31E-01 PA4706 3.49 7.89E-30 PhuV 0.02 9.93E-01 -0.01 9.96E-01 PA4707 3.39 1.08E-35 PhuU -0.41 6.70E-01 -0.81 7.79E-02 PA4708 5.39 1.53E-53 Heme-transport protein, PhuT -0.52 6.99E-01 -2.36 2.41E-10 PA4709 5.90 1.16E-65 Putative heme degradation protein -0.02 9.93E-01 -3.38 1.16E-24 PA4710 4.84 7.25E-44 Heme/haemoglobin uptake outer membrane receptor PhuR precursor 0.54 4.94E-01 -3.23 1.71E-14 Pyochelin biosynthesis genes PA4221 4.87 2.61E-47 Fe(iii)-pyochelin outer membrane receptor precursor (FptA), outer membrane receptor for ferric coprogen and ferric-rhodotorulic acid -0.68 5.02E-01 0.03 9.80E-01
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 92 PA4224 4.22 1.24E-15 Pyochelin biosynthetic protein PchG -0.09 9.85E-01 0.78 1.60E-01 PA4225 3.93 6.86E-43 Pyochelin synthetase, PchF -0.09 9.49E-01 0.91 3.03E-02 PA4228 4.67 1.24E-33 Pyochelin biosynthesis protein PchD -0.80 7.48E-01 1.24 2.61E-02 PA4229 4.17 1.96E-09 Pyochelin biosynthetic protein PchC -0.15 9.86E-01 1.10 1.59E-01 Pyoverdine biosynthesis PA2385 7.67 3.25E-116 PvdQ -0.30 9.13E-01 1.14 8.79E-04 PA2386 8.52 7.18E-123 PvdA, l-ornithine n5-oxygenase -0.23 9.15E-01 0.55 3.22E-01 PA2389 3.95 3.04E-25 PvdR -0.41 7.48E-01 0.83 5.36E-02 PA2390 3.95 9.09E-37 PvdT, pyoverdine biosynthetic process -0.73 3.15E-01 0.58 1.98E-01 PA2391 4.24 2.65E-42 Probable outer membrane protein precursor, OpmQ: pyoverdine biosynthetic process -0.59 6.23E-01 0.60 2.23E-01 PA2392 8.58 7.12E-173 PvdP -0.27 8.92E-01 0.28 6.48E-01 PA2393 6.00 9.19E-78 Putative dipeptidase: pyoverdine biosynthetic process, dipeptidase activity 0.68 5.75E-01 0.96 1.39E-02 PA2394 6.12 9.67E-70 PvdN -0.78 6.99E-01 1.19 3.05E-03 PA2395 8.17 1.43E-61 PvdO 1.02 6.99E-01 0.42 5.80E-01 PA2396 8.10 6.06E-132 PvdF, pyoverdine synthetase f 0.04 9.88E-01 -0.10 9.10E-01 PA2397 4.92 1.68E-35 PvdE, pyoverdine biosynthesis protein 0.11 9.61E-01 1.54 4.06E-04
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 93 PA2399 8.03 4.72E-237 PvdD, pyoverdine synthetase d -0.07 9.60E-01 0.23 7.36E-01 PA2400 7.46 1.05E-170 PvdJ, pyoverdine biosynthetic process -0.35 6.57E-01 0.52 2.46E-01 PA2424 8.29 1.40E-211 Pvdl -0.22 8.58E-01 0.50 3.08E-01 PA2425 8.53 5.51E-74 PvdG -0.82 8.46E-01 -0.72 9.94E-02 PA2426 7.85 1.96E-09 PvdS, sigma factor 0.70 8.41E-01 0.49 3.98E-01 PA2413 10.46 5.29E-159 PvdH, l-2,4-diaminobutyrate:2-ketoglutarate 4aminotransferase 0.25 9.34E-01 -0.15 8.37E-01 Table S3.2Upregulated genes in MCCM medium comparatively to LB medium. These genes have various functions related with bacterial pathogenicity and virulence. Interesting upregulated genes in MCCM vs LB at 0 min LB (5 vs 0 min) MCCM (5 vs 0 min) Gene Log2Fold padj Name Log2Fold padj Log2Fold padj T3SS system PA1706 2.35 2.28E-06 Type III secretion protein, PcrV -0.76 3.08E-01 -0.44 7.59E-01 PA1708 4.21 2.83E-06 Translocator protein PopB: Type III Secretion pathway -0.12 9.47E-01 -0.31 9.50E-01 PA1709 3.61 0.002 Translocator protein PopD: Type III Secretion 0.13 9.68E-01 -0.29 9.54E-01 PA1718 3.21 9.58E-16 Type III export protein, PscE -0.63 6.77E-01 0.07 9.72E-01 PA1720 2.02 0.0003 Type III export protein, PscG -0.35 7.66E-01 -0.24 9.00E-01 PA1721 2.90 1.94E-10 Type III export protein, PscH -0.20 9.15E-01 0.41 7.55E-01
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 94 PA1722 2.33 1.25E-07 Type III export protein, PscI -0.35 7.56E-01 0.36 8.03E-01 PA1725 2.71 2.35E-09 Type III export protein Psc: Flagellar biosynthesis/Type III secretory pathway protein FliH -0.35 7.03E-01 -0.19 9.03E-01 PA0044 2.49 0.01 Exoenzyme T, ExoT -0.05 9.77E-01 -0.44 9.21E-01 PA1714 2.28 8.15E-07 ExsD -0.41 4.51E-01 0.33 6.60E-01 PA2191 2.33 2.33E-06 Adenylate cyclase, ExoY -0.71 2.53E-01 -1.53 5.09E-01 PA3841 3.99 1.72E-06 Exoenzyme S 0.03 9.89E-01 -1.17 7.03E-01 PA1713 2.28 1.36E-18 Transcriptional regulator ExsA: AraC-Type DNAbinding domain and AraC-containing proteins -0.44 4.58E-01 0.04 9.83E-01 PA1984 3.71 3.96E-19 NAD+ dependent aldehyde dehydrogenase, ExaC -0.56 7.95E-01 1.74 4.51E-04 Other secretion system PA0423 4.01 1.83E-51 PasP. Polyisoprenoid-binding periplasmic protein YceI, Secreted Factors (toxins, enzymes, alginate) 0.69 2.95E-02 0.16 8.63E-01 PA1249 3.22 9.05E-15 Alkaline metalloproteinase precursor, AprA: Apr Type I secretion system 0.05 9.81E-01 -0.08 9.62E-01 PA2862 3.63 2.19E-09 Lactonizing lipase precursor, LipA: metabolism and protein secretion by the Type II secretion system 1.73 1.27E-01 -0.19 9.02E-01 Type IV secretion system PA2360 2.98 5.26E-11 HsiA3: Predicted component of the Type VI protein secretion system 0.41 8.42E-01 -0.22 8.73E-01 PA2361 2.91 7.75E-16 IcmF3 0.53 6.40E-01 -0.24 7.95E-01 PA2362 3.33 2.84E-06 DotU3: Bacterial secretion system -0.07 9.90E-01 -0.23 8.97E-01 PA2363 3.35 9.93E-15 HsiJ3 0.33 8.27E-01 -0.39 7.16E-01
