Community-Acquired Methicillin Resistant Staphylococcus aureus:Effects Of Subinhibitory Concentrations of Antibiotics on the Proteome Profile USA300. Pathogeneses and susceptibility

TESE DE DOUTORAMENTO Community-Acquired Methicillin-Resistant Staphylococcus aureus: Effects Of Subinhibitory Concentrations of Antibiotics on the Proteome Profile USA300. Pathogeneses and susceptibility. Community-Acquired Methicillin Resistant Staphylococcus aureus: Effects Of Subinhibitory Concentrations of Antibiotics on the Proteome Profile USA300. Epidemiology and susceptibility. Eva Torres Sangiao Departamento de Microbioloxía DEPARTAMENTO DE MICROBIOLOXÍA TESE DE DOUTORAMENTO Community-Acquired Methicillin-Resistant Staphylococcus aureus: Effects Of Subinhibitory Concentrations of Antibiotics on the Proteome Profile USA300. Pathogeneses and susceptibility. Memoria presentada por EVA TORRES SANGIAO para optar o grao de Doctor en Farmacia Universidade de Santiago de Compostela Departamento de Microbioloxía & Universitas Bergen Department of Sciences, The Gade Research Group DEPARTAMENTO DE MICROBIOLOXÍA TESE DE DOUTORAMENTO Carlos Garcia Riestra, Professor of Microbiology of the School of Medicine of the University of Santiago, Support that the PhD thesis entitled "Community-Acquired Methicillin Resistant Staphylococcus aureus: Effects Of Subinhibitory Concentrations of Antibiotics on the Proteome Profile USA300. Pathogeneses and susceptibility". that Eva Torres Sangiao, BD in Pharmacy and Clinical Specialist/Consultant Microbiology and Parasitology, presents, it has been conducted under the co-direction and cosupervision of the University of Santiago, is considered completed and is authorized for submission, to be defended in the appropriate court, considering that it meets the requirements in article 34 of the regulation of the doctoral studies, and that as codirector do not incur the causes for abstention laid down in law 30/1992. Santiago de Compostela, March 2015 Signs Prof. Carlos García Riestra Esta tesis se ha realizado con el apoyo económico de SERGAS ISCIII SEIMC USC UiB Los estudios han sido financiados por ASPIRE (Pfizer) SEIMC UiB AGRADECIMIENTOS / ACKNOWLEDGE Al Dr. Carlos García Riestra, Profesor y Facultativo de Microbiología Clínica, por ser codirector y tutor de esta tesis, GRACIAS, GRACIAS, GRACIAS.............gracias por brindarme esta oportunidad, sin tu apoyo incondicional y tus buenos consejos esta pequeña locura no hubiera llegado a su fin. MUCHAS GRACIAS. Professor Harald G Wiker, Professor of Microbiology, co-director and co-director. TUSEN TAKK, THANKS & GRACIAS!! I should thank you for a lot of things, besides the English that I have learned with you , I would highlight your support, help, patience and understanding in every different circumstance that I have spent in all this time. And also, because you have tried to teach me to get the critical vision necessary to carry out an investigation, tusen takk. A mis compañeros de laboratorio del INIBIC, Son muchas las personas y muchos los agradecimientos!, gracias por ayudarme y por todos estos años donde se han compartido tantas cosas. Por esas pequeñas pero grandes soluciones. Gracias a todo el personal del INIBIC, a los que seguís y especialmente a los que ya no estais, Al Dr. Nelson Soares da Cruz: "Darling, gostaria de que você estivesse aqui para ver o que você realizou. Moito obrigado". A Carlos "Charlie", Vanessa por esas cervezas hablando de Ciencia....? A la familia de la Fuente por una obviedad bastante evidente!. Y a mis Dra. Cillero Pastor y Dra. Cartelle Gestal, AMIGAS y compañeras, gracias por lo que nos queda por vivir juntas!. The Gade Institute, It's been almost two years, two wonderful years, Many thanks to everyone of Bacteriology group, especially for Heidi, Tøbe, Cecilia, I've learned a lot from you. Åse, ....... !! Mamma mia, simply "du er rett og slett super & fantastisk". Professor Audun Nerland and Vidar Bakken, jeg takker hjertleng for deres hjelp og støtte. Veronika, I do not know how say "diky" to you... I will never express enough thanks for your endurance and support during this time. A la Dra. Sonia Pérez, por su humildad y ayuda. Siempre te daré las gracias, Sonia. Introduction Introduction: Staphylococcus aureus: The Microorganism 5 1 Staphylococcus aureus : The microorganism. Staphylococcus aureus belongs to, Phylum 2 of Bacteria , class Bacilli , of the order Bacillales , family Staphylococcaceae , genus Staphylococcus. The genus includes 46 species, some of which are opportunistic pathogens for humans and animals (http://www.bacterio.cict.fr/s/staphylococcus.html). The term Staphylococcus comes from the Greek staphyle = a bunch of grapes and kokkus = grains, the name was originally given by Ogston around 1883 although both Koch and Pasteur had also observed.1 This are gram-positive cocci, non-spore former, low G+C content,2 of 0.5-1.5μm in diameter, and a feature presentation in irregular clusters similar to grapes because of their ability to divide in three planes (Figure 1a-b). The type specie, Staphylococcus aureus or "golden staphylococci" (Figure 1c), to produce carotenoids during growth, and have all the typical features of the genre. It is aerobes or facultative anaerobes, stationary, mesophilic and amino acids and vitamins are needed to grow. It is able to ferment glucose and mannitol with acid production, and tolerates highly variable environmental conditions. Thus, it can grow in any temperature between 6-46ºC (optimum 30-37ºC), pH between the values of 4.0-9.8 (optimum setting to neutrality). It is also tolerant to salt concentrations resisting up to 20% NaCl, and this is a property used to create selective culture conditions for its isolation and propagation.3 This characteristic allows it to grow in foods with very low water activity. It is also quite resistant to drying, freezing and heat, but not as much as the endospores of sporeforming bacteria. S. aureus is the most virulent and pathogenic specie for human, due to its potential to cause a wide spectrum of infections and to be implicated in major outbreaks or hospital Figure 1 . S. aureus morphology. (a) Gram stain of gram-positive coco grouped in clusters. (b) Scanning micrograph of S. aureus . (c) Growth in blood-agar-plate of S. aureus ´s golden colonies. (a) (b) (c) Introduction: Staphylococcus aureus: The Microorganism 6 and community epidemics. However it can be also found colonizing the skin and mucous membranes.2 1.1 Habitat and epidemiology. Staphylococcus is ubiquitous colonizers of the skin and mucosa of all animals including mammals and birds. Some species have their preferential ecological niche as indicated by their names, i.e., S. epidermidis and S. capitis are constantly colonizing the skin, and hair & scarp, respectively.2 S. aureus is widespread among primates, but not restricted to them. In humans, the primary reservoir of S. aureus is the anterior nostrils, mainly in adults, though it can be isolated from multiple locations. Approximately 20% of healthy people are chronic nasal carriers of S. aureus , 30% intermittent carriers and 50% non-carriers. Colonization by S. aureus is more common in hospitals, especially in immunodeficient patients (undergoing dialysis, insulin-dependent diabetics, HIV seropositive), or intravenous drug users and patients with skin lesions.4 Methicillin-resistant S. aureus (MRSA) colonization has been increasing during the last decade. Factors associated with MRSA carriage include, prolonged hospitalization, prior use of antibiotics, surgery, intensive care units (ICUs) stay, living-in a nurse-home and close coexistence with a carrier of MRSA or infected patient.5 Obviously, MRSA nasal carriers have an increased risk of infection with this microorganism, and have a higher morbidity and mortality compared with patients infected with methicillin sensible (MSSA).6 Hence, to determine the clonality of MRSA in these patients, it is important to differentiate sporadic cases of MRSA and epidemic situation by a single clone. In addition, though community acquire- (CA-)MRSA has increased, the prevalence rate of MRSA among people without risk factors is relatively low, and most colonization and infection by MRSA, still develops in hospitals and/or health-care associated settings and in close contact with carriers.2 1.2 Microbiological diagnosis: Culture and Identification. S. aureus grows well on non-selective culture media such as trypticase-soy agar supplemented with 5-10% sheep-blood (blood-agar-plate) or poor liquid medium such as trypticase-soy broth. In cultures from clinical samples, which may have gram-negative bacteria, it is advisable to use a selective medium to isolate S. aureus . For diagnosis from normally sterile samples, besides solid-agar medium, enrichment and thioglycolate broths should be used.7 Introduction: Staphylococcus aureus: The Microorganism 7 The selective media used in most clinical laboratories to isolate S. aureus is the mannitolsalt agar medium (Chapman medium), which allows a presumptive identification based on the acquirement of a characteristic yellow coloration for colonies, due to mannitol fermentation with acid production, as a result the medium turns from pale pink to yellow (Figure 1.2.A). In recent years, it has been developed culture media incorporating a chromogenic substrate and so allowing direct identification of S. aureus, even MRSA. In the presence of specific enzymes and substrates, these chromogens are modified to colored colonies (Figure 1.2.B). Although the cost of these resources is high, they let you generate the isolation and identification of S. aureus and MRSA directly.8-10 The identification of S. aureus can be performed by a few conventional biochemical tests. The workflow, from my point of view, could be, catalase detection to differentiate the genus Staphylococcus (catalase-positive) from the general Streptococcus and Enterococcus (catalase-negative), fermentation of glucose to differentiate among the genus Micrococcus (non-ferment anaerobically) and Staphylococcus (yes-ferment),7 and coagulase, which remains the most commonly test used for identification of S. aureus . The coagulase test differentiates S. aureus (coagulase-positive) from the rest of staphylococci (coagulase-negative, CoNS) and it is based on the ability of S. aureus to produce the extracellular enzyme, which coagulates the plasma. (a) (b) In addition, small colony morphology variants (SCVs) of S. aureus have been described growing on blood-agar-plate as colonies approx. 1/10 of the usual size morphotype. These colonies are not pigmented and non-haemolytic, and require at least 48 hours of incubation to develop. They are mutants in the respiratory chain with low membrane potential, auxotrophic for haemin, use fewer carbohydrates and are resistant to aminoglycosides. In culture medium, these SCVs may occur alone or together with the Figure 2.1. Culture and identification. (a) S. aureus on mannitol-salt agar medium. (b) MRSA on selective-differential medium. Introduction: Staphylococcus aureus: The Microorganism 8 usual morphotype, giving the impression of a mixed culture. After subculturing, they may be stable or revert to the wild-morphotype, especially if the medium are supplemented with haemin or thymidine and incubated in a CO2 atmosphere.11,12 SCVs were recovered from the sputa of up to 20% of patients with cystic fibrosis carrying S. aureus or osteomyelitis infections, and were associated with prior treatment with trimethoprimsulfamethoxazole13 and aminoglycosides. Clinical laboratories generally have commercial phenotypic identification systems. These manual or automatic systems use various dried substrates which allow the identification of different staphylococci spp. with a reliability ranging from 70 to over 90% depending on the system.14 Currently some clinical laboratories have mass spectrometry technology, such as MALDI-TOF (Matrix Assisted Laser Desorption / IonizationTime of Flight), allowing identification scores > 2. 1.3 Molecular diagnosis. Although laborious and very costly, molecular diagnosis plays an increasing role in rapid detection of microbial pathogens and identification of drug-resistance determinants so that results can be obtained within a few hours. Identification techniques based on molecular probing15 fluorescent detection of 16S rRNA with a peptide nucleic acid probe (peptide nucleic acid fluorescence in situ hybridization, PNA-FISH). It identifies S. aureus in positive blood cultures in less than 3 hours with > 95% sensitivity and specificity.16 The method allowed rapid discrimination between S. aureus and potential contaminant coagulase-negative, thus improving therapeutic decision-making.17 Another technique developed to quantify organisms directly in clinical sample has been multiplex real-time PCR, which amplified simultaneously genes of species and resistance mechanism.18,19 A wealth of molecular techniques is being developed and proposed for routine use. Further high-throughput techniques, including RNomics and proteomics, might provide a comprehensive picture of the “goodcoagulase-negative” and the “badaureus” staphylococci and help to decide which of them must be considered for therapeutic intervention. 1.3.1 Molecular Typing. Molecular typing systems are one of the great microbiological diffusion contributions in recent years. These systems consist of a variety of techniques, which are intended to compare the composition of the nucleic acids of two or more microorganisms, thereby can be recognized the relationship between epidemiologically linked isolates, and therefore a recent derived from a common precursor microorganism. In addition, Introduction: Staphylococcus aureus: The Microorganism 9 techniques must be able to differentiate unrelated isolates, irrespective of their belonging to the same microbial species or taxon.20,21 The application of these techniques is essential in the study of nosocomial infections, especially in hospitals where there are available ICUs, neonatal units, burn units, hematology or oncology, wherever inpatients are more capable of acquiring nosocomial severe infections, particularly MRSA (59% for ICUs patients, 55% in non-ICU patients, and 48% in outpatients),22 which usually appears to be highly clonal. The main typing methods underlying this comprehension are briefly presented subsequently. 1.3.1.1 Pulsed Field Gel Electrophoresis (PFGE). Pulsed field gel electrophoresis (PFGE) is a highly discriminative molecular typing technique that is used in epidemiological studies worldwide. Actually it is the most widely used method and is extremely useful for following given clones, although it does not provide accurate information on the genealogy of the organism. PFGE is a restriction-fragment length technique to separate large chromosomal fragments generated by digestion with the low-frequency cutting enzyme Sma1 for S. aureus . The generated fragments are separated, yielding banding patterns specific for particular clones. Banding comparison has allowed identification of the major epidemic clones, which represented 70% of more than 3000 MRSA isolates recovered worldwide.22 However, the length of chromosomal fragments, and thus the clonespecific banding, may be modified with acquisition or loss of mobile DNA (MGEs) such as transposons, prophages, or pathogenicity islands. The new banding pattern may identify a different clone, which is in fact the same bacterium that has gained or lost MGEs. If the new organism has acquired properties important for successful spread, it may indeed behave as a new clone with its proper behaviour. Nevertheless, the phylogenetic relation between the new clone and the parent persists. 1.3.1.2 Secuenciación: “Multilocus Sequence Typing” (MLST). Multilocus sequence typing (MLST) is a technique designed to track clones and/or clonal lines and is a good molecular marker of long-term epidemiology. However, its discriminatory power is lower than other techniques such as PFGE, and it is a demanding technique in terms of methodology and expensive. MLST is a sequencebased method that allows the unambiguous assignment of the ancestral phylogeny of the staphylococcal population.39 The technique consists of sequencing a total of seven housekeeping genes: Introduction: Staphylococcus aureus: The Microorganism 10 arcC: carbamate kinase. aroE: shikimato dehydrogenase. gmk: guanylate kinase. pta: phosphate acetyltransferase tpi: triosephosphate isomerase yqiY: acetyl coenzyme A acetyltransferase. glpF: glycerol kinase. and submitting the sequences to a central database (www.mls.net) where they are checked and matched. The classification is based on allelic profile diversity based on approximately 300-700-bp internal gene fragments. Thousands of sequences have been submitted, generating numerous sequence types (STs). Organisms that share all seven alleles are defined as clones, those that share five of seven identical alleles are defined as clonal complexes (CC), and those that share less than seven alleles are defined as unrelated. Since the implementation of this technique in S. aureus , over 2000 year, it has been found that MRSA strains have a clonal structure highly well conserved compared with MSSA, and that a small number of clones are able to spread.23,24 Also the data obtained by MLST, indicate that S. aureus has a low level of genetic recombination and clonal diversity is more frequently caused by point mutations than genetic exchange processes. 1.3.1.3 “Spa typing” y “double locus spa-clfb typing”. Spa typing and double-locus spa-clfb typing , are sequencing-methods, based on PCR amplification of strain-specific regions of hypervariable segments of the spa (protein A) or clfB (clumping factor B) genes, respectively.25 The variable regions are made of 24 nucleotides repeats in spa (http://spa.ridom.de/) and serine-aspartate repeats in clfB , the length of which may vary from duplication or accidental loss of DNA material. Although less discriminative, these simpler methods generate unambiguous data sets that can be compared in multicenter studies. Typing has become an important part of the comprehension of the S. aureus epidemiology. However, no specific types can be attributed to disease producing versus colonizing strains, as yet.25 Introduction: Staphylococcus aureus: Pathogenesis 11 2 Staphylococcus aureus : Pathogenesis. S. aureus can cause a wide range of infections, from superficial skin infections to deepseated infections, from which it spread through the blood stream. The wide range of infections caused, is due to expression of several proteins, which may be surfaceassociated or secreted. Many of these proteins are involved in colonization of host tissues, lysis of host cell membranes, promotion of bacterial spread within host tissues and survival in phagocytes. These accessory proteins are expressed coordinately during growth, and are controlled by several regulatory systems, such as two component regulatory systems (two-CS) (e.g., agr , arlRS, saeRS, srrAB, vraRS ) and transcriptional regulatory systems (e.g., sarA family, sigB ).26 Currently, thirty-five complete S. aureus genomes are available in public databases. The S. aureus genome is circular and contains approximately 2.8 million bp that represents approximately 2700 coding sequences (2600 proteins),27 plus structural and regulatory RNAs. These belong either to, a core genome containing mostly housekeeping genes, which is quite conserved along various staphylococcal species and accounts for about 80% of the whole DNA, or an accessory genome that carries mobile genetic elements (MGEs) that contains most S. aureus pathogenic and drug-resistance features, which may vary between different species and strains. Genome evolution is driven by random point mutations that lead to single nucleotide polymorphism (SNP), larger variations in core genes (e.g., deletions or duplication of repeat regions) that may differ between lineages, and MGEs that include insertion sequences (IS), transposons (Tn), viruses, and pathogenicity and genomic islands.2 2.1 Pathogeneses: Regulation. At least three families of regulatory elements intertwine to adjust gene expression to specific environmental conditions: (1) two-component regulatory systems (two-CS); (2) DNA-binding proteins or transcriptional regulatory system, largely represented by the Sar family of proteins; and (3) small regulatory RNAs. 2.1.1 Two-Component Regulatory System. Bacterial two-component systems (two-CS) are signaling pathways, which permit the creation of a communication bridge to the external environment, allowing the cell to translate an external stimulus into an intracellular change in gene expression. The defining components are a membrane-associated sensor histidine kinase and a cytoplasmic response regulator. After activation from an external signal, the histidine Introduction: Staphylococcus aureus: Pathogenesis 18 Figure 2.1.2.1. Regulatory network of agr and sar family of DNA-binding proteins. Intertwining of activation (green arrows) and repression (red arrows) underlines complexity of system. gene expression is further modulated by additional factors ( sigB , arls , sae and srrAB ), which can act on agr promoters or directly on specific genes. Gene promoters are denominated P1, P2, and P3 and represented by dark blue arrows. Figure adapted from Mandell [2] . The sar A locus, is complex and includes three promoters, which drive the production of three transcripts ( sarB , sarC and sarA ). The sar A locus is known to up-regulate the synthesis of fibronectin and fibrinogen binding proteins, haemolysins, enterotoxins, TSST-1 (toxic shock syndrome toxin) and capsule biosynthesis genes, and to downregulate proteases, protein A and a collagen binding protein. SarA has been also shown to bind to several regulatory and target gene promoter regions (e.g., Agr, SarS, Rot, SarV, SarT, Hla, Fnb, Spa, Cna, Bap, IcaRA) to modulate gene transcription, thus implicating both agr -dependent and agr -independent pathways, as well as direct and indirect mode for SarA-mediated regulation.62 Interestingly, the inactivation of sarA has been consistently shown to result in a reduced capacity to form a biofilm, the opposite to that of agr ,63 suggesting that the role of sarA in biofilm formation is independent to its regulation of agr . On the other hand, the impact of SarA on exotoxin production has been shown to be heavily influenced by SaeR/S, with an impact on α-haemolysin and PSMs.64 In addition, in the same way as agr , inactivation of sarA has been shown to Introduction: Staphylococcus aureus: Pathogenesis 19 attenuate virulence in multiple animal models of S. aureus infection, including septic arthritis, osteomyelitis, and endocarditis.65 This, together with the impact of SarA on expression of agr , provides direct indications of the interactive role of SarA in S. aureus regulatory circuits. SarR down-regulates the expression of SarA, and its maximum expression is in the exponential phase of growth. Inactivation of sar R has positive effects on the transcription of the agr locus and also on the maximal transcription of aureolysin and serine protease in S. au reus.62 SarS, repressed by Agr and SarA, and activated by SigB (σB), is an activator of protein A and a repressor of α-haemolysin.66 SarT, repressed by Agr, also represses hla (via sae ),67 and induces expression of protein A (indirectly via sarS ). SarU, repressed by SarT, whose inactivation results in a reduction of both RNAII and RNAIII expression, suggests a positive effect of SarU on agr, and a key role during biofilm-associated infections by modulating agr . SarV is involved in regulation of autolysis, which may be part of the common pathway through which SarA and MgrA control autolysis.68 SarX also acts as a repressor of the agr locus and can therefore regulate other genes via Agr. Its maximal expression is during the stationary phase of growth,69 and could be involved in a regulatory cascade that promotes polysaccharideintercellular-adhesion (PIA)-dependent biofilm formation in S. aureus .70 SarZ positively regulates the expression of agr and mgrA but negatively of sarA . Its expression is growth phase dependent, with maximum expression during early exponential phase, and affects surface proteins, toxins and biofilm by modulating the aforementioned global regulators, as well as direct activation on SspA protease.71 2.1.2.2 Others Transcriptional Regulatory Systems. Sigma factors (Sig, σ), are other major mechanism of response to environmental stimuli. There are currently four identified sigma factors (Sig) in S. aureus : SigA, responsible for transcription of housekeeping genes; SigB (σB), responsible for the transcription of stress-response genes; SigS, controls expression of genes required for overall fitness and survival; and SigH, involved in competence and more recently, prophage integration and excision.72 The alternative SigB σB operon contains: σB, anti-σB factor RsbW, anti-anti-σB factor RsbV, and RsbU, a Mn2+-dependent phosphatase that positively controls σB activity by dephosphorylating RsbV (Figure 2.1.3.2). This alternative transcriptional factor SigB σB is an essential part of the complex regulatory network controlling the expression of over 200 genes involved in virulence, cell wall metabolism, membrane transport processes, and microbial response to a variety of stress (temperature, energy Introduction: Staphylococcus aureus: Pathogenesis 20 depletion,..) and chemical stimuli.2 It has been also demonstrated to aid in heat tolerance and resistance to cell wall active antibiotics,73 and contribute to pathogenesis in animal models of infections. More recently, it has been shown that inactivation of SigB has an indirect impact on the agr ‘ quorum sensing ’ system by enhancing RNAIII expression,74 as well as to regulate several extracellular virulence factors and capsule via SpoVG. Hence, it is demonstrating the role in virulence as a response to stress.75 MgrA, member of the multiple antibiotic resistance regulator MarR and SarA family proteins, which positively regulates sarX gene expression, plays a key role in regulating the expression of major virulence factors in S. aureus, including capsule and sortase. Several studies have provided evidence that MgrA regulates hla and spa expression by agr -dependent and independent pathways, and plays an important role in S. aureus sepsis, even increasing mortality and accelerating the onset and development of sepsis.76 In addition, MgrA has been found to repress biofilm formation, in part, by agr -dependent pathway and DNA release, probably by affecting LytS/R and the antiholin-like protein Lrg AB .77 Figure 2.1.3.2 Post-transcriptional regulation of SigB. After stressinduction, RsbU de-phosphorylates RsbV, which can then bind specifically to RsbW thereby removing RsbW from SigB. Phosphorylated RsbV is inactive and therefore cannot bind RsbW. RsbW also promotes phosphorylation of RsbV to maintain its inactivity. RsbW binds to SigB to inhibit transcription by preventing SigB from complexing with the RNA polymerase (RNAP). Once SigB is free from inhibition by RsbW, it can complex with RNAP forming the holoenzyme and activate transcription of target genes. Active proteins are highlighted with yellow. Figure adapted from [ Junecko ME et al. , 2012, World J Clin Infect Dis ]. 71 August 25, 2012 | Volume 2 | Issue 4 | WJCID | www.wj gnet.com lence [157] . Thus, CodY is able to regulate virulence via direct binding of virulence gene promoters and via inhibition of metabolic regulatory pathways providing another regulatory link between metabolism and virulence [70,156] . CodY is repressed by the intracellular chaperone ClpC[40] , possibly via ClpC-induced proteolytic degradation in association with ClpP. Although CodY acts as a repressor of virulence genes, it can also be negatively regulated under various environmental conditions, eliminating the repressive effect of CodY on virulence genes. Sigma factors Sigma factors are highly conserved among bacterial species. They provide promoter specificity to the RNA polymerase, and are highly regulated by anti-sigma factors via direct binding of the protein[158] . There are currently four identified Sigma factors in S. aureus: SigA, which is responsible for transcription of housekeeping genes; SigB, which is responsible for the transcription of stress-response genes; SigS, which controls expression of genes required for overall fitness and survival [2]; and SigH, which has a demonstrated involvement in competence and more recently, prophage integration and excision[159,160] . The most thoroughly studied of these is SigB, which is transcribed from the four-gene operon rsbUVWsigB that encodes an anti-sigma factor (RsbW), anti-anti-sigma factor (RsbV) and RsbU, an anti-RsbV phosphatase [95,161] . The regulation of SigB is very tightly controlled by RsbW, RsbV and RsbU (Figure 2). SigB controls expression of an array of genes responsible for the survival of hydrogen peroxide-induced stress and desiccation as well as production of the carotenoid staphyloxanthin and extracellular proteases [161-164] . SigB has also been demonstrated to aid in heat tolerance and resistance to cell-wall active antibiotics [165,166] . The repressive effect of SigB on V8 proteases positively regulates biofilm formation [162] because the presence of extracellular proteases has been correlated with the inability to form a biofilm [164] . SigB regulates its target genes either by recognizing a conserved sequence or by downstream regulators. For example, SigB effect on sarA or agr expression has been reported[95,167] . More recently, SigB has been shown to regulate several extracellular virulence factors and capsule through SpoVG [168,169] , demonstrating a role for SigB in virulence as a response to stress. CONCLUSION In this review, we describe several regulators involved in virulence regulation. These represent only a fraction of all regulators encoded in the S. aureus genome. S. aureus is a pathogen that can cause a wide range of diseases and can infect almost every tissue. It is thus not surprising that a large number of regulators are needed to modulate the production of various virulence factors in different environmental conditions in the host. What is surprising is the high degree of complexity of the interactions among the regulators. Compounding the complexity is the finding that virulence genes in different strains often are regulated differently. The molecular mechanisms underlying some of the strain differences have been illustrated but most have not. Nonetheless, significant progress has been made toward understanding virulence gene regulation. However, most of the results have been obtained by in vitro studies. The big challenge that lies ahead would be to test the in vitro results in suitable animal models to better