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 95 PA2364 2.93 1.27E-14 Lip3 -0.21 9.18E-01 -0.27 7.77E-01 PA2365 2.74 6.62E-05 HsiB3 -0.29 9.37E-01 -0.59 6.67E-01 PA2366 3.17 8.55E-14 HsiC3: Predicted component of the Type VI protein secretion system 0.24 9.05E-01 0.02 9.90E-01 PA2367 2.56 5.58E-09 Type VI protein secretion system component Hcp (secreted cytotoxin), Hcp3 0.07 9.87E-01 -0.56 5.78E-01 PA2368 4.50 0.01 HsiF3 1.46 8.49E-01 -1.31 6.33E-01 PA2369 3.35 1.04E-10 HsiG3 0.27 9.33E-01 0.26 8.28E-01 PA2370 2.66 0.02 HsiH3 0.89 8.21E-01 0.65 7.73E-01 PA2371 3.66 1.00E-13 ClpV3, Type iV secretion system -0.76 6.63E-01 0.05 9.71E-01 PA2373 2.23 8.95E-07 VgrG3. Implicated in Type VI secretion and phage assembly 0.11 9.63E-01 0.69 3.11E-01 PA2374 2.70 0.0005 TseF -1.06 7.74E-01 -0.05 9.85E-01 Two-component system PA0176 2.46 4.03E-14 Aerotaxis transducer Aer2: Methyl-accepting chemotaxis protein -0.09 9.67E-01 0.31 6.92E-01 PA0177 2.30 9.51E-08 Probable purine-binding chemotaxis protein: Chemotaxis signal transduction protein 0.05 9.87E-01 0.08 9.57E-01 PA0178 2.22 2.81E-05 Probable two-component sensor 0.12 9.63E-01 0.10 9.46E-01 PA0179 2.72 1.19E-07 Probable two-component response regulator 0.41 8.45E-01 0.19 8.96E-01 PA0180 2.20 6.30E-11 Chemotactic transducer for trichloroethylene [positive chemotaxis], CttP -0.25 8.34E-01 -1.04 6.23E-02
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 96 PA0929 2.36 1.30E-08 Two-component response regulator: DNAbinding response regulator, OmpR family, contains REC and winged-helix (wHTH) domain -0.20 8.81E-01 -3.95 1.81E-20 PA0930 2.73 1.06E-15 Two-component sensor: Signal transduction histidine kinase, HAMP domain -0.69 3.53E-01 -2.55 8.29E-12 PA1342 3.33 3.57E-09 AatJ: ABC-Type amino acid transport/signal transduction system, periplasmic component/domain 1.06 2.60E-03 0.75 8.29E-01 PA0676 2.04 0.002 Sigma factor regulator, VreR. It is involved in signal transduction controlling virulence functions -0.55 8.03E-01 -1.07 3.21E-01 PA2383 2.22 2.49218E-08 DNA-binding transcriptional regulator, LysR family -0.60 6.77E-01 0.24 8.11E-01 PA2571 2.26 1.58E-11 Probable two-component sensor: Signal transduction histidine kinase regulating C4dicarboxylate transport system -0.35 7.58E-01 -0.69 2.33E-01 PA2572 2.45 0.0001 Probable two-component response regulator 0.64 7.30E-01 0.28 7.87E-01 PA2573 2.49 8.15E-18 Probable chemotaxis transducer 0.26 8.04E-01 -0.31 6.81E-01 PA2686 3.35 6.22E-22 Two-component response regulator, PfeR -0.21 9.05E-01 -4.25 1.91E-13 PA2687 2.74 1.86E-15 Two-component sensor, PfeS -1.10 1.07E-01 -1.94 2.82E-05 PA4290 2.74 2.62E-10 Probable chemotaxis transducer 2.31 1.07E-09 -0.76 3.18E-01 PA4296 2.80 7.54E-07 Two-component response regulator, PprB 0.26 9.13E-01 0.27 7.83E-01 PA4520 2.26 4.25E-19 Probable chemotaxis transducer 0.09 9.42E-01 -0.99 2.06E-02 PA1992 2.42 5.22E-10 ErcS: signal transduction 0.91 1.62E-01 -0.14 9.24E-01 Regulators PA3622 3.55 7.73E-30 Sigma factor, RpoS 0.10 9.34E-01 -1.54 7.95E-06
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 97 PA0985 3.71 4.29E-18 Pyocin S5 0.56 6.85E-01 0.28 7.47E-01 Virulence factor PA1041 5.72 1.61E-75 Probable outer membrane protein precursor: Outer membrane protein OmpA and related peptidoglycan-associated (lipo)proteins 0.34 8.21E-01 -1.03 6.29E-03 Biofilm involved PA1955 2.47 0.02 FapB 1.13 7.15E-01 -1.55 4.33E-01 Quorum sensing PA0602 3.06 4.10E-21 Probable binding protein component of ABC transporter: quorum sensing 1.26 1.18E-04 -0.54 3.93E-01 PA0997 2.56 8.70E-23 3-oxoacyl-[acyl-carrier-protein] synthase III, PqsB 0.81 3.36E-02 -0.82 5.92E-02 PA0998 2.28 1.07E-22 3-oxoacyl-[acyl-carrier-protein] synthase III, PqsC 0.66 5.28E-02 -0.42 4.93E-01 PA1000 2.47 2.04E-16 Quinolone signal response protein, PqsE 0.57 2.26E-01 -0.29 7.36E-01 PA1001 2.37 3.86E-19 Anthranilate synthase component I, PhnA 0.65 9.13E-02 -0.24 7.31E-01 PA1002 2.22 6.95E-12 Anthranilate synthase component II, PhnB: Anthranilate/para-aminobenzoate synthase component II 0.44 4.60E-01 0.00 9.98E-01 PA3478 3.07 2.64E-08 Rhamnosyl transferase chain B: in pathways like quorum sensing and biofilm 0.20 9.05E-01 -0.84 4.48E-01 Multidrug resistance PA0424 2.02 1.90E-10 Multidrug resistance operon repressor MexR:DNA-binding transcriptional regulator, MarR family -0.18 8.84E-01 0.23 7.88E-01 PA0958 2.28 4.24E-10 Basic amino acid, basic peptide and imipenem outer membrane porin OprD precursor 0.75 4.20E-03 0.42 6.67E-01 PA1908 4.12 6.09E-05 Probable major facilitator superfamily (MFS) transporter: Tetracycline resistance protein signature 0.45 9.55E-01 0.54 7.14E-01
Chapter III: Transcriptomics of Pseudomonas LUZ19 phage infected bacteria under in vitro conditions __________________________________________________________________________________ Brandão, A.|2022 98 PA5542 3.64 3.66E-23 Pseudomonas imipenem beta-lactamase PIB-1: response to antibiotic 1.05 7.27E-03 0.15 8.70E-01 PA4218 4.48 0.0004 AmpP -1.09 5.78E-01 1.13 1.77E-03 PA4219 5.47 1.07E-31 Uncharacterized iron-regulated membrane protein, AmpO -0.08 9.87E-01 1.39 6.88E-04 PA3038 2.87 5.28E-14 OpdQ 1.24 4.97E-03 0.28 7.55E-01 PA3186 4.56 1.94E-28 Glucose/carbohydrate outer membrane porin OprB precursor 0.62 1.84E-01 0.68 5.46E-01