understand virulence gene regulation in pathogenesis. With the rise of antibiotic resistance and the prevalence of multi-drug resistant isolates, fully understanding the virulence regulation in pathogenesis may provide sound rationale for identifying regulators as potential targets for anti-staphylococcal drug therapies. Targeting a cellular factor not absolutely required for survival, such as a virulence regulator, may lessen selective pressures, and therefore resistance, while still attenuating virulence of the organism [170,171] . REFERENCES 1 Felden B, Vandenesch F, Bouloc P, Romby P. The Staphylococcus aureus RNome and its commitment to virulence. PLoS Pathog 2011; 7: e1002006 2 Shaw LN, Lindholm C, Prajsnar TK, Miller HK, Brown MC, Golonka E, Stewart GC, Tarkowski A, Potempa J. Identi • caSigB Stress Target gene transcription SigB SigB RNAP RsbV RsbV RsbW RsbV P RsbU Figure 2 Post-transcriptional regulation of SigB. After stress-induction, RsbU de-phosphorylates RsbV, which can then bind speci • cally to RsbW thereby removing RsbW from SigB. Phosphorylated RsbV is inactive and therefore cannot bind RsbW. RsbW also promotes phosphorylation of RsbV to maintain its inactivity. RsbW binds to SigB to inhibit transcription by preventing SigB from complexing with the RNA polymerase (RNAP). Once SigB is free from inhibition by RsbW, it can complex with RNAP forming the holoenzyme and activate transcription of target genes. Active proteins are highlighted with yellow. SigB Junecko JM et al . Regulatory elements in S. aureus RsbW Introduction: Staphylococcus aureus: Pathogenesis 21 Repressor of toxins Rot, is another global regulator that belongs to the Sar family, which mediates modulation of several genes involved in virulence (especially autolysins).78 Its transcription is growth-phase dependent,79 however its translation is regulated by the agr ‘ quorum sensing ’ system.80 Rot has also been shown to repress hla production by repressing the SaeR/S two-CS, and both, Rot and the Sae-two-CS, had been proposed to work in opposition of one another on their target genes. Otherwise now, it is known that both regulators work in concert to activate promoters.81 Other similar transcriptional regulatory system, teicoplanin-associated locus regulator TcaR, belongs to MarR family, appeared to be a weak negative regulator of transcription of the ica (intercellular adhesion) locus, necessary for biofilm production, as well as a further activator of sar S, and a modulator of sas F expression. Obviously, the inactivation of the tca RAB operon leads to teicoplanin resistance. And, AraC/XylS family, involves in biofilm formation82 and virulence in S. aureus. 83 Finally, the GTP-sensing transcriptional pleiotropic repressor CodY , well conserved within the low G+C Gram-positive, has been shown to be an important regulator of metabolism and virulence. CodY acts repressing toxin production during times of plentiful nutrition by directly and indirectly via agr . It functions by sensing intracellular levels of branched chain aminoacids and GTP during growth, and responds by repressing genes involved in starvation behaviors in nutrient-rich conditions (amino acid transport, sporulation…). When these levels decline (branched chain-aminoacids and GTP), CodY loses its affinity for DNA binding, bringing about de-repression of target genes, and as a result, a physiological transition from growth and division to amino acid metabolism and stress tolerance.84 Briefly, apart from direct regulation of virulence genes, CodY also affects metabolic regulation in S. aureus via carbon flow, nitrogen assimilation, amino acid synthesis and transport systems.85 Moreover, CodY is also repressed by the intracellular protease/chaperone ClpC, possibly via ClpC-induced proteolytic degradation in association with ClpP, eliminating the repressive effect of CodY on virulence genes.86 “CodY is activated in nutrient replete environments, repressing virulence factors and metabolic synthesis genes” . 2.1.3 Small regulatory RNAs systems (sRNA). S. aureus also uses, besides these signaling pathways, around 250 regulatory RNAs to coordinate the expression of the numerous virulence genes for growth and survival. Most S. aureus sRNAs are located within the core genome, but a few are expressed from Introduction: Staphylococcus aureus: Pathogenesis 22 the pathogenicity islands and from plasmids. The sRNAs expressed from the core genome are probably involved in wider biological functions. Most of the few sRNAs, whose physiological roles have been determined, control the expression of genes involved in response to quorum sensing, in central metabolisms, and on virulence by pairing to target mRNAs to modulate their translational activities and stabilities. Several sRNAs encode and express small peptides that may play important roles in virulence or in bacterial growth control. Most of these well-characterized sRNAs act as fine-tuning regulators by repressing the translational level of only one gene, but it is probably that one gene is regulated by different sRNAs.74 "Multiple sRNAs controlling the expression of a similar component from a regulatory network allows the sharp regulation of virulence genes. It is most probably that S. aureus expresses many other sRNAs that deeply interact with this network to influence bacterial virulence" .74 SbrA and SbrB, highly conserved among Staphylococci, encode putative basic peptides, which are potential virulence factors. Another SbrC, encodes for an ABC transporter dedicated in the uptake of manganese, crucial element for defense systems against oxidative stress and contributes to the virulence of S. aureus .74 RsaA, RsaD, and RsaF, are differently transcribed in response to environmental stress, heat, cold, osmotic and oxidative stress, as well as acidic pH. Recently, RsaE, sRNA conserved in all S. aureus strains, has been shown to regulate several metabolic pathways, exactly down-regulates the synthesis of enzymes from the Krebs cycle (tricarboxylic cycle, TCA) and from the folate-dependent one-carbone metabolism. However its expression profile is a subject of controversy,87,88 because of some S. aureus strains. RsaE is expressed at late exponential phase and repressed at stationary phase, it could facilitate the transition of energy metabolisms, the purine biosynthesis, and amino acid transport in response to the nutrients’ availability. Moreover, the RsaE expression seems to be dependent on the agr ‘ quorum sensing ’ system and SigB activity (Figure 2.1), suggesting that it could modulate the metabolism profile according to stress responses and/or virulence.43 Small stable RNAs (SSRs) are RNAs specifically produced and/or stabilized in response to various environmental conditions. For example, SSR42, expressed during the stationary phase, is involved in host erythrocyte lysis, resistance to human polymorphonuclear leukocyte killing, and pathogenesis in a murine model of bacterial infection.74,89 Introduction: Staphylococcus aureus: Pathogenesis 23 Figure 2.1. The agr system and its two main intracellular effectors AgrA and RNAIII. The agr system regulates the expression of multiple genes in an RNAIII-independent manner via AgrA and in an RNAIII-dependent manner. RNAIII-independent regulation: AgrA activates the synthesis of several peptides by binding to their promoter regions and represses metabolic enzymes by an unknown mechanism. RsaE regulates enzymes of the central metabolism. RNAIII-dependent regulation: RNAIII encodes hld (δ-haemolysin) and the secondary structure of RNAIII and three of the hairpin domains containing a redundant UCCC motif (gray color). The 3’ non-coding region of hld, which contains the three redundant hairpin loops, binds to the ribosome binding sites of coa mRNA encoding coagulase, rot mRNA encoding repressor of toxins rot, and spa mRNA encoding protein A. The 5’ non-coding region of hld binds to hla mRNA to facilitate ribosome binding, and to activate translation. Dashed bars and dashed arrows are for transcriptional regulation while black bars and arrows are for posttranscriptional regulation. Bars are for repression and arrows for activation.90 Reprint from [90] with permission from Taylor & Francis Group LLC. Copyright © 2012 Landes Bioscience. All Rights Reserved. Introduction: Staphylococcus aureus: Pathogenesis 24 Small pathogenicity island rNA D9 (SprD), is expressed from the genome of a converting phage, a horizontally-acquired pathogenicity island, being the repository of superantigens, toxins, adherence, invasion factors, and secretion systems, playing important roles during host infection. SprD down-regulates, the expression of the immunoglobulin binding (Sbi) immune evasion molecule, located on the core genome.74 Finally, RNAII encoding a ‘quorum sensing’-cassette and an agr -locus-two-CS [see above], and RNAIII encoding δ-haemolysin (Hld). (Figure 2.1) RNAIII is one fascinated regulator and structured mRNAs that regulates multiple targets involved in virulence and peptidoglycan metabolism.74,90 RNAIII is responsible for post-transcriptional regulation of multiple virulence factors, by mediating a switch from expression of cellsurface associated proteins, such as staphylococcal protein A and fibronectin-binding proteins A and B (FnbA and FnbB), to secreted toxins, such as α-haemolysin and δhaemolysin, PSMs, and leukocidins such as Panton–Valentine leukocidin (PVL).36,37,91 Recently, a new sRNA, named ArtR (AgrA-repressed, toxin-regulating sRNA), has been reported to activate α-haemolysin (Hla) expression by binding to the sar T mRNA.92 Although both, RNAIII and ArtR, similarly regulate hla expression, in contrast to RNAIII, ArtR transcription is repressed by agr A, suggesting that up-regulation of hla mediated by ArtR could be enhanced in agr -deficient strains.74 2.2 Pathogenesis: Virulence determinants. S. aureus is known due to its virulence, which is multifactorial and dependent on a series of toxins, adhesion proteins, another virulence determinants and immune system evasion; the same is true for MRSA strains. The molecular basis of infection by S. aureus , has been an active field of research for several decades. However, now such research has further intensified to become decisive, with the recent emergence of highly pathogenic CA-MRSA strains that combined antibiotics resistance, rapid ability for spreading and exceptional virulence.93 The last decade has been decisive for the identification of yet unrecognized S. aureus virulence factors such as the PSMs,94 as well as the characterization of the pathogenic role of long-known toxins such as the PVL.95,96 This virulence can be gained via acquisition of new toxin genes by horizontal transfer, such as the phage-borne pvl genes, or can be gained via overexpression of core genomeencoded toxins, such as PSMs or α-haemolysin.94,97,98 Moreover, to this already intricate scheme of complexity, it has also been added, the observation that the acquisition of new genes, can be harbored by MGEs.99 (Figure 2.2) Introduction: Staphylococcus aureus: Pathogenesis 25 Figure 2.2 Pathogenic factors of S taphylococcus aureus, with structural and secreted products, both playing roles as virulence factors. (a) , surface and secreted proteins.(b) & (c) , cross-sections of the cell envelope. Reprint from [100,101] with permission from Lowy, FD (author) and Oxford University Press. Copyright © 2008 Copyright Clearance Center, Inc. All rights reserved. 2.2.1 Cell Surface Determinants Involved in Pathogenesis. Cell surface proteins are proteins embedded in cell membrane with a multiple important roles in S. aureus pathogenesis, such as key functions in bacterial cell wall metabolism. They bind to host tissue, facilitate internalization and immune evasion, and are involved in bacterial aggregation and biofilm formation. Most surface proteins are encoded on the core genome.102 2.2.1.1 Teichoic, Lipoteichoic Acids and peptidoglycan. Teichoic acids are bacterial polysaccharides constituted of polyribitol-phosphate polymers cross-linked to N-acetylmuramic acid residues of the peptidoglycan, and decorated with D-alanine and N-acetylglucosamine residues. They represent up to 50% of the dry weight of purified staphylococcal walls, and play an important physiologic role in cell wall metabolism, probably to be a site of attachment of cell wall for active enzymes and other proteins. Teichoic acids have also been involved in adherence to nasal epithelia, nevertheless, their role in invasive infection and host inflammatory response is unclear.2,102 (Figure 2.2.1.1) by guest on February 2, 2012http://cid.oxfordjournals.org/Downloaded from Introduction: Staphylococcus aureus: Pathogenesis 26 Figure 2.2.1.1. Cell wall organization Lipoteichoic acids are the major constituent of cell wall in gram-positive, with a plasma membrane-bound counterparts of teichoic acids. They have a similar general structure to teichoic acids, except that they contain polyglycerol-phosphates and are linked to a diacylglycerol-moiety, which serves as a plasma membrane anchor. Lipoteichoic acids can act like receptors, and have been implicated in inflammation via triggering the release of cytokines by macrophages and other players of the innate immune system. In particular, the stereochemistry of the D-alanine and the presence of the diacylglycerol lipid anchor, were shown to be determinants for host recognition and subsequent inflammation. Certainly, lipoteichoic acids may facilitate bacterial recognition by host innate immunity, but at the same time, they protect bacteria from killing by cationic antimicrobial peptides, which are produced by professional phagocytes. Native lipoteichoic acid is polyanionic (negative charge) and therefore, attracts cationic antimicrobial peptides. To circumvent the problem, lipoteichoic acids become decorated with D-alanyl residues (positive charge) by the dlt ABC gene products, which render the structure more positively charged, and thus repulse cationic antimicrobial peptides. Indeed, mutants impaired in dlt ABC are also less adherent to endothelial cells and less able than wild type to produce experimental endocarditis in rabbits. Hence, the microbial cover is not an amorphous scaffold that only ensures bacterial shape, it is also a sophisticated structure indispensable to mediate adherence, sensing, and growth in complex environments.2 (Figure 2.2.1.1). Peptidoglycan, also known as murein, is a critical cell structure highly conserved constituent of both the gram-positive and gram-negative, however in gram-positive Introduction: Staphylococcus aureus: Pathogenesis 27 bacteria is a thick structure while in gram-negative is thin. It is constituted of glycan chains made of N-acetylglucosamine and N-acetylmuramic acid disaccharide subunits, in which the N-acetylmuramate moiety is linked to highly conserved pentapeptide or tetrapeptide stems (L-alanine–D-isoglutamine–L-lysine–D-alanine–[D-alanine]). In S. aureus , the peptidoglycan is cross-linked via a characteristic pentaglycine interpeptide bridge, piece that comprises 1 to 5 glycine residues. The addition of glycines to the wall precursors is driven by fem ABC and fmh B genes. These determinants are implicated in the plasticity of the wall and are indirectly implicated in staphylococcal resistance to methicillin and vancomycin (see subsequent Antibiotic Resistance section).2 Peptidoglycan is the major scaffold for anchoring most MSCRAMMs (microbial surface component recognizing adhesive matrix molecule), thus it plays a key role in pathogenesis. However, peptidoglycan is recognized by the innate immune system and triggers cytokine release and inflammation, therefore it is made important for the microorganisms to be able to hide these structures (peptidoglycan and lipoteichoic acids) from host recognition. The objective is achieved by producing antiphagocytic components such as a capsule, or protein A. 2 2.2.1.2 Capsule and biofilms. More than 90% of clinical isolates of S. aureus elaborate a polysaccharide capsule. Heretofore, 11 serotypes have been reported, of which capsule type 1 and 2 produce large quantities of polysaccharides and appear mucoid on culture plates, though they are rarely found in human clinical samples. Nevertheless, capsule type 5 and 8 are responsible for up to 75% of clinical infections, indeed antibodies against these capsular types are protective in animal models of sepsis, and naturally occurring antibodies are detected in normal human serum. This could be because of, both capsule type (type 5 and 8) are antiphagocytic and can increase virulence in several animal models. Capsule type 5 and type 8 are made of various sugars, including mannose and fucose. Thus, the capsule is an antiphagocytic constituent that might be a promising target for vaccination. Actually, in patients for haemodialysis, a conjugate vaccine addressed against type 5 and 8 capsules has been shown temporally effective, however, no definitive human studies are available on this issue.2 Biofilms are surface-attached bacterial agglomerations embedded in extracellular matrix. The production of a series of surface molecules that promote extracellular matrix formation, allow Staphylococci to be known as a very good biofilm formers.102 Biofilm-formation evolves in two steps, starting with nonspecific adherence of Introduction: Staphylococcus aureus: Pathogenesis 34 Luk GH exhibits potent cytolytic activity towards neutrophils acting in synergy with the PVL in vitro .104 γ-haemolysin Hlg , also named leukocidin , can lyse white blood cells as well as other cells. It is encoded by two distinct operons, one that encodes a unique HlgA (S protein) and another encodes for HlgC (S protein) and HlgB (Table 2.2.2.2). Both proteins (S and F) have to be assembled to form membrane-perforating complexes. Active Hlg is encoded in the core genome, and exists in two bioactive forms, haemolysin-c (Hlg) and haemolysin-c2 (Hlg2). Hlg is present in 99% of S. aureus strains and appears to contribute to septic arthritis with weight loss in mice, and to endophthalmitis in rabbits.104 Table 2.2.2.2 Leukotoxins of S. aureus. 104 # PMN: polimorphonucleares. Leukotoxins Components Localization Prevalence Cell specificity Class F subunit Class S subunit Haemolysin-c (Hlg) HlgB (Hlg1,LukF) HlgA (Hlg2) hlg gene cluster; genome ∿99% Erythrocytes from humans and other mammalian species Haemolysing-c2 (Hlg2)/Leukocidin (Luk) HlgB (Hlg1, LukF) HlgC (LukS) Human and rabbit PMN and rabbit erythrocytes Panton-Valentine Leukocidin (PVL) LukF-PV LukS-PV pvl locus; phage 0-5% Human and rabbit PMN LukDE LukD LukE pathogenicity island 30-87% Murine and rabbit PMN LukFM LukF`-PV LukM phage 0% Bovine PMN LukGH (LukAB) LukG (LukB) LukH (LukA) n.d. n.d. Human PMN Panton-Valentine leukocidin PVL, two-component leukocidin, belongs to a β-barrel forming family of cytolytic toxins, comprising also other leukocidins, γ-haemolysin (Hlg) and α-haemolysin (Hla). PVL is encoded by the prophage-encoded adjacent lukS and lukF genes, which produce the two toxin parts. LukS and LukF, both are needed for the cytolytic activity of the toxin. Since 1932, when Panton and Valentine noted an association between PVL production and abscess formation,105 the interest in PVL has been increased enormously due to an epidemiological association between the presence of the lukSF genes and CA-MRSA.111 Most CA-MRSA strains have both lukSF genes, while their frequency in MSSA is much lower, and they are absent from predominant hospital acquire (HA)-MRSA clones. Introduction: Staphylococcus aureus: Pathogenesis 35 PVL is lytic toxin to human neutrophils at concentrations between 0.3-2 μg/ml.105 Concentrations of PVL reaching or exceeding that range were demonstrated in human skin abscesses and in some clinical specimens from different infection types.105 In laboratory experiments, the contribution of PVL to the lytic activity towards human neutrophils when assayed with CA-MRSA culture filtrates proved strongly dependent on the type of growth media used.112 For these reasons, such studies are barely conclusive when judging the role of PVL in CA-MRSA virulence.105 (Figure 2.2.2.2) On the other hand, animal infection models have yielded conflicting results, which has been attributed to differences between models, inoculum sizes, and, very importantly, the host species.104,105 Actually, the leukotoxic activity of PVL differs dramatically between species: human and rabbit neutrophils are lysed by very low toxin concentrations, whereas 1000-fold higher amounts are required for the lysis of mouse or java monkey neutrophils.113 Consequently, and the same way as with many other toxins, it is not clear whether pro-inflammatory effects are of benefit to the bacteria, as they may cause excessive phagocyte infiltration and tissue damage, or whether they serve the human innate immune system to recognize infiltration bacteria and launch a defensive response. Most probably, both mechanisms contribute to infection out-come and it depends on the specific scenario, which has a stronger impact. Finally, it needs to be noted that an increasing number of CA-MRSA clones have been found that do not contain lukSF genes.98 For example, in Korea114 and the United Kingdom,115 and Li et al .,97 have showed in rabbit skin infection studies that these clones are o average not less virulent than lukSF containing CA-MRSA clones. γ-haemolysin (HlgC/B) and PVL probably target the same cell types. Both toxins are similarly potent at lysing granulocytes and human macrophages. Whereas, PVL and ϒhaemolysin are extremely potent, leukocidins LukAB/GH and LukED are only active at concentrations 100-fold higher. The relative secretion of the different toxins in different conditions remains unknown.110 2.2.2.3 Phenol-soluble modulins. Phenol-soluble modulins PSMs are a family of amphipathic α-helical peptides produced by staphylococci.94 Many members of the PSMs family have pronounced cytolytic activity towards a variety of human cells, including neutrophils and erythrocytes.94 In addition, PSMs trigger inflammatory responses by interaction with the formyl peptide receptor 2 (FPR2).116 The α-type PSMs, ∼20–25 amino acids in length, may have strong cytolytic activity, and β-type PSMs are barely cytolytic and with a ∼40–45 amino acid long.94 In particular, the PSMα peptides of S. aureus , PSMα1–PSMα4, are encoded in the psm α Introduction: Staphylococcus aureus: Pathogenesis 36 operon that contains the potent cytolysin PSMα3. This PSMα3 or δ-haemolysin (Hld), is a moderately potent yet often strongly expressed. Three different mechanisms have been proposed to explain the haemolytic activity of δ-haemolysin, (i) bind to the cell surface and aggregate to form transmembrane pores; (ii) bind to the cell surface and affect the membrane curvature, thereby destabilizing the plasma membrane; or (iii) at high concentration, act as a detergent to solubilize the membrane.110 PSMs also contribute to staphylococcal biofilm formation by forming fibril-like structures, structure biofilms, and cause biofilm detachment, resulting in the dissemination of biofilm-associated infection.105 Actually, in S. epidermidis have been demonstrated to participate in the maturation of the biofilm structure, specifically intrabiofilm channels, and at high concentrations, in mediating bacterial detachment from the biofilm. Hence and by extension, probably performing the same biofilm regulatory functions in S. aureus. 110 (Figure 2.2.2) CA-MRSA strains produce high amounts of PSMs, whereas production is on average lower in typical HA-MRSA strains (USA100 and USA200 strains) where the psm-mec gene is located on a chromosomal cassette.117 94 This might, at least in part, be due to the facts that (i) Agr virulence regulator exerts an exceptionally strict control over PSMs expression,91 and (ii) HA-MRSA strains often show low, while CA-MRSA strains commonly have high, Agr activity.117 Briefly, the PSMα peptides have a significant impact on CA-MRSA virulence, in experimental skin infection using mice or rabbits and bacteremia in mice.37,94 Notably, PSMα peptides, especially PSMα3, are responsible for the increased neutrophil killing capacity that distinguishes CAfrom HAMRSA strains.118 Recent findings suggest that PSMs are expressed after neutrophil ingestion of the bacteria, in the neutrophil phagosome as a result of agr induction, identifying PSMs as the main mediator of quorum- (or diffusion-) sensing-induced neutrophil killing, after S. aureus ingestion.119 Moreover, the last research are setting towards the possibility that production of α-haemolysin could be modulated by PSMs expression, suggesting one coordinated action among Hla and PSMs in host tissue during early pathogenesis, confirming a major role for α-haemolysin in epithelial injury during S. aureus infection.120 2.2.3 Other toxins and enzymes. Others secreted S. aureus proteins, exactly proteases, are enzymes that degrade host molecules in a broad fashion, or interfere with host metabolic or signaling cascades, leading to tissue destruction, and may also have a more specific impact. The protease Introduction: Staphylococcus aureus: Pathogenesis 37 aureolysin , cleaves many proteins including insulin B, with a preference of cleaving after hydrophobic residues, or even can have a major impact on the pathogenesis of osteomyelitis because of inactivating PSMs. Up-regulated by Agr at the end of exponential phase, aureolysin also leads to maturation of another non-specific S. aureus exoprotease, such as the glutamyl endopeptidase SspA (o V8 protease), which cleaves after glutamate residues. Aureolysin, glutamyl endopeptidase, and the cysteine proteases staphopain B SspB , all interfere with complement factors, leading to evasion of complement-mediated bacterial killing. The biological function of further S. aureus proteases, a series of serine proteases , is not well understood, except for the exfoliative toxin serine proteases [see below]. Finally, S. aureus may produce a protease that degrades collagen, called collagenase .109 Figure 2.2.2 Non-lytic functions of haemolysins, bicomponent toxins, and PSMs. (1) α-hly binds ADAM10 and relocalizes it to E-cadherin-containing micro-domains. ADAM10 cleaves E-cadherin, leading to loss in epithelial barrier function. (2) Exposure of the underlying glycosaminoglycans-rich extracellular matrix to LukS-PV signal peptide favors adhesion. (3) psm - mec RNA controls the transcription of virulence factors. (4) β-haemolysin binds DNA, forming nucleoproteins nucleating S. aureus biofilm. PSMβ acts as surfactant, promoting the formation of intra-biofilm tunnels and bacterial dissemination from biofilms. psm-mec enhances biofilm formation (5) in addition, PSMs have antibacterial activity. (6) Finally, PSMs in synergy with β-haemolysin participate in phagosome lysis and escape into the host cytosol [see below Figure 2.4]. # α-hly: α-haemolysin; ADAM10: disintegrin and metalloproteinase domain containing. Reprint from [110] with permission from Frontiers Group. Copyright © 2007 - 2015 Frontiers Media S.A. Creative Commons Attribution License. All Rights Reserved. Introduction: Staphylococcus aureus: Pathogenesis 38 Some S. aureus secreted host-damaging factors, such as the fibrinogen-binding protein Efb and staphylococcal complement inhibitor SCIN (scn) , which are potent inhibitors of the function of convertase C3, a crucial enzyme in the complement pathway.109 Staphylokinase (Sak) is enzyme that activates plasminogen to plasmin, which degrades fibrin clots, whose biological significance is to diminish the function of the fibrin meshwork in keeping a staphylococcal infection localized. It also cleaves the complement factor C3b, adding to the broad attack of other staphylococcal proteases and further molecules, such as Efb (fibrinogen-binding protein) and SCIN (staphylococcal complement inhibitor), on complement function. Aside staphylokinase facilitates bacterial penetration through the skin barrier, while it decreases the severity of skin infections by leading to drainage.109 S. aureus further produces two coagulases, staphylocoagulase and von Willebrand factor , which contribute to the formation of fibrin clots after binding to prothrombin (forming a complex called staphylothrombin) and several other plasma proteins, thereby triggering the conversion of fibrinogen to fibrin. This leads to fibrin clots on the surface of S. aureus cells, inhibiting phagocytosis, causing abscess formation and adhesion of S. aureus to catheters during biofilm-associated infection.109 Moreover, S. aureus produces other proteins such as lipases and nucleases, whose functions in pathogenesis are poorly understood. Possibly, nucleases may decrease the antibacterial activity of neutrophil extracellular traps, which consist of DNA released from lysed neutrophils.109 On the other hand, S. aureus also harbors up to 15 enterotoxins (SEs, A, B, Cn, D, E, G, H, I, J, K, L, M, N, O), which are defined as superantigens, and able to produce gastrointestinal symptoms that include vomiting and diarrhea. For example, SEA, SEB, and SEC are the most frequent enterotoxins associated with food poisoning. Although many of these toxins have potential superantigen activity, others do not have a clear role in human disease, and the mode of action or mechanism at the surface of the intestinal mucosa is unclear. SEB can traverse the mucosa via transcytosis121,122 nevertheless, SEA, which is one of the first causes of food intoxication, apparently cannot. Likewise, SEB and SEC are associated with non-menstrual TSS. Finally , Staphylococcal scalded skin syndrome (SSSS) is a superficial skin disorder by toxigenic S. aureus that produces either exfoliative toxin A (ETA) or B (ETB), encoded by the eta and etb genes, respectively. These toxin genes are located either on a Introduction: Staphylococcus aureus: Pathogenesis 39 phage (eta)123 or on a plasmid (etb) . Two additional isoforms of SSSS toxins (exfoliative toxins C [ETC] and D [ETD]) were isolated through pathologic observations in animals and with genome screen.124 A recent study indicates that the proportion of S. aureus carrying eta or etb in overall staphylococcal nasal carriers or clinical isolates is low (0 to 2% of isolates),125 which may explain the rarity of the disease and its clustering in favorable milieus. 