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 105 Successful rRNA depletion was verified on a Bioanalyzer using the DNA High sensitivity kit (Agilent) and the final concentrations were determined using the Qubit 4 Fluorometer (Invitrogen). Samples were processed immediately, according to Illumina Stranded Total RNA Prep with RiboZero Plus TruSeq Stranded mRNA Library Prep recommendations. In the end, cDNA libraries were evaluated on a Bioanalyzer (Agilent) to confirm average fragments between the range of 200-300bp, and final concentrations were measured on a Qubit. The libraries’ normalization and pooling were performed according to MiniSeq System Denature and Dilute Libraries Guide: protocol A from Illumina. For each run, a library of three samples with equimolar amounts was combined with PhiX control (spike-in of 0.52%) and paired-end sequenced (2×75bp) on an in-house Illumina Miniseq sequencer. After a first sequencing run, specific samples were re-sequenced to increase the sequencing depth of the phage and bacterial transcriptomes and improve the statistical power to detect differentially expressed genes. 4.2.7 Data analysis The quality of the cDNA reads was analysed using FastQC (0.11.8) [35]. Adapters and poorquality sequences were trimmed using Trimmomatic (v0.39) [36]. Paired-end reads were aligned to the LUZ19 (NC_010326.1), P. aeruginosa PAO1 (NC_002516.2), and Human reference genomes (assembly GRCh38.p13) using the BWA-MEM aligner [37] and SAMtools (v1.9) [38] was used to process the alignment files and assess mapping quality. Next, the aligned read pairs (fragments) were assigned to the genomic features of the phage and the host using featureCounts [39]. Alignment visualization was performed using Integrative Genomics Viewer (IGV) [40]. Reads that mapped to the human genome and bacterial rRNA reads (60-80%) were removed in silico . Subsequently, fragment counts were first normalized by gene length and next by the number of non-ribosomal read pairs in each sample to obtain FPKM (fragments per kilo base of transcript per million mapped fragments) values. Samples variance was analysed using PCA in R. Differential gene expression analysis was performed using a negative binomial distribution test from the DESeq2 Bioconductor package in R [41]. An ANOVA test was performed to identify the most significantly up or downregulated genes. The bacterial transcriptome during LUZ19 phage infection when adhered to human airway epithelial cells was analysed to elucidate bacterial major responses triggered by the phage under human physiological conditions. Sequencing of the RNA samples revealed that, despite the depletion of ribosomal RNA (rRNA) and mammalian RNA, a high proportion of reads mapped to the human genome and rRNA, (Table S4.1 and Figure S4.1). The reduced sequencing depth of the bacterial transcriptome constitutes
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 106 a limitation of using the triple RNA-seq technique where most of the reads map to the mammalian host cells, which can be overcome by using high-throughput sequencers systems. Considering this technical limitation, to increase the number of reads that map to the bacterial genome, it was re-sequenced three samples (4_5, 7_5, 5_10, and 4_15) to achieve at least ≈500,000 prokaryotic non-rRNA reads per sample, sufficient for reliable data analysis. The data that support the findings of this study are deposited into GEO database with an access number GSE213159. 4.3 Results 4.3.1 Pseudomonas LUZ19 phage transcriptome is consistent for standard LB growth medium, mammalian cells culture medium, and lung epithelium presence The phage transcriptional landscape during infection of bacteria adhered to epithelial cells is very similar to the LUZ19 transcriptomes previously observed in LB or MCCM media [16]. More specifically during infection of bacteria adhered to epithelial cells monolayer at 5min-post infection, the reads were mapped at regions located in the beginning of phage genome (position 0 > ≈10,000bp), then at 10minpost infection reads were also mapped at beginning and middle regions of phage genome (0 > ≈25,000bp), and at 15min-post infection a high number of reads were already mapped also in regions located closer to the end of the phage genome (25,000 > 45,000bp), as the infection progresses, as visualized in Figure 4.2. Under these conditions, the number of reads was significantly reduced (Figure S4.2) as a consequence of performing triple RNA-seq. In addition, the burst size was reduced (Figure S4.3) because of a lower bacterial cell growth rate in these conditions.