2.2.4 Superantigens. Aforementioned staphylococcal enterotoxins (SEs), and TSST-1 are the paradigm of a large family of pyrogenic exotoxins, called superantigens (SAgs) and denominated SET1 to SET15.126-128 Superantigens are proteins that do not activate the immune system via normal contact between antigen-presenting cells and T-lymphocytes. All of them share a common architecture, though quite some variation exists in the primary structure of many superantigens. They consist of A and B globular domains, β-sheet barrels and αhelices, rejoined by a discrete linking piece. A genealogy study of superantigens built on the base of their sequence homologies, has segregated them into five groups. Group I represented only by TSST-1, and group III contains only staphylococcal superantigens (SEs H, I, K, L, and P). Groups II and V contain both staphylococcal and streptococcal superantigens (staphylococcal SEs B, C, and G and SEs I, K, L, and P, respectively), and group IV only streptococcal superantigens. This underlines the likelihood of horizontal gene transfer between these two genera, a fact that is becoming increasingly apparent with genome comparisons.129,127 Recall that toxin genes are dispensable elements, not necessary for growth in rich media and in the absence of competition, but obviously provide a way for the bacterium to escape host immunity. Thereby, superantigens can result in harm of the host immune system, however they are not ultimate bacterial weapons, as they only affect to restricted subgroup of anergic patients.123,126,129 This survival advantage of provoking allergic diseases, including rhinitis, asthma130 and Kawasaki syndrome,131 is true but less intuitive. For example, TSST-1 and few SEs (A,B and C), have been involved in the etiology of psoriasis and atopic dermatitis,132 where toxin-induced skin modification could promote bacterial survival. TSST-1, associated with the toxic shock syndrome, is secreted locally by toxigenic strains, and can cross the mucosal membrane, and then disseminate throughout the body. An experimental study suggests that TSST-1 could activate directly epithelial cells and the innate immune system to promote its translocation.121 Actually, the clinical relevance of this multiplicity of toxins is not entirely understood. Introduction: Staphylococcus aureus: Pathogenesis 40 In short, CA-MRSA and CA-MSSA USA300 do not produce the superantigens SEB and SEC, but instead produce SEl-Q (and others superantigens including occasionally SEA and often SEl-K). Also it produces an apparently a N-terminal one-half deletion variant of TSST-1, whose activity is incompletely characterized, and has recently been associated with a newly described illness, extreme pyrexia syndrome, in which patients rapidly developed fevers in excess and quickly succumbed and died.133 USA300 also secretes the recently described superantigen SEl-X, which has been associated with necrotizing pneumonia caused by USA300 strains.134 2.3 Pathogenesis: Genomic and mobile elements. Mobile genetic elements (MGEs) can be defined as DNA fragments, which are able to encode one or more virulence and/or resistance determinants, including enzymes that mediate for their own transfer and integration into another DNA. Thus, they play a central role in the adaptation process, and are a means to transfer genetic information (DNA) between and within bacterial species. MGEs are called a ‘‘mobilome’’ because they are able to display a mobility in the same cell and between cells.135 MGEs can insert through vertical gene transfer phenomena various size of DNA sequences, such as phages, transposons, pathogenicity islands, plasmids and chromosome cassettes. “ This extra-chromosomal DNA elements play a crucial role in the plasticity of the genome, allowing bacteria to adjust readily to new environments ”.136 2.3.1 Plasmids, transposons and bacteriophages. The definition of Plasmids could be, any small-DNA-molecule able to auto-replicate in the own genome within a suitable host, and with the ability to transfer high frequency both resistance and virulence determinants, from one bacterium to another (even of another species) via horizontal gene transfer. For S. aureus have been classified into three different classes, (1) class I , composes of small (1.3–4.6 kbp), multicopy (10–55 copies per cell) plasmids with either cryptic or caring a single (rarely two) resistance determinants (pT181, pC194, pE194); (2) class II , are larger (15–46 kbp) and exist in lower copy numbers (4–6 per cell), however, this group includes most of the penicillinase and aminoglycoside/trimethoprim resistance plasmids (e.g. pSK1); and (3) class III consists of most large (30–60 kbp) plasmids caring a determinant of transfer ( tra ) by conjugation and in most of them, a combination of resistance markers; this group includes glycoside-resistance plasmids (pGO1 or pCRG1600) and usually possess one or two transposons and many copies of insertion sequences.135 In addition, staphylococcal plasmids encode resistance to a variety of organic and inorganic ions, Introduction: Staphylococcus aureus: Pathogenesis 41 such as cadmium, mercury, arsenate, etc., which are highly toxic for living cells, as well as molecules involved in metabolism.136 Insertion sequences (IS) are transposable elements, responsible for the recombination and stabilization of some resistance genes, which carry only that genetic information required for transposition, although it does not code for any resistance. Their presence is really important in the evolution of bacterial genome by inducing changes in the chromosomal genes expression,135 and can even inactivate several genes either by direct insertion or by polar effect on nearby genes transcription. For example, IS256 and IS257 are mediated by Tn4001 and Tn4003, and form a hybrid pair promoting resistance to aminoglycoside ( aac Aaph D) and trimethoprim ( dfr A), respectively.135,136 Staphylococcal transposons (Tn) are probably the smallest elements that encode resistance genes predominantly. The smaller transposons are usually presented in multiple copies in the genome, either inserted into the chromosome or into MGEs, such as SCC or plasmids. The larger transposons are present in single copies and encode resistance to antibiotics, such as tetracycline, trimethoprim, aminoglycosides, or vancomycin.136 Transposon Tn552 carries bla for penicillinase. Tn554 carries resistance to erythromycin, spectinomycin and macrolide–lincosamide-streptogramin B, presented in multiple copies, which can be found integrated into SCC (staphylocociccal cassette chromosome), plasmids or on the chromosome.137 Tn5801, found as unique conjugative transposon in Mu50, carries tet M encoding the resistance to both tetracycline and minocycline.138 Transposon Tn4291 carries resistance gene to methicillin, on the penicillinase plasmid (pI524).135 Tn1546 encodes the van A operon, ( van A, van H, van X, van S, van R, van Y and van Z) to vancomycin resistance, within a conjugative plasmid, whose gene expression occurs only in the presence of vancomycin, a process mediated by a two-CS-signal transduction encoded by van S and van R.136 Van Y and van Z encode an accessory protein that could play a role in teicoplanin resistance.139 Transposon Tn1546, was likely transferred to MRSA from vancomycinresistant Enterococcus (VRE) during co-infection.136 Bacteriophages (phages) or bacterial viruses, perhaps, have the greatest impact on staphylococcal diversity and evolution. All phages are classified into one of three distinctive groups: lytic, chronic and temperate. Lytic phages ( Myoviridae family) have been used in phage therapy, as they lyse completely to bacteria during release of progeny phages. Bacteria infected with chronic phages, release progeny into the extracellular environment without killing the host, which allows bacteria to grow and Introduction: Staphylococcus aureus: Pathogenesis 42 divide. Temperate phages ( Siphoviridae family), the most numerous group, have the ability to lyse bacteria after infection, but they typically form a long-term relationship with the host cell, whereby the phage DNA integrates into the staphylococcal genome as a prophage. Anyhow, phages can impact expression of virulence determinants by lysogenic conversion, positive or negative. Following positive lysogenic conversion, bacteria express prophage-encoded virulence determinants. Negative lysogenic conversion occurs when there is insertional inactivation of genes (e.g., hlb of S. aureus ) by integration of the phage DNA into the bacterial chromosome. Although there is a loss of β-haemolysin during lysogeny, these prophages contain genes encoding immune-modulator proteins, such as staphylokinase, staphylococcal inhibitor of complement (Scn), and chemotaxis inhibitory protein (Chp). Other S. aureus prophages encode virulence molecules such as enterotoxins and PVL. Prophages and prophage-encoded molecules also work together with other MGEs within staphylococci. They, for example, can create mobility for some staphylococcal pathogenicity islands, i.e., helper phage 80a mediates excision and transfer of pathogenicity island SaPI1 to other staphylococci. And others, have also the ability to transfer antibiotic resistance by transduction of plasmids or plasmid elements previously incorporated into chromosomal DNA, i.e., plasmid pS194 with a chloramphenicol resistance determinant and pI258.135,136 Figure 2.3.1 Acquisition of MGEs by S. aureus. (1) Incorporation of plasmids or plasmid elements into genomic DNA. (2) Plasmids can be maintained as free circular DNA. (3) Suicide plasmid. (4) Transfer of a transposon or an insertion sequence between plasmid and genomic DNA. (5) Transfer of a transposon or an insertion sequence between plasmids within the cell. (6) Transfer of a transposon or an insertion sequence from genomic DNA to another plasmid. Reprint from [136] with permission from Springer. Copyright © 2010 Copyright Clearance Center, Inc. All Rights Reserved. Introduction: Staphylococcus aureus: Pathogenesis 43 2.3.2 Pathogenicity and genomic Islands. Pathogenicity and genomic islands are continuous structures with variety in size (approx. 15 kb to 70 kb) that can harbour many virulence or resistance genes. They mostly contain heterologous DNA pointing out exogenous acquisition. A common feature of these elements is that they are bracketed by direct or inverted repeats and carry recombinase genes. These repeats serve as attachment site (att) for integration into homologous regions of the bacterial chromosome. The recombinase, which is often an integrase, catalyzes integration into the chromosome.2 Staphylococcal pathogenicity islands (SaPIs) are MGEs of 14–17 kb of which, at least 23 have been sequenced, and SaPI1 is considered as the prototype.136,135 SaPIs family core genes are highly conserved,136 and include two open reading frame (ORF) encoding transcriptional regulatory proteins, and a region encoding integrase ( int ), replication initiator with helicase activity ( rep ), phage interference function ( pif ) and phage terminase small subunit homologue ( ter S). SaPIs are integrated in one of six different specific sites on the chromosome ( att s) and each is always in the same orientation. In addition to core genes, almost all SaPIs encode integrase, resistance and virulence genes, and other superantigen-related diseases, such as food poisoning or host adaptation. SaPIs can be mobilized following infection by certain staphylococcal bacteriophages or by induction of endogenous prophages,135,136 such as induced excision of SaPI1 by phage 80a. Six kinds of superantigens genes ( tst -1, seb , sec , sel k, sel l and sel q) are harbored by several kinds of SaPI.136 Agr-regulated enterotoxin genes ( seb and sec) are carried on SaPI3.135 On the other hand, several hypotheses to explain the origin and evolution of SaPIs have been proposed, between them, Yarwood et al . propose the existence of a common ancestral genetic element, probably, a prophage for all SaPIs that then generated diversity of islands through modular recombination events.140 Staphylococcal genomic islands are larger segment of DNA (10–200 kb) commonly acquired by horizontal gene transfer.136 The families of genomic islands among the S.aureus strains whose genomes have been sequenced, are named ʋSAα, ʋSAβ and ʋSAϒ, and are flanked by a broken transposase gene (up-) and partial restrictionmodification system type I (down-). Both flanking DNA segments contribute to the stability of genomic islands within the S. aureus chromosome.135,136 Upstream, the direct-repeat genes are commonly associated with phages and plasmids and encode for both integrases and transposases.136 Introduction: Staphylococcus aureus: Pathogenesis 50 To avoid complement system components, gram-positive pathogens employ several proteolytic strategies through specific proteases capable of cleaving C5a or degradation of complement system proteins. Notable between these, is the serine protease V8153 and the metalloprotease aureolysin,154 which degrade key complement system components, including the opsonin C3b and the chemoattractant C5a. (Figure 2.4) Alternatively to the degradation of the complement-derived chemoattractant C5a, the neutrophil recruitment can be blocked by antagonizing the chemoattractant receptors. This is possible by antagonizing of formyl-methionyl-leucylphenylalanine (fMLP) peptides, one powerful neutrophil chemoattractants that arise from bacterial protein degradation, and can activate high-affinity fMLP peptide receptors on neutrophils. How? Chemotaxis inhibitory protein (Chp) is secreted by the pathogen and binds specifically to the fMLP peptide (and C5a) receptors to impair their sensing function. Likewise, S. aureus releases FLIPr, another virulence factor capable of antagonizing the fMLP peptide receptor to impair leukocyte responses to the bacterial-derived chemoattractant agonists.155 (Figure 2.4) The next steps to avoid the phagocytic activity, gram-positive bacteria are not sensed effectively by extracellular Toll-like receptors (TLRs), but intracellular detection is important. TLR family recognition S. aureus invasion triggers a complex mechanism through the activation of specific receptors for pathogen associated molecular patterns, eventually starting innate immunity response. Recently, a novel mechanism for staphylococci to escape the host immune system via interference with recognition by immune cells has been identified. Actually, the “phagocytized S. aureus”, have been shown to evade detection by limiting phagosome acidification or phagosome fusion with granules or lysosome, or by escaping the phagosome altogether, as mediated by toxins such as leukocidins.155 In all of this mechanism, Staphylococcal superantigens-like proteins play an important role. These proteins family of molecules are able to interfere with multiple components of host immunity, including humoral immunity, opsonization, and trafficking of leukocytes.150 Other mechanisms of evading intracellular killing include using protein mimicry to immune signaling. On the other hand, S. aureus can be observed intracellularly in non-professional phagocytes, membrane bound in a compartment or in the cytosol. Respect to this last point, the escape into the host cytosol would involve a synergism between δ-haemolysin or PSM and β-haemolysin.152 Curiously, in Cystic Fibrosis patients, α-haemolysin is involved in phagosomal membrane lysis in CFTRdeficient cells.156 (Figure 2.4) Introduction: Staphylococcus aureus: Pathogenesis 51 Howsoever, S. aureus is able to reply the bacteria to phagocytosis, by inducing the oxidative burst response, a key host microbicidal process in which oxygen is consumed, and toxic ROS are generated. This pathway is catalyzed by two major reactions in the cell. To survive the oxidative burst response, gram-positive pathogens have mechanisms to either prevent the production of or dispose of harmful oxidants. Invasive gram-positive species like S. aureus , encode a bacterial superoxide dismutase (SOD)157 that accelerates conversion of O2  H2O2, and catalase (Kat) is a common mechanism to oxidize 2H2O2  O2 + 2H2O. Whereas, reducing pathways such as thioredoxin systems provide electrons to small molecules that can react with H2O2, the tripeptide glutathione can reduce H2O2 directly.155 Shortly, other relevant concepts related such as the autophagy, which serves for degradation of organelles or self-digestion during nutrient limiting conditions (starvation), presumably to constitute a cellular survival mechanism. During bacterial infections, eventually, the bacteria escape from their intracellular confinement into the host cell cytoplasm in an agr -dependent manner (Figure 2.4), and consequently the host cell death is induced. Indeed, agr -deficient S. aureus fails to induce autophagy, which results in maturation of bacteria containing phagosomes followed by lysosomal degradation of the pathogens. As stated , S. aureus -induced autophagy resulted in a vacuolization of the host cell cytoplasm (“Swiss cheese phenotype”). And even more, αhaemolysis is able to permeabilize membranes for Ca2+, an inducer of autophagy.156 The phagocytised bacterial pathogens, also can evade lysosomal killing, e.g ., by disintegration of the organelle membrane in order to translocate into the host cell cytoplasm. This translocation into the cytoplasm of host cell, and the growing without an immediately ensuing cell death, illustrates that phagosomal escape is not identical with cytotoxicity. The virulence factors required for apoptosis by S. aureus induced in endothelial cells, also depend on agr and the alternative stress–response SigB (σB), independently of SarA. Aside from apoptosis S. aureus is also able to induce pyronecrosis (Figure 2.4). Introduction: Staphylococcus aureus: Pathogenesis 52 ADAM: Metalloprotease and Disintegrin; Arp2/3: Actin-Related Protein 2 And 3; Atl: Autolysin; Cytc, Cytochrome C ; Eap: Extracellular Adherence Protein; Fak: Focal Adhesion Kinase; Fnbp: FibronectinBinding Protein; Hla: α-haemolysin; Hsp: Heat Shock Protein; Il: Interleukin; Nfκb: Nuclear Factor Κb; Nwasp: Neural Wiskott–Aldrich Syndrome Protein; Pax: Paxillin; Sr: Scavenger Receptor; Vcl: Vinculin; WTA: Wall Teichoic Acid. Figure 2.4. A map of intracellular fates of S. aureus . (1) α5β1 integrins are sequestered by Fnbp-dependent fibronectin cross-linking at focal adhesions. (2) Bacteria are eventually endocytosed. (3) Assembly of Hla pores on the plasma membrane of host cells leads is dependent on ADAM10. Hla-pores are permeable for cations. [Ca2+ has been reported to induce macroautophagy]. (4a) Bacteria are disinfected by phagolysosomes or (4b) survive and grow within endosomes or (4c) in the cytoplasm after phagosomal escape. (5a) Phagosomal escape can be mediated by Hla, in Cystic Fibrosis cells, and (5b) also by a combination of PSMs and phospholipases. (6) Cytoplasmic S.aureus peptidoglycan is recognized by NOD2. (7) The mode of cell death induced by S.aureus is not completely understood. While caspaseindependent cell death exists, Hla is capable of inducing extrinsic apoptosis. Up on Hla induced K+ efflux caspase2 has recently been shown to lead to mitochondrial outer membrane permeabilization. (8) PVL has been reported to permeabilize mitochondrial outer membrane thereby releasing cytochrome c and thus inducing the apoptosome in a Bax-independent pathway of intrinsic apoptosis. Caspase 9 subsequently activates executioner caspases. (9) Cathepsin release from permeabilized phagosomes activates the inflammasome. Activated caspase1 leads to Il1β maturation and inflammatory pyro-necrotic cell death. (10) Toxin-permeabilized endocytic vesicles are targeted by autophagy. During autophagy isolation membrane engulfs leaky endosomes or cytoplasmically located bacteria. Within these autophagosomes bacterial replicate and eventually escape the organelle ultimately lead into host cell death. Reprint from [156] with permission from Frontiers Group. Copyright © 2007 - 2015 Frontiers Media S.A. Creative Commons Attribution License. All Rights Reserved. Introduction: Staphylococcus aureus: Pathogenesis 53 “The spread of highly virulent CA-MRSA is a major concern. Increased prevalence of leukotoxins, such as PVL, and increased expression of membrane-damaging factors, such as α-haemolysin and PSMs, have been observed in these strains” .110 Over decades, haemolysis and leukotoxicity have been highlighted as the major role of these virulence factors, and increasingly it is clear that the membrane-damaging toxins and peptides could have other functions besides killing host cells. Certainly, in vitro studies, suggest that these virulence factors are secreted in response to different environmental signals or at different stages during colonization or infection, and that this differential diffusion of the toxins and peptides within tissues could lead to different functions.110 Study of the interaction between these virulence factors and host cells in more physiological models should provide us novel important findings. 3 Antibiotic Resistance. S. aureus is a magnificent example of evolving pathogen, this organism has been shown an effective adaptation to the changing conditions of the surrounding medium, and therefore, from the point of view of the pathogenicity and the evolution to antimicrobial resistance, is regarded as the evolutionary paradigm progression. S. aureus has been able to develop resistance to virtually all antibiotic classes available for clinical use. These encompass cell wall inhibitors such as β-lactams and glycopeptides; ribosomal inhibitors that include macrolide-lincosamide-streptogramin B (MLSB resistance), aminoglycosides, tetracyclines, and the new oxazolidinones; the RNA polymerase inhibitor rifampicin; the DNA gyrase blocking quinolones; the antimetabolite trimethoprim-sulfamethoxazole; and the newer lipopeptides and lipoglycopeptides.2 The main resistance mechanisms are summarized in Table 3. Some are discussed subsequently. Introduction: Staphylococcus aureus: Antibiotic Resistance 54 Table 3. Antimicrobial resistance genes and mechanism in S.aureus. ANTIBIOTIC RESISTANCE GENE(S) GENE PRODUCT(S) MECHANISM(S) OF RESISTANCE RESISTANCE TO LOCALIZATION Inhibitor(s) of cell wall synthesis β-lactams blaZ β-lactamase Enzymatic hydrolysis of β-lactam ring. Inactivation. Penicillins Plasmid: Transposon mecA, mecC144 PBP2a PBP2 modified with reduce affinity. Modified Target. Penicillins, cephalosporins, monobactams, carbapenems Chromosome: SCCmec Glycopeptides Mutations in vraRS, rpoB? Altered peptidoglycan Trapping of vancomycin in the cell wall. Cell wall thickness: VISA. Vancomycin Chromosome vanA, vanH? D-Alanine-D-Lactate Reduce affinity. Modified target: VRSA Vancomycin Plasmid: Transposon Lipoglycopeptides vanA, vanZ? D-Alanine-D-Lactate Unknown Oritavancin Plasmid: Transposon Lipopeptides Mutation in mpfR, dltA. Multifactorial Change in the cell membrane charge. Decrease drug binding. Daptomicin Chromosome Bacitracina158 braDE, vraDE ABC transporters BraS/R-two-CS (Bacitracin resistance associated) responds to low bacitracin concentrations. Bacitracin Chromosome Fosfomycin159 fosA, fosB, fosX Glyoxilases Ring opening of the epoxide motif. Inactivation Fosfomycin Chromosome and Plasmid Inhibitor(s) Proteins Synthesis Aminoglycosides ant(4´)-Ia O-Adenyltransferases (ANT) Catalyzes ATP-dependent adenylation of hydroxyl group Tobramycin, amikacyn Plasmid. Plasmid: Transposon aph(3´)-IIIa O-Phosphotransferases (APH) Catalyzes ATP-dependent phosphorylation of a hydroxyl group Kanamycin, neomycin, amikacyn, gentamicin, Introduction: Staphylococcus aureus: Antibiotic Resistance 55 aac(6´)- Ie+aph(2´´) N-Acetyltransferases (AAC) Catalyzes acetyl CoA-dependent acetylation of an amino group Gentamycin, tobramycin, amikacyn Macrolidelincosamidestreptogramin msrA, msrB MsrABC pump Active efflux Macrolides, streptogramin B (MSb) Plasmid or chromosome lnu Nucleotidyltransferase Inactivation Lincosamines vgb Phosphorilases Inactivation. Macrolides, streptogramin B (MSb) ermA, ermB, ermC, (ermT or ermY). Erythromycin methylase Methylation of ribosome. Reduce binding to the 23s ribosomal. Macrolides, lincosamines, streptogramins (MLSb) Plasmid or chromosome Quinoprustin /Dalfopristin vat Acetyl transferases Enzymatic modification of dalfopristin Streptogramin A Plasmid vga ATP-binding cassette (ABC) transporters Active efflux. Oxazolinones rrn 23sRNA Mutations in domain V of 23S rRNA component of the 50S ribosome. Interferes with ribosomal binding. Linezolid Chromosome cfr 23S rRNA methylase Methylation of ribosome. Modified target. Linezolid, chloramphenicol, streptogramins, lincosamides Plasmid Tetracyclines tetK, tetL Efflux pump Active efflux. Tetracyclines Plasmid or chromosome tetM, tetO Ribosome protecting protein Protection of drug target Tetracyclines, minocyclines Fusidic Acid160 Mutation in fusA, fusE Elongation factor EF-G (fusA) and RNApol RplF (fusE) Reduce affinity. Modified target. Fusidic ac. Chromosome fusB, fusC, fusD Protection of drug target Plasmid Introduction: Staphylococcus aureus: Antibiotic Resistance 56 Chloramphenicol cat Chloramphenicol-acetyl transferase Inactivation Chloramphenicol Plasmid or chromosome fexA Efflux pump Active efflux of phenicoles. Florphenicol, chloramphenicol Chromosome Mupirocin161 Mutation in ileS Isoleucyl tRNA synthetase Low resistance (MuL) Reduce affinity. Modified target. Mupirocin Chromosome mupA, mupB162 High resistance (MuH) Plasmid Inhibitor(s) of metabolites Sulfonamides Mutations in dpsA, Dihidro-pholate synthetase (DHPS) DHPS with reduce affinity. Modified target. Overproduction of PABA (p-aminobenzoic ac) Sulfonamides (sulfamethoxazole, etc) Chromosome. Plasmid? Trimethoprim/ sulfamethoxazole Mutations in dfrA Dihidro-pholate reductase (DHFR) DHFR with reduce affinity. Modified target. Trimethoprim Chromosome, Plasmid Inhibitor(s) of nucleic acids function or synthesis Quinolones163 Mutations in grlA (parC), grlB (parE) parC and parE, components of topoisomerase IV Mutations in the quinolones-resistance-determiningregion QRDR, reducing affinity of enzyme-DNA complex for quinolones. Modified target. Stepwise resistance. 1st generation of quinolones (nalidixic ac, etc) 2nd generation, fluorquinolones (norfloxacin, ciprofloxacin, etc) Chromosome Mutations in gyrA, gyrB gyrA, gyrB component of gyrase norA, norB, norC Efflux pump Active efflux of hydrophilic quinolones. Quinolones, fluorquinolones Chromosome Rifampicin Mutations in rpoB β-subunit of RNA polymerase RNA polymerase with reduced affinity Rifampicin, vancomycin? Chromosome Introduction: Staphylococcus aureus: Antibiotic Resistance 57 3.1 β-Lactams. β-lactams are inhibitors of bacterial growth. They act by irreversible inhibiting of penicillin-binding proteins (PBPs) via preventing the final cross-linking (transpeptidation) step in the synthesis of the peptidoglycan, and thus, they disrupt the cell-wall synthesis. Penicillin and other β-lactams are steric analogs of the cell wall precursors D-alanineDalanine terminal. They compete with it for binding to the active site of the membranebound transpeptidase and act as mechanism-based inhibitors of PBP coined for these enzymes.2 In S. aureus , three different mechanisms of resistance has been identified, (i) enzymatic inactivation by β-lactamases or penicillinases, (ii) alteration on PBPs, and (iii) tolerance phenomena which affect to all β-lactams too.1 Tolerant microorganisms have reduced autolytic activity, and in the case of staphylococci, the experiments indicate that this is due to an excess of an autolysin inhibitor. Hence, tolerance is manifested as an increased resistance to the lethal, rather than the inhibitory, action of β-lactams.164 3.1.1 Resistance to Penicillin. The most common resistance mechanism of S. aureus to β-lactams is penicillinase production, encoded by the bla Z gene, usually carried on a plasmid. The gene is inducible and proceeded by the blaR1 and blaI regulatory determinants. Penicillinase is a secreted enzyme that hydrolyses penicillin and other penicillinase-susceptible compounds into inactive penicilloic acid (Figure 3.1.a). Penicillinase-producing S. aureus emerged rapidly after penicillin was introduced as a therapeutic agent in the mid-1940s. It is prevalent both, in the hospital and in the community, where it represents >95% of the isolates.2,165 3.1.2 Mechanism of Methicillin Resistance. The first penicillinase-stable β-lactams such as semisynthetic methicillin, nafcillin or cephalosporins became available in the late 1950s. Ironically, the first MRSA was described at about the same time. The prevalence of MRSA progressively increased thereafter, and nowadays MRSA constitutes 25–50% of clinical isolates in the North America, Europe and Asia.165 Higher-level β-lactam resistance (MRSA) results from the acquisition of the mecA gene, which encodes the penicillin-binding protein 2a (PBP2a) whose genes is controlled by the MecR-MecI-MecA regulatory systems,165 which responds to β-lactam antibiotics in a fashion similar to that of the regulation of blaZ . PBPs are membrane-bound enzymes Introduction: Staphylococcus aureus: Antibiotic Resistance 58 that catalyze the transpeptidation reaction that is necessary for cross-linkage of peptidoglycan chains. Their activity is similar to that of serine proteases, from which they appear to have evolved. PBP2a substitutes for the other PBPs and, because of its low affinity for all β-lactam antibiotics, enable staphylococci to survive exposure to high concentrations of these agents. Thus, resistance to methicillin confers resistance to all β-lactam agents, including cephalosporins.141 (Figure 3.1.b) Figure 3.1 (a) Induction of staphylococcal β-lactamase synthesis in the presence of the β-lactam antibiotic penicillin. (i) The BlaI binds to the operator region, repressing RNA transcription from both blaZ and blaR1-blaI . In the absence of penicillin, β-lactamase is expressed at low levels. (ii) Binding of penicillin to the transmembrane sensor-transducer BlaR1 stimulates BlaR1 autocatalytic activation. (iii)–(iv). Active BlaR1 either directly or indirectly (via a second protein, BlaR2) cleaves BlaI into inactive fragments, allowing transcription of both blaZ and blaR1-blaI . (v)–(vii) β-lactamase extracellular (v), hydrolyzes the β-lactam ring of penicillin (vi), thereby rendering it inactive (vii). (b) Mechanism of S. aureus resistance to methicillin. Synthesis of PBP2a proceeds in a fashion similar to that described for β-lactamase. β-lactam antibiotic induces MecR1 synthesis. MecR1 inactivates MecI, allowing synthesis of PBP2a. MecI and BlaI have co-regulatory effects on the expression of PBP2a and β-lactamase. Reprint from [141] with permission from American Society for Clinical Investigation. Copyright © 2003 Copyright Clearance Center, Inc. All Rights Reserved. Introduction: Staphylococcus aureus: Antibiotic Resistance 59 Provision of this adequate substrate to PBP2a requires the functionality of numerous accessory genes implicated in the normal wall building,165 some of which, femABCD essential factors for the expression and fmhB, are responsible for adding the glycine residues critical for the PBP2A function.1 Any alteration in these elements decreases the expression of methicillin resistance, even though PBP2a is present. Another fragility of PBP2a is that it carries