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 107 Figure 4.2Pseudomonas phage LUZ19 transcriptional landscape on P. aeruginosa PAO1 adhered to Nuli-1 epithelial cells. 5min samples, 10 in samples and 15min samples represent the phage transcripts at the early middle, and late infection stage, respectively. Considering similarities between expression patterns obtained in LB, MCCM [16] and in the conditions reported in this study, a PCA analysis was performed (Figure 4.3) of the overall transcriptomes. Before phage exposure (t=0min) the bacteria have very distinct gene expression patterns when comparing the different media conditions, yet the presence of Nuli-1 epithelial cells introduces a variable that leads to a very distinct transcriptome from both MCCM medium and LB medium. Looking closer to the samples that are under phage exposure, the transcriptomes in different media begin to converge over time until the end of the infection. Overall, phage transcription mechanisms seem to proceed efficiently with minimal changes.
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 108 Figure 4.3Principal Component Analysis of total transcriptomes from samples acquired at early (5min), middle (10min), and late infection (15min) in the presence of LB media, MCCM media, and Nuli-1 epithelial cells. The control samples (without phage presence) in each respective media are also included (t0). (Figure contains data described in chapter III). 4.3.2 P. aeruginosa PAO1 Differential Expressed Genes (DEGs) during phage infection in the presence of Nuli-1 epithelial cells The transcriptome of P. aeruginosa PAO1 in the presence of Nuli-1 epithelial cells was analysed during phage infection and compared to the transcriptome of uninfected bacteria. Thresholds used to find DEGs were placed at -1.5≤Log2Fold≤1.5, and padj≤0.05. Under these conditions, 21 DEGs were obtained 5min post infection, 39 DEGs at 10min, and 129 DEGs at 15min, as shown in Figure S4.2 and Figure 4.4. Generally, DEGs upregulated during early infection were associated with lipid A modification, glycerol metabolism, translation, the prokaryotic degradosome, and phosphate acquisition, whereas aromatic amino acids transport DEGs were downregulated. At 10min, genes involved in sulphate transport, phosphate transport, spermidine biosynthesis, glycine metabolism, pyochelin biosynthesis, and protein folding are upregulated. By contrast, the aromatic amino acids transport, arginine/ornithine transport, lipid A modification, lactate metabolism, and several transcriptional regulators are relatively
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 109 downregulated at 10min post infection. In the late infection stage, a larger number of differentially transcribed genes were upregulated: genes involved in sulphate transport and sulphur metabolism, spermidine biosynthesis, fatty acid biosynthesis, pyoverdine biosynthesis, metabolism of specific amino acids (glycine, serine), transcription, protein folding, ribosomal proteins and RNA, taurine metabolism, phosphate transport, EPS biosynthesis, pyochelin biosynthesis, two-component systems, and antibiotic precursors (β-lactamase, isopenicillin N); and downregulated: genes associated with amino acids transport (branched amino acids, acidic amino acids), amino acids metabolism (tyrosine, glutamine, isoleucine/leucine, valine, histidine), carbon metabolism, lactate oxidation, oxidative phosphorylation, acetyl-coA metabolism, LPS synthesis, type IV pili synthesis, and virulence transcriptional regulators. To understand if the transcriptional responses obtained from RNA-seq of samples acquired during lung epithelial cells exposure are specific to Nuli-1 cells growth condition or if they are caused only by the phage regardless of the bacterial growth conditions, the data was compared with the previously published bacterial transcriptomes in LB medium and MCCM medium [16]. In total, 17 genes were found to be differentially expressed in all growth conditions, 78 genes are specifically differentially transcribed in the Nuli-1 condition, 242 are specific for LB condition, and 721 for MCCM (Figure 4.5). Interestingly, among the 17 genes that are differentially expressed in all media, four are annotated as probable phage proteins, although their role during phage infection is unknown. Besides phage-related genes, genes involved in glycine cleavage, spermidine biosynthesis, post-translational modification (tpbA), tyrosine catabolism, glutathione metabolism, and leucine catabolism were also found to be commonly differentially transcribed in all growth conditions (Table S4.3). Genes exclusively differentially transcribed in the Nuli-1 condition can be seen in Table S4.4.
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 111 Figure 4.5Venn diagram showing genes that are exclusively differentially transcribed in each media and the genes that are commonly expressed in each condition. The intersection of each condition represents the number of shared genes that are differentially expressed. 4.3.3 Conserved induced phage responses between the different bacterial growth conditions To understand the transcriptional changes that occurred in the different growth conditions during the phage infection, it was combined previously obtained complementary data from Chapter III [16] with new data reported here and represented the Log2Fold values over time (5, 10, 15min) as a heat map (Figure 4.6). The first common transcriptional changes that appear to be triggered during phage infection regardless of the growth conditions are the genes related to spermidine biosynthesis. spE2 is among the most significant differentially expressed genes. This gene is significantly upregulated in MCCM and Nuli1 conditions and is downregulated in LB. Another consistently triggered transcriptional change was related with the increasing upregulation of hypothetical phage proteins and hypothetical prophage Pf1 genes in all bacterial growth conditions as phage infection progresses. On the other hand, the opposite is observed for the operon that comprises genes hisF2, hisH2, wzx, wzy . These genes are involved in the biosynthesis of the heteropolymer o-specific B-band O-antigen of LPS, and are increasingly downregulated over time in all growth conditions. In addition, during the early stage of phage infection and specifically on bacteria adhered to lung epithelium and grown on MCCM, glycerol metabolism is induced, although upregulation of genes associated with this metabolism becomes less pronounced over time. Lastly, alginate-associated biosynthetic genes are also triggered after phage infection. Whereas the alginate-related genes are upregulated in the presence of LB medium and Nuli-1 epithelium, it was observed a drastic shift from downregulation to upregulation in MCCM medium (with the exception of gene algItranscriptional regulator). The transcriptional profile for type IV pili and flagella-associated genes is less pronounced,
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 112 although in general, these appear to be upregulated during early infection (for MCCM and Nuli-1 conditions) and subsequently become less transcribed over the course of infection. In all growth conditions, rsaL (control of QS cascade) and dpsI (virulence-related) genes also appear to be common phage targets, as they are downregulated over the course of infection, with exception of dpsI in LB medium. Figure 4.6Heat maps of genes Log2Fold values for each categorical group of genes at different stages of infection (5-, 10-, and 15min post-infection) in bacteria adhered to Nuli1 epithelial cells monolayer, grown on MCCM media, and grown on LB media. Log2Fold values are always relative to the expression level of uninfected bacteria at each respective growth condition. 4.3.4 Specific phage induced responses in the presence of lung epithelial cells Even though the phage efficiently progresses with its infection and leads to major changes in the bacterial transcriptome, which seem to be common for all tested media, some specific transcriptional changes unique to the lung epithelial cell condition were observed (Figure 4.7). Among these differences,
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 113 the upregulation of sulphate-related genes over the infection time was observed only for the Nuli-1 condition, which indicates a requirement of this nutrient during phage infection , perhaps by providing a phage fitness advantage under this condition [42,43]. In addition, the transcription of the arn operon, which is associated with the modification of the lipid A component of LPS, is upregulated during infection when bacteria adhere to Nuli-1 epithelial cells. Likewise, the genes that positively regulate the arn operon, the pmrA/pmrB belonging to the two-component system, are also upregulated during infection. Figure 4.7Heat maps with Log2Fold values for each group of genes that are specifically targeted at Nuli-1 at different stages of infection (5-, 10-, and 15min post-infection) in bacteria adhered to Nuli-1 epithelial cells monolayer, grown on MCCM media, and grown on LB media. Log2Fold values are always relative to the expression level of uninfected bacteria at each respective growth condition. Siderophore pyochelin synthesis-associated genes are also significantly differentially expressed in the Nuli-1 condition during phage infection, as well as in LB medium, although slightly attenuated compared to the Nuli-1 environment. Considering the importance of iron homeostasis in P. aeruginosa, the expression of genes involved in iron acquisition was evaluated in the three growth conditions (Figure S4.4). In the Nuli-1 condition the overall set of iron-related genes do not seem to be differentially
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 114 transcribed with the exception of pyochelin. By contrast, iron-related genes are generally upregulated in LB and a strong downregulation is observed in the MCCM medium, with exception of bfd (bacterioferritinassociated ferredoxin). To better understand these differences between media, the transcription of the same genes at each medium without phage exposure was also examined. Comparison between the different conditions showed that bacteria are highly transcribing iron-related genes in the MCCM medium relative to the Nuli-1 and LB growth conditions, and the same genes are upregulated in the Nuli-1 condition compared to LB medium. Therefore, bacteria are increasing expression of these genes in the following sequence: MCCM>Nuli-1>LB, indicating that the bacteria are showing the most iron starvation in MCCM, followed by the Nuli-1 condition. Another interesting transcriptomic modulation during phage infection of bacteria adhered to lung epithelium is the consistent upregulation of genes involved in T3SS, which in LB and MCCM seem to be downregulated during infection, relative to uninfected bacteria. Also, non-coding RNAs (ncRNAs) rsmY and rsmZ were found to be downregulated during infection when bacteria is adhered to Nuli-1 cells. The Log2Fold values of their transcriptional regulators gacA/gacS , as well as rsmYZ ’s final target ( rsmA ) and other genes such as hfq and pnp involved in rsmYZ stability and degradation were also evaluated. The results demonstrated that pnp is upregulated during infection, especially in Nuli-1 and MCCM, and that hfq expression is reduced over the course of infection. RsmA, a global virulence regulator, is exclusively downregulated in the Nuli-1 condition, although its transcriptional regulators ( rsmY and rsmZ ), which are both downregulated throughout infection. On the other side, gacA/gacS seem to not be differentially expressed during infection, which is likely related to the non-transcription of rsmY and rsmZ. In addition, pnp which encodes an PNPase and is involved in the rsmY and rsmZ degradation, is upregulated in both MCCM and Nuli-1 conditions. This might explain the decay in rsmYZ transcripts since hfq (involved in rsmYZ stabilization) is also decreasing over the infection, especially in Nuli-1 condition. As a final remark, another Nuli-1 specific transcriptional change was observed for the gene mvaT, which is increasingly downregulated over the infection. 4.4 Discussion Unveiling phage-bacteria interplay under conditions that mimic human physiology is a crucial step in phage biology research. Previously, it was demonstrated that bacterial media conditions significantly impact the phage-bacteria transcriptome [16]. Here, it was increased the design complexity by tracking transcriptional changes under conditions that more closely mimic the human lung epithelium
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 121 10. Hurwitz BL, U’Ren JM: Viral metabolic reprogramming in marine ecosystems. Curr Opin Microbiol 2016, 31:161–168. 11. Rosenwasser S, Ziv C, Creveld SG Van, Vardi A: Virocell Metabolism : Metabolic Innovations During Host – Virus Interactions in the Ocean. Trends Microbiol 2016, 24:821– 832. 12. Hendrix H, Kogadeeva M, Zimmermann M, Sauer U, De Smet J, Muchez L, Lissens M, Staes I, Voet M, Wagemans J, et al.: Host metabolic reprogramming of Pseudomonas aeruginosa by phage-based quorum sensing modulation. bioRxiv 2019, doi:10.1101/577908. 13. De Smet J, Zimmermann M, Kogadeeva M, Ceyssens PJ, Vermaelen W, Blasdel B, Bin Jang H, Sauer U, Lavigne R: High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection. ISME J 2016, 10:1823–1835. 14. Santos SB, Costa AR, Carvalho C, Nóbrega FL, Azeredo J: Exploiting Bacteriophage Proteomes: The Hidden Biotechnological Potential. Trends Biotechnol 2018, 36:966–984. 15. Lavigne R, Lecoutere E, Wagemans J, Cenens W, Aertsen A, Schoofs L, Landuyt B, Paeshuyse J, Scheer M, Schobert M, et al.: A Multifaceted Study of Pseudomonas aeruginosa Shutdown by Virulent Podovirus LUZ19. MBio 2013, 4:1–13. 16. Brandão A, Pires DP, Coppens L, Voet M, Lavigne R, Azeredo J: Differential transcription profiling of the phage LUZ19 infection process in different growth media. RNA Biol 2021, 00:1–13. 17. Wicke L, Ponath F, Coppens L, Gerovac M, Lavigne R, Vogel J: Introducing differential RNAseq mapping to track the early infection phase for Pseudomonas phage ɸKZ. RNA Biol 2020, doi:10.1080/15476286.2020.1827785. 18. Shah M, Taylor VL, Bona D, Tsao Y, Stanley SY, Pimentel-Elardo SM, McCallum M, BondyDenomy J, Howell PL, Nodwell JR, et al.: A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa . Mol Cell 2021, 81:571-583.e6. 19. Zhao X, Chen C, Jiang X, Shen W, Huang G, Le S, Lu S, Zou L, Ni Q, Li M, et al.: Transcriptomic and metabolomic analysis revealed multifaceted effects of phage protein Gp70.1 on Pseudomonas aeruginosa . Front Microbiol 2016, 7:1–14. 20. Zhong Q, Yang L, Li L, Shen W, Li Y, Xu H, Zhong Z, Chen M, Le S: Transcriptomic Analysis Reveals the Dependency of Pseudomonas aeruginosa Genes for Double-Stranded RNA Bacteriophage phiYY Infection Cycle. iScience 2020, 23:101437.
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 122 21. Zhao X, Shen M, Jiang X, Shen W, Zhong Q, Yang Y, Tan Y, Agnello M, He X, Hu F, et al.: Transcriptomic and metabolomics profiling of phage-host interactions between phage PaP1 and Pseudomonas aeruginosa . Front Microbiol 2017, 8:1–10. 22. Blasdel BG, Ceyssens P-JJ, Chevallereau A, Debarbieux L, Lavigne R: Comparative transcriptomics reveals a conserved Bacterial Adaptive Phage Response (BAPR) to viral predation. bioRxiv 2018, doi:10.1101/248849. 23. Blasdel BG, Chevallereau A, Monot M, Lavigne R, Debarbieux L: Comparative transcriptomics analyses reveal the conservation of an ancestral infectious strategy in two bacteriophage genera. ISME J 2017, 11:1988–1996. 24. Zhao X, Chen C, Shen W, Huang G, Le S, Lu S, Li M, Zhao Y, Wang J, Rao X, et al.: Global Transcriptomic Analysis of Interactions between Pseudomonas aeruginosa and Bacteriophage PaP3. Sci Rep 2016, 6:1–12. 25. Gerovac M, Wicke L, Chihara K, Schneider C, Lavigne R, Vogel J: A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions. MBio 2021, 12. 26. Putzeys L, Boon M, Lammens E-M, Kuznedelov K, Severinov K, Lavigne R: Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages. Comput Struct Biotechnol J 2022, 20:2624–2638. 27. Kordes A, Preusse M, Willger SD, Braubach P, Jonigk D, Haverich A, Warnecke G, Häussler S: Genetically diverse Pseudomonas aeruginosa populations display similar transcriptomic profiles in a cystic fibrosis explanted lung. Nat Commun 2019, 10. 28. Rossi E, Falcone M, Molin S, Johansen HK: High-resolution in situ transcriptomics of Pseudomonas aeruginosa unveils genotype independent patho-phenotypes in cystic fibrosis lungs. Nat Commun 2018, 9:1–13. 29. Damron FH, Oglesby-Sherrouse AG, Wilks A, Barbier M: Dual-seq transcriptomics reveals the battle for iron during Pseudomonas aeruginosa acute murine pneumonia. Sci Rep 2016, 6:1–12. 30. Kumar SS, Tandberg JI, Penesyan A, Elbourne LDH, Suarez-Bosche N, Don E, Skadberg E, Fenaroli F, Cole N, Winther-Larsen HC, et al.: Dual Transcriptomics of Host-Pathogen Interaction of Cystic Fibrosis Isolate Pseudomonas aeruginosa PASS1 With Zebrafish. Front Cell Infect Microbiol 2018, 8:1–15.
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 123 31. Zabner J, Karp P, Seiler M, Phillips SL, Mitchell CJ, Saavedra M, Welsh M, Klingelhutz AJ: Development of cystic fibrosis and noncystic fibrosis airway cell lines. Am J Physiol Cell Mol Physiol 2003, 284:L844–L854. 32. Lammens E, Ceyssens PJ, Voet M, Hertveldt K, Lavigne R, Volckaert G: Representational Difference Analysis (RDA) of bacteriophage genomes. J Microbiol Methods 2009, 77:207–213. 33. Ceyssens PJ, Mesyanzhinov V, Sykilinda N, Briers Y, Roucourt B, Lavigne R, Robben J, Domashin A, Miroshnikov K, Volckaert G, et al.: The genome and structural proteome of YuA, a new Pseudomonas aeruginosa phage resembling M6. J Bacteriol 2008, 190:1429–1435. 34. Azeredo J, Sillankorva S, Pires DP: Pseudomonas Bacteriophage Isolation and Production. In Pseudomonas Methods and Protocols: Methods in Molecular Biology . Edited by Filloux A, Ramos J-L. Humana Press; 2014:23–32. 35. Andrews S: FastQC A Quality Control tool for High Throughput Sequence Data. Babraham Bioinforma 2010, 36. Bolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30:2114–2120. 37. Li H, Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 2010, 26:589–595. 38. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R: The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009, 25:2078–2079. 39. Liao Y, Smyth GK, Shi W: FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 2014, 30:923–930. 40. Thorvaldsdóttir H, Robinson JT, Mesirov JP: Integrative Genomics Viewer (IGV): Highperformance genomics data visualization and exploration. Brief Bioinform 2013, 14:178–192. 41. Love MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014, 15:1–21. 42. Jacobson TB, Callaghan MM, Amador-noguez D: Hostile Takeover : How Viruses Reprogram Prokaryotic Metabolism. 2021, 43. Kieft K, Breister AM, Huss P, Linz AM, Zanetakos E, Zhou Z, Rahlff J, Esser SP, Probst AJ, Raman S, et al.: Virus-associated organosulfur metabolism in human and environmental systems. Cell Rep 2021, 36:109471. 44. Mangalea MR, Duerkop BA: Fitness Trade-Offs Resulting from Bacteriophage
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 124 Resistance. Am Soc Microbiol 2020, 45. Periasamy S, Nair HAS, Lee KWK, Ong J, Goh JQJ, Kjelleberg S, Rice SA: Pseudomonas aeruginosa PAO1 exopolysaccharides are important for mixed species biofilm community development and stress tolerance. Front Microbiol 2015, 6:1–10. 46. Chung IY, Kim B o., Han JH, Park J, Kang HK, Park Y, Cho YH: A phage protein-derived antipathogenic peptide that targets type IV pilus assembly. Virulence 2021, 12:1377–1387. 47. Daniels JB, Scoffield J, Woolnough JL, Silo-Suh L: Impact of glycerol-3-phosphate dehydrogenase on virulence factor production by Pseudomonas aeruginosa . Can J Microbiol 2014, 60:857–863. 48. Tralau T, Vuilleumier S, Thibault C, Campbell BJ, Hart CA, Kertesz MA: Transcriptomic analysis of the sulfate starvation response of Pseudomonas aeruginosa . J Bacteriol 2007, 189:6743–6750. 49. Wagemans J, Blasdel BG, Van den Bossche A, Uytterhoeven B, De Smet J, Paeshuyse J, Cenens W, Aertsen A, Uetz P, Delattre AS, et al.: Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa . Cell Microbiol 2014, 16:1822–1835. 50. Ames BN, Dubin DT, Rosenthal SM: Presence of Polyamines in Certain Bacterial Viruses. Science (80-) 1958, 127:814 LP – 816. 51. Yu T-Y, Schaefer J: REDOR NMR characterization of DNA packaging in bacteriophage T4. J Mol Biol 2008, 382:1031–1042. 52. Firpo MR, Mounce BC: Diverse functions of polyamines in viruses infection. Biomolecules 2020, 10. 53. Gutu AD, Rodgers NS, Park J, Moskowitz SM: Pseudomonas aeruginosa high-level resistance to polymyxins and other antimicrobial peptides requires cprA , a gene that is disrupted in the PAO1 strain. Antimicrob Agents Chemother 2015, 59:5377–5387. 54. Adamiak JW, Jhawar V, Bonifay V, Chandler CE, Leus I V., Ernst RK, Schweizer HP, Zgurskaya HI: Loss of RND-Type Multidrug Efflux Pumps Triggers Iron Starvation and Lipid A Modifications in Pseudomonas aeruginosa . Antimicrob Agents Chemother 2021, 65. 55. Breazeale SD, Ribeiro AA, Mcclerren AL, Raetz CRH: A Formyltransferase Required for Polymyxin Resistance in Escherichia col i and the Modification of Lipid A with 4-Amino-4deoxyL -arabinose. J Biol Chem 2005, 280:14154–14167. 56. Nairz M, Schroll A, Sonnweber T, Weiss G: The struggle for iron - a metal at the host-
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 125 pathogen interface. Cell Microbiol 2010, 12:1691–1702. 57. Moradali MF, Ghods S, Rehm BHA: Pseudomonas aeruginosa lifestyle: A paradigm for adaptation, survival, and persistence. Front Cell Infect Microbiol 2017, 7. 58. Penner JC, Ferreira JAG, Secor PR, Sweere JM, Birukova MK, Joubert LM, Haagensen JAJ, Garcia O, Malkovskiy A V., Kaber G, et al.: Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration. Microbiology 2016, 162:1583–1594. 59. Bonnain C, Breitbart M, Buck KN: The Ferrojan Horse Hypothesis: Iron-Virus Interactions in the Ocean. Front Mar Sci 2016, 3. 60. Browning C, Shneider MM, Bowman VD, Schwarzer D, Leiman PG: Phage pierces the host cell membrane with the iron-loaded spike. Structure 2012, 20:326–339. 61. Minandri F, Imperi F, Frangipani E, Bonchi C, Visaggio D, Facchini M, Pasquali P, Bragonzi A, Visca P: Role of iron uptake systems in Pseudomonas aeruginosa virulence and airway infection. Infect Immun 2016, 84:2324–2335. 62. Sultan M, Arya R, Kim KK: Roles of two-component systems in Pseudomonas aeruginosa virulence. Int J Mol Sci 2021, 22. 63. Jakobsen TH, Warming AN, Vejborg RM, Moscoso JA, Stegger M, Lorenzen F, Rybtke M, Andersen JB, Petersen R, Andersen PS, et al.: A broad range quorum sensing inhibitor working through sRNA inhibition. Sci Rep 2017, 7:1–12. 64. Burrowes E, Baysse C, Adams C, Gara FO, O’Gara F: Influence of the regulatory protein RsmA on cellular functions in Pseudomonas aeruginosa PAO1, as revealed by transcriptome analysis. Microbiology 2006, 152:405–418. 65. Murray TS, Kazmierczak BI: Pseudomonas aeruginosa exhibits sliding motility in the absence of type IV pili and flagella. J Bacteriol 2008, 190:2700–2708. 66. Zhang X, Yin L, Liu Q, Wang D, Xu C, Pan X, Bai F, Cheng Z, Wu W, Jin Y: NrtR Mediated Regulation of H1-T6SS in Pseudomonas aeruginosa . Microbiol Spectr 2022, 10:1–14. 67. Chen R, Weng Y, Zhu F, Jin Y, Liu C, Pan X, Xia B, Cheng Z, Jin S, Wu W: Polynucleotide phosphorylase regulates multiple virulence factors and the stabilities of small RNAs RsmY/Z in Pseudomonas aeruginosa . Front Microbiol 2016, 7:1–12. 68. Liu W, Li M, Jiao L, Wang P, Yan Y: PmrA/PmrB two-component system regulation of lipA expression in Pseudomonas aeruginosa PAO1. Front Microbiol 2018, 8:1–13.
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 126 69. McMackin WAW, Marsden AE, Yahr TL: H-NS Family Members MvaT and MvaU regulate the Pseudomonas aeruginosa Type III secretion system. J Bacteriol 2019, 201. 70. Li Y, Liu X, Tang K, Wang P, Zeng Z, Guo Y, Wang X: Excisionase in Pf filamentous prophage controls lysis-lysogeny decision-making in Pseudomonas aeruginosa . Mol Microbiol 2019, 111:495–513. 71. Li C, Wally H, Miller SJ, Lu CD: The multifaceted proteins MvaT and MvaU, members of the H-NS family, control arginine metabolism, pyocyanin synthesis, and prophage activation in Pseudomonas aeruginosa PAO1. J Bacteriol 2009, 191:6211–6218. 72. Shen DK, Filopon D, Chaker H, Boullanger S, Derouazi M, Polack B, Toussaint B: High-celldensity regulation of the Pseudomonas aeruginosa type III secretion system: Implications for tryptophan catabolites. Microbiology 2008, 154:2195–2208. 73. Wagemans J, Delattre AS, Uytterhoeven B, De Smet J, Cenens W, Aertsen A, Ceyssens P-J, Lavig: Antibacterial phage ORFans of Pseudomonas aeruginosa phage LUZ24 reveal a novel MvaT inhibiting protein. Front Microbiol 2015, 6. 4.7 Supplementary data Table S4.1Absolute values of reads mapping for each sample of P. aeruginosa infected with LUZ19 phage under the presence of Nuli-1 epithelial cells. Samples, where it was made a second sequencing to increase the depth power, are highlighted in bold. Sample Sample ID Total reads Reads mapped to PAO1 Reads mapped to PAO1 w/o rRNA Reads mapped to LUZ19 5-B t0 NuLi 1 22,655,247 1,164,875 587,178 44 6-B t0 NuLi 2 21,143,209 1,758,163 841,096 28 7-B t0 NuLi 3 25,952,330 2,400,326 1,113,004 47 4-5 merged t5 NuLi 1 34,036,980 1,143,479 589,164 71,559 5-5 t5 NuLi 2 26,876,170 2,604,199 1,459,406 90,603 7-5 merged t5 NuLi 3 33,455,113 1,390,562 462,542 15,831
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 129 Figure S4.4Heat map representing the expression of iron-related genes (Log2Fold values relative to uninfected bacteria) during each stage of phage infection, under the presence of different growth conditions. Table S4.2DEG of P. aeruginosa PAO1 adhered to Nuli-1 epithelial cells monolayer during phage LUZ19 infection at different stages of infection using as control uninfected bacteria. Locus Tag Log2Fold padj Name Biological role Nuli-1 (5min vs 0min) PA0865 -1.61 3.19E-03 hpd tyrosine catabolism PA0866 -1.63 4.07E-03 aroP2 aromatic amino acid transport PA2445 1.97 5.45E-03 gcvP2 glycine cleavage system PA3049 -1.72 2.48E-03 rmf ribosome modulation factor PA3266 1.95 5.32E-07 capB cold acclimation protein B PA3280 2.64 2.21E-05 oprO Pyrophosphate-specific outer membrane porin OprO precursor PA3554 2.31 2.30E-03 arnA beta-L-Ara4N-lipid A biosynthetic process
Chapter IV: Impact of phage predation on P. aeruginosa adhered to human airway epitheliumassessing the major transcriptomic changes in metabolism and virulence-associated genes __________________________________________________________________________________ Brandão, A.|2022 130 PA3582 1.80 5.45E-03 glpK glycerol metabolism PA4541.3 1.53 4.06E-02 tRNA-Asn Aminoacyl-tRNA biosynthesis and tRNA charging PA4740 1.52 1.31E-05 pnp prokaryotic degradosome Nuli-1 (10min vs0 min) PA0280 2.24 1.79E-04 cysW sulphate transport PA0281 1.67 1.98E-02 cysA sulphate transport PA0866 -1.75 2.08E-03 aroP2 aromatic amino acid transport PA1431 -1.96 5.54E-04 rsaL regulatory protein PA1687 1.70 1.45E-02 speE spermidine biosynthetic process PA2445 2.24 3.00E-03 gcvP2 glycine cleavage system PA2942.1 -2.10 7.45E-03 P15 non-coding RNA PA3552 -2.07 2.51E-03 arnB beta-L-Ara4N-lipid A biosynthetic process PA3621.1 -2.06 9.08E-04 rsmZ regulatory RNA PA4225 1.79 4.67E-02 pchF Pyochelin biosynthesis PA4226 1.97 1.52E-02 pchE Pyochelin biosynthesis PA4230 2.23 1.31E-02 pchB Pyochelin biosynthesis PA4231 2.01 2.55E-02 pchA Pyochelin biosynthesis PA4315 -1.58 6.23E-04 mvaT transcriptional regulator MvaT, P16 subunit PA4572 1.59 1.20E-03 fklB protein folding PA4771 -1.63 3.79E-02 lldD L-lactate dehydrogenase PA5170 -1.88 3.85E-03 arcD arginine/ornithine antiporter PA5360 1.89 2.25E-03 phoB two-component response regulator PA5361 1.84 1.31E-02 phoR two-component response regulator PA5367 1.67 1.46E-03 pstA phosphate transport PA5368 2.09 1.75E-05 pstC phosphate transport PA5369 2.35 3.61E-05 pstS phosphate transport Nuli-1 (15min vs 0min) PA0280 2.56 2.4E-08 cysA sulphate transport PA0282 1.61 4.8E-03 cysT sulphate transport PA0296 -1.76 1.2E-05 spuI Glutamylpolyamine syntheses PA0298 -1.57 1.4E-04 spuB Glutamylpolyamine syntheses PA0527,1 -1.98 5.8E-03 rsmY Regulatory RNA