only a transpeptidase domain and misses a transglycosidase activity. Thus, for successful assembly of the peptidoglycan, PBP2a needs to hijack the transglycosidase domain of normal staphylococcal PBP, namely PBP2.166 This is a salient example of heterologous protein cooperation in antibiotic resistance, but also represents the ’Achilles’ heel of the system. According to that, successful treatment of experimental endocarditis from MRSA achieved with an array of older and newer βlactams with good PBP2a affinity could be explained. This approach is a driving force for the development of new anti-MRSA compounds, or the re-using of older β-lacatms.2 As noted, the mecA gene is invariably part of a larger unique MGE, SCC mec , which may also contain additional genes for antimicrobial resistance and insertion sequences, as well as genes of uncertain function.141 These elements include, transposons such as Tn 554 , which carries resistance genes for spectinomycin and erythromycin, or integrated plasmids such as pUB110, which encodes tobramycin and kanamycin resistance among others.167 The different situation about the full-resistance to β-lactams, classified different types of strains: (i) higher-producing to β-lactamases, (ii) borderline oxacillin resistant mecA (+) strains, BORSA showing heterogeneous resistance, (iii) intermediate level of resistance to methicillin due to production of modified (PBP1, PBP2) or normal (PBP4) PBPs, achieving a reduced affinity for beta-lactams, and mec A (-), called MODSA,168 (iv) cefoxitin/oxacillin susceptibles mec A (+) OS-MRSA,169 and (v) the own MRSA. 3.2 Glycopeptides. In susceptible strains, glycopeptides inhibit cell wall through hydrogen bond interactions with the terminal D-alanyl-D-alanine moieties of the N-acetyl-muramic acid/N-acetylglucosamine (NAM/NAG)-peptides, and block both transpeptidation and transglycosylation. As a general rule, current glycopeptides are less effectives than βlactams against MSSA. Thus, they should not be used as first-line treatment against βlactam–susceptible organisms. However, vancomycin is still a "gold standard" against severe MRSA infections, until proof is found of newer anti-MRSA drugs, such as daptomycin, linezolid and anti-MRSA β-lactams. Introduction: Staphylococcus aureus: Antibiotic Resistance 66 resistance to oritavancin if all precursors terminating in D-Alanine are eliminated, and the expression of the van Z gene of the van A gene cluster also results in resistance to oritavancin (MIC > 8 μg/ml) via an unknown mechanism. Anyhow, the selection of dalbavancin resistance among staphylococci is slower than that with vancomycin. Dalbavancin and telavancin are active against VISA and oritavancin is active against both VISA and VRSA.193 Daptomycin is a lipopeptide and an amphophilic molecule that requires calcium to solubilize as octamer-micelles in liquid phases,194 necessary to interact with the plasma membrane and destabilize its electric potential.195 It is proposed as a replacement for vancomycin against MRSA, especially in right-sided infective endocarditis.2 The mechanisms of resistance to daptomycin might involve more than one mechanism, including (i) increased cell membrane fluidity, (ii) significantly reduced susceptibility to cationic host defense peptides of platelet and leukocyte, and (i) altered expression of two key determinants of net positive surface charge, either during exponential or stationary growth phases. DltA gain-in-function , reflected by a significant increase in teichoic acid D-alanylated content, and MprF gain-in-function , reflected by a heightened elaboration of lysinylated phosphatidylglycerol. Taken together, S. aureus appears to involve multi-factorial and strain-specific adaptive mechanisms for resistance to daptomycin. This issue is especially relevant to MRSA strains in the context of invasive endovascular infections.196 4 Characteristics of CA-MRSA. The emergence of CA-MRSA as an important pathogen has occurred over the past 15–20 years.197,198 The first cases described, 1997-1999, were associated with necrotizing pneumonia or pulmonary abscesses and sepsis100 with a resolution rapidly fatal. The strain responsible was USA400 (also known as the MW2 strain).199 Subsequently, clonal outbreaks of skin and soft-tissue infections caused by CA-MRSA were also reported.100 This time, the strain responsible was USA300.200 These CA-MRSA, both ST8, appear to have enhanced virulence, enhance capacity to colonize multiple body sites and to survive on environmental surfaces,98,146 as well as one important characteristic, their transmissibility due to their small SCC mec IV. Although cases of pyomyositis, purpura fulminans with toxic shock syndrome, and Waterhouse-Friderichsen syndrome,100 were described, their fatality is due to necrotizing fasciitis and necrotizing pneumonia, associated especially to USA300. Introduction: Staphylococcus aureus: Characteristics of CA-MRSA 67 Pointing out, CA-MRSA is different from health care-acquired (HCA)-MRSA, from both epidemiologic and molecular points of view, as well as for clinical syndromes.201 Health care-acquired -MRSA is associated with risk factors that included recent hospitalization or surgery, living in a nursing-home, or carrying an indwelling catheter or device, producing mostly hospital-related pneumonia and bacteremia. Otherwise, CA-MRSA is not associated with any risk factors, and produces primarily skin and soft tissue infections, recently necrotizing fasciitis and bone and joint infections and sometimes rapidly fatal necrotizing pneumonia.202 In addition, health care-acquired -MRSA is multiresistant and highly clonal, whereas CA-MRSA is pauciresistant and seemingly more polyclonal,111 except for USA300 clone.203 Thus, health care-acquired -MRSA and CAMRSA are not alike. Practically, MRSA in patients at risk is likely to be of the multiresistant hospital type, whereas MRSA in patients without risks is likely to be more susceptible but more invasive. 4.1 Genetics of methicillin resistance in S. aureus and CA-MRSA clones. Aforementioned in a previous chapter, the main characteristic of CA-MRSA is the acquisition of a MGE SCC mec of smallest size , 204 usually SCC mec IV (sometimes SCC mec V), and especially the strains that cause virulent infections. The capacity to acquire novel elements have been and is essential to the success of these clones, so much as to its transmissibility, as well as to its adaptation or evolution, which has also permitted them to hold over time (Figure 5.1). " The evolution of virulence in S. aureus clones and lineages is frequently only analyzed in terms of acquisition or loss of virulence-associated genes. However, significant changes in virulence may arise from minute changes in the genome and all of these changes contribute to the evolution of highly virulent CA-MRSA. The peculiar in all CA-MRSA, is the higher expression of PSMs and α-haemolysin (Hla) compared to HA-MRSA strains, 117 likely or at least in part due to the high activity in CA-MRSA of Agr, which controls expression of most S. aureus toxins and has a strong impact on CA-MRSA virulence ".205 4.2 CA-MRSA transmissibility and fitness. CA-MRSA isolates carry SCC mec elements of type IV or V, which may be associated with lower fitness costs105 due to smaller size, so that they may also show increased transmissibility and colonization characteristics. Although, the "in vitro206 vs in vivo"202 studies have been dissimilar in this aspect about the fitness. Actually, the fact that all Introduction: Staphylococcus aureus: Characteristics of CA-MRSA 68 CA-MRSA strains detected so far harbour one type of the novel, short SCC mec type IV or V, indicates that they played a crucial role in the evolution of CA-MRSA. On the other hand, although there is only limited data, this potentially increased colonization capacity present in CA-MRSA is related to the presence of the ACME, exactly ACMEspeG gene, which encodes a spermidin acetyltransferase gene that transfers resistance to spermidin and other polyamines molecules.147 Thus, the synthesis of this speG , could serve to explain augmented colonization capacity in USA300, since the exceptional sensitivity towards polyamines is only abolished in ACME-containing strains. Briefly, SpeG detoxifies polyamines and may, thus enhance survival of USA300 on the human skin.105 4.3 CA-MRSA virulence. Many studies about the virulence of CA-MRSA, where MSSA infections vs MRSA infections are compared, have been published. To date, there is no compelling evidence that MRSA, in general, is more virulent than MSSA. Although the issue remains unresolved, invasive MRSA infection is associated with greater costs, limited treatment options100 and worse Figure. 4.1. Evolution of CA-MRSA. The acquisition of SCC mec Type IV (or V) by virulent strains appears to have been a common first step in the evolution of CA-MRSA strains. In some strains, USA300, additional steps were required. These additional steps may have involved adaptations of gene expression. Uptake of SCC mec type IV by a virulent ST8 strain resulted in a virulent MRSA strain (USA500), whose fitness was further improved by uptake of ACME. Acquisition of PVL-encoding genes appears to have increased virulence at least in some infection types. Reprint from with [105] permission from Elsevier. Copyright © 2013 Copyright Clearance Center, Inc. All Rights Reserved. Introduction: Staphylococcus aureus: Characteristics of CA-MRSA 69 long-term outcomes after MRSA bacteremia vs those after MSSA bacteremia.207 The major virulence of CA-MRSA is a fact. These strains exhibit high capacity to circumvent the first line of defence of the human body against staphylococcal infections, even the killing by human neutrophils. Hence, it is exactly the accountable for the ability of CA-MRSA strains to infect healthy people.102 In general S. aureus is not commonly known for producing a plethora of toxins, some of which are found virtually in all S. aureus , while others are linked to MGEs and restricted to a subset of strains. Among these toxins, some are involved in evasion of neutrophil killing, which was at least one of the predominant factors assumed to be associated with that enhanced virulence. In short, staphylococcal leukocidins, a specific toxin repertoire or an enhanced production of toxins appeared as a likely basis for the enhanced virulence characteristics of CA-MRSA.105 α-haemolysin (Hla) is a cytolysin that has demonstrated a big impact on virulence in many infection models. Hla contributes to the penetration of the epithelial barrier during skin infection by USA300,106 and it could contribute to the observed systemic severity by CA-MRSA. Thus, α-haemolysin is a critical virulence factor provable in numerous animal infection models, for example, brain abscesses, skin and soft tissue infections, and has a significant effect on morbidity and mortality in CA-pneumonia.107 The PSMs peptides have a significant impact on CA-MRSA virulence, due to being responsible for the increased neutrophil killing capacity that distinguishes CA-MRSA from HA-MRSA strains.94 And the leukocidin PVL, a virulence factor whose role is more controversial. Because, depending on specific infection types or scenarios (animal model) and even of strains, could significantly contribute to the severity of the disease caused by CA-MRSA.105 Actually, its effect keeps on being studied in different experimental models,96,97, 114 because an increasing number of CA-MRSA clones, not containing luk SF genes are found equally virulent.98 On the other hand, the contribution of lytic activity in this PVL toxin towards human neutrophils is studied in laboratory experiments.112 Therefore, perhaps initially overestimated their virulence. CA-MRSA, especially USA300, has been shown to overexpress a number of core-genome– encoded virulence factors, such as α-haemolysin (Hla) and PSMs. PVL and Hla seem to be required for early lung involvement via haematogenous spread. α-haemolysin, but not Introduction: Staphylococcus aureus: Characteristics of CA-MRSA 70 PVL, significantly impact severe sepsis-related mortality. PVL is the predominant factor determining late-stage bone abscesses.208 Reprint from [161] with permission from American Society for Microbiology. Copyright © 2010 Copyright Clearance Center, Inc. All Rights Reserved. Table 4. Plasmid and characteristics MGEs of S. aureus USA300. Size (bp) Characteristics genes Function Mobile genetic elements SCCmec type IV - mecA Methicillin resistance ACME type I - arc cluster + opp3 cluster Arginine desaminase + oligopeptide permease system SaPIs - seq2, sek2 Pyrogenic toxin superantigens ϕSA2USA - lukS-PV, lukF-PV PVL ϕSA3USA - sak, chp Fibrin-specific-blood-dot dissolving enzyme, chemotaxis inhibitory factor Plasmids pUSA01 3125 - Cryptic pUSA02 4439 tetK Resistance to tetracycline pUSA03 37136 ermC, ileS Resistance to macrolides, lincosamides, streptogramins B an mupirocin Figure 4. Hypothetical virulence factors in USA300 and other CA-MRSA strains. on October 4, 2014 by guesthttp://cmr.asm.org/Downloaded from State-of-the-Art State –of-the-Art 73 Since 1900s we have known about mass spectrometry and, since a lack of suitable ionization techniques for high mass biomolecules, proteins remained inaccessible to MS analysis for decades. Due to the introduction of soft ionization techniques, such as Matrix Assisted Laser Desorption Ionization (MALDI) and Electrospray Ionization (ESI), mass spectrometry at the end of the 1980s209,210 and protein analysis by mass spectrometry underwent a rapid phase of development. Nowadays, has become a widely established technique for analyzing chemical structures in low quantities to trace levels. In parallel, an increasing number of full genome sequences for a variety of organisms are now available and numerous protein databases were constructed from this information. Well-annotated, high-quality protein databases built the ground on which high-output of protein identification with mass spectrometry can be performed. Another major approach has been the combination, or the modular arrangement of different types of mass analyzers (MALDIor ESI), which has resulted in a wide variety of different mass spectrometric instrumentation (e.g., MALDI-TOF, ESI-Q-TOF, ESI-ion trap, MALDITOF/TOF, etc.). Although they always required additional sample preparation techniques, all of these mass spectrometry techniques allowed the determination of the primary structure of a protein. Now, modern mass spectrometers are combined with high sensitivity, to improve mass accuracy, mass resolution, and rapid analysis as well as getting sophisticated data handling in a system-dependent manner. In addition to these technical aspects in mass spectrometry, greatly improved sample separation and preparation techniques have also lead to enhanced sensitivity. Currently, the multienzyme digestion (MED) filter-assisted sample preparation strategy (FASP) approach appears to be especially useful for the analysis of samples available only in minute amounts, significantly increasing the number of identified proteins and their sequence coverage. The MED-FASP offers efficient exploration of previously unused sample material, increasing depth of proteomic analyses and sequence coverage.211 Quantitative proteomics yields comprehensive data on protein expression levels enabling the detailed characterization of biological processes, by comparison of various physiological states or different biological entities (such as different bacterial strains or samples from healthy and diseased organisms), on a proteome-wide scale. Mass spectrometry-based label-free quantification (LFQ) can be achieved by spectral counting212 or by determination of peptide signal intensities.213 However, the reliable quantification via these approaches requires a highly reproducible chromatographic sample separation, which has been resolved with the pre-treatment of the samples by nano-LC (Liquid Chromatography). Although it is known that LFQ techniques suffer from the fact that peptide signal intensities are not strictly proportional to the peptide State –of-the-Art 74 concentration owing to ion suppression effects,214 LFQ is a great alternative a labeltechniques such as ITRAQ for these proteogenomic studies. The applicability of label-free approaches was shown in different proteomic studies215 and even in the challenging field of biomarker discovery.216 Furthermore, bioinformatics software’s such as, MaxQuant software suite, developed for data acquired by high-resolution instrumentation, which permits the identification of proteins and analysis later, linked to the large "online" bioinformatics tools. Thus, nowadays, an exhaustive-wide and in depth proteogenomic analysis, is possible. The proteogenomic studies give us information of each protein within its own functionality and its adaptation in different environments or contexts. Background Manuscript Manuscript 85 Quantitative Proteomic Analysis of Community-Acquired Methicillin-Resistant Staphylococcus aureus USA300 in Response to Subinhibitory Concentrations of Antibiotics Torres-Sangiao, Eva (1,2), Kucharova, Veronika (1), de Souza, Gustavo Antonio (3), Bou, Germán (2) García Riestra, Carlos (4) and Wiker, Harald G (1)* (1) The Gade Research Group for Infection and Immunity, Department of Clinical Science, Faculty of Medicine and Dentistry, University of Bergen, Norway, (2) Clinical Microbiology Laboratory, University Hospital Complex of A Coruña – INIBIC, Spain, (3) Proteomics Core Facility, Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital, Norway, (4) Department of Microbiology, University Hospital Complex of Santiago de Compostela - IDIS-, University of Santiago de Compostela, Spain *Corresponding author: Wiker, Harald G The Gade Research Group for Infection and Immunity, Department of Clinical Science, Faculty of Medicine and Dentistry at University of Bergen, Norway Laboratory building 5th floor, Jonas Lies vei 65, 5021 Bergen Email: Harald[email protected].no Office number: +47 95 07 11 95 FAX number: +47 55 97 58 17 Keys words: USA300, MRSA, pathogenesis, LFQ proteomics, virulence factors The version of this manuscript may undergo minor modifications for publication. Manuscript 87 Synopsis This study gives an overview of the expressed proteome of a hypervirulent, community acquired and resistant Staphylococcus aureus isolate, S. aureus USA300 strain, and its response to antibiotic pressure. Unlike previous genome-wide transcriptome studies, which monitored changes on mRNA level, we show qualitative analysis of USA300 cell at the protein level that directly reflects physiologically relevant adaptations. We focused on several groups of main proteins regulatory system, response to stress (include mechanism of resistance) and virulence. Here we provide evidence of the S. aureus USA300 cells adaptation to different antibiotics, and the possible therapeutic effect on the S. aureus USA300 strain of subinhibitory concentrations of several clinical used antibiotics. This study shows how the bacteria adapts to overcome low-level antibiotic stress and what proteins are involved. Manuscript 89 Summary Antimicrobial resistance of the human pathogen Staphylococcus aureus is an ongoing problem in the healthcare sector. Community-acquired methicillin-resistant S. aureus , USA300 strain, has the ability to infect healthy individuals because of its high virulence. By using tandem mass spectrometry, we investigated the expressed proteome of USA300 in response to two subinhibitory concentrations of linezolid, tigecycline, oxacillin and vancomycin, and compared it to a control condition without antibiotic. Analysis of the respective USA300 cell extracts identified over 40% of USA300 predicted proteome in total. Analysis of expression patterns of virulence and pathogenesis proteins showed that protein synthesis inhibitors had the most effect during the conditions used and cell-wall synthesis inhibitors had less effect. The treatment with protein synthesis inhibitors, linezolid and tigecycline, increased the expression of ribosomal protein RplC. Tigecycline showed a dose-dependent bactericidal activity, exhibited by a strong decrease in proteins expression, particularly of α-haemolysin Hla. Oxacillin and vancomycin inhibit cell-wall synthesis. Treatment with oxacillin led to an extensive lysis of the bacterial cells. Nevertheless, oxacillin increased the expression of virulence factors such as phenolsoluble-modulins (PSMs) and Panton-Valentine Leukocidin toxin (PVL), but not Hla. Therefore, the USA300 strain appeared to have a capacity to adapt itself to subinhibitory concentrations of antibiotics, by regulating its metabolism, redirecting the energy, and biofilm formation. Manuscript: Material and Methods 91 Material & Methods Bacterial strains and reagents. The pvl(+) CA-MRSA S. aureus strain USA300 (SCC mec IV-ST8)200 was kindly provided by Dr. DeLeo (National Institutes of Health, Hamilton) and genotyped as able to release the PVL toxin.146 The strain was grown in tryptic-soy broth. Linezolid, tigecycline, oxacillin and vancomycin were all obtained from Sigma Chemicals Co. (St. Louis, MO). Antibiotic dilutions were made fresh prior to each experiment. S. aureus strains ATCC 29213 and ATCC 25923 served as controls for MIC determinations. Determination of MICs. Antibiotic MICs were determined for each isolate using the broth microdilution method recommended by Clinical Laboratory Standards Institute (CLSI) in Mueller–Hinton II medium (bioMérieux, Marcy l’Etoile, France),14 and by E-test® following the manufacturer’s instructions (bioMérieux). The medium was inoculated with 5x105 colony-forming units per milliliter (CFU/ml) (equivalent to 0.5 McFarland) and incubated for 18-20 h and until 24 h for vancomycin, at 37 °C without shaking. Experiments were done in triplicate and the results are given in Supplementary Table S1. The E-test® method was used for determining vancomycin MICs, because it is considered to be more reliable in predicting treatment response, and also showed higher correlation with population analyses profile-area under the curve (PAP-AUC).231-233 Culture conditions for proteomic analysis. To prepare stock cultures, a single colony of S. aureus USA300 was inoculated into 5 ml of sterile tryptic-soy broth and incubated overnight at 37 °C with rotary aeration (200 rpm). 100 μl of the overnight culture was used to inoculate fresh TSB (dilution 1:100, volume ratio 5:1, OD600 = 0.04 + 0.005), and incubated for 4 h at 37 °C with shaking (250 rpm). The growth was monitored spectrophotometrically until OD600 = 0.75 + 0.05 (early stationary phase). At this point, concentrations of 0.25 and 0.5 of MICs of each antibiotic were added to 2 ml of culture tubes (final volume and same conditions, volume ratio 5:1). The concentrations used were: 0.25 and 0.5 μg/ml for linezolid, 0.12 and 0.25 μg/ml for tigecycline, 8 and 16 μg/ml for oxacillin, 0.5 and 1 μg/ml for vancomycin, respectively. Cultures with or without antibiotics were re-incubated for 4 h at 37 °C with shaking (200 rpm) until OD600 reached at 2 + 0.1 (stationary phase). Subsequently, an aliquot of each culture was plated on blood agar to determine CFU/ml. Experiments were performed in triplicate (Supplementary Figure S1.A). Manuscript: Results and Discussion 98 cellular localization for the identified proteins from the UniProt database (Table 1). Treatment with linezolid and vancomycin preserved the cellular localization distribution of proteins with respect to the control. In samples treated with 0.5 MICs of oxacillin or tigecycline, the numbers of identified membrane proteins were decreased more than 40 %, especially proteins involved in transport processes, whereas extracellular proteins were increased more than 40 %. Extracellular proteins,30 cell surface proteins or proteins associated with the membrane,100,243 are often associated with pathogenesis, colonization or antibiotic resistance. Therefore, a loss of transporters could point to a loss of viability of cell wall function, and the great number of extracellular proteins could be associated with an increase in virulence factors and / or a stress response. Second, we performed qualitative analysis by DAVID software program, to predict associated biological processes and metabolic pathway enrichments. As expected, 0.5 MICs gave the most pronounced differences, while 0.25 MICs produced only minor changes, compared to the control. Hence according to the literature, the antibiotic pressure led to a redirection of the cell metabolism by regulation of the energy, amino acid or purine / pyrimidine metabolism pathways, to overcome the influence of the antibiotics244 (Supplementary Table S3). In addition, changes in the metabolic pathways observed for 0.5 MIC of linezolid was concordant with the study of Bernando et al .,245 and the downregulation of metabolic pathways observed for tigecycline could be associated with bactericidal activity242,246 (Supplementary Table S3). On the other hand, linezolid showed a dose-dependent increase in a number of ABC transporters, which could be related to an early stage of developing resistance.247,248 Whilst, oxacillin increased the number of stress response proteins and pathogenesis proteins.247 (Supplementary Table S4). Indeed, the treated S. aureus USA300 cells had a substantial number of proteins involved in response to stress (Supplementary Figure S2). Therefore, there were considerable changes in the protein profiles in response to 0.5 MICs of the antibiotics. The effects were particularly pronounced for tigecycline and oxacillin. Manuscript: Results and Discussion 99 Figure 1. Proteome comparison in response to antibiotic treatments. Venn Diagrams showing shared and exclusive proteins among control samples and both subinhibitory concentrations (0.25 MIC and 0.5 MIC) of linezolid, tigecycline, oxacillin and vancomycin. Table 1. Cellular localization distribution of identified proteins in USA300 proteome, under absence (control) or presence of subinhibitory concentrations of antibiotics. CELLULAR LOCALIZATION ANTIBIOTIC Control Linezolid Tigecycline Oxacillin Vancomycin Total 0.25 μg/ml 0.5 μg/ml 0.12 μg/ml 0.25 μg/ml 8 μg/ml 16 μg/ml 0.5 μg/ml 1 μg/ml CELL WALL 8 6 6 5 4 6 6 7 7 10 MEMBRANE 56 49 53 25 21 40 27 45 51 67 EXTRACELLULAR 30 31 32 23 33 36 35 30 30 42 CYTOPLASM 247 241 245 169 189 239 216 219 258 291 UNKNOWED 433 388 395 196 219 407 332 334 418 570 Total proteins 774 715 731 418 466 728 616 635 764 980 Manuscript: Results and Discussion 100 Quantitative analysis of differentially expressed proteins in response to 0.5 MICs To ascertain that the identified proteins originating from extracts of S. aureus USA300 cells showed good correlation, the LFQ intensities of identified proteins were used to calculate Pearson correlation coefficient R, determined by Perseus software (Supplementary figure S3). The scatter plots showed good correlations, comparing each antibiotic condition with the control (Figure 2, a - d), or by comparing the two different antibiotic concentrations. The culture supernatants had relatively few identified proteins, except for oxacillin, which resulted in a much higher number of proteins in the supernatant than the other antibiotics (Figure 2, e - f). The LFQ intensities of identified proteins in the culture supernatants of the two concentrations of oxacillin, correlated well but poorly to the control. These “extra” proteins found in the oxacillin supernatants, were mainly identified as typical intracellular proteins, inferring extensive bacterial lysis of S. aureus USA300 cells.249 For the comparative analysis of S. aureus USA300 specific response to each antibiotic, we only considered proteins differentially expressed in samples treated with 0.5 MICs. To analyze these differentially expressed proteins, we took into consideration the experiment design and the detection limitations. The differentially expressed proteins not identified under some condition, antibiotic or control, with a normalized LFQ intensities ( N -LFQ intensity) less than 0.20 (LFQ intensity < 106), we decided not to consider them. Therefore, among differentially expressed proteins in control, 26, 205, 117 and 21 were not observed with linezolid, tigecycline, oxacillin and vancomycin treatments, respectively, whereas 19, 32, 39 and 17 proteins were differentially expressed with linezolid, tigecycline, oxacillin and vancomycin, respectively, which were not observed in the control. For the differentially expressed proteins in treated and untreated-control S aureus USA300 cells, the statistical analysis revealed 4, 37, 24 and 3 proteins showing significant statistical differences for linezolid, tigecycline, oxacillin and vancomycin, respectively (Figure 3). We only investigated in depth proteins predicted to Manuscript: Results and Discussion 101 Figure 2. Individual Plots correlation by Pearson coefficient R. Scatter plots showing each Pearson coefficient R, and the correlation between LFQ intensities means ( LFQi) of, (a-d) proteins identified in the pellet fraction under 0.5MICs ( y axis), plotted against proteins identified in the control ( x axis), and (e-f) proteins identified in supernatant fraction under 0.5 MIC of oxacillin ( y axis), plotted against proteins identified in control and 0.25MIC of oxacillin, respectively ( x axis). LFQ intensities means were calculated as the average among the six, for the control and three, for antibiotics, replicates. BLUE = proteins from pellet; BLACK = shared proteins between both fractions; RED = proteins from supernatant; GREEN = proteins are not within control. Manuscript: Results and Discussion 102 be involved in mechanism of action, pathogenesis and response to stress, including mechanism of resistance. Hence, we could analyze regulatory systems or proteins that regulate virulence factors in S. aureus USA300, e.g ., repressor CodY250 or Clp protease family,86 and proteins involved in pathogenesis such as phenol-soluble-modulins (PsmA), α-haemolysin (Hla/Hly) and PVL (LukF-PV / LukS-PV). Phenol-soluble-modulins, Hla/Hly and PVL are the main virulence toxins characteristic of hypervirulent S. aureus USA300.251 We can also analyze proteins predicted to be involved in mechanism of resistance to vancomycin (RpoB, RpoC),252 β-lactam family (BlaZ, PBP2a/MecA) or aminoglycosides (AphA(3')III). The differentially expressed proteins in control but not observed with antibiotics are summarized in Table 2, the differentially expressed proteins under antibiotic conditions in Table 3, and the statistical analysis is summarized in Supplementary Table 5 and is shown by volcano plots (Figure 3). Figure 3. Volcano Plots. Volcano Plots generated by Perseus (v.1.5.1.6) showing the most significant differences for proteins differentially expressed under 0.5MIC-conditions of, (a) linezolid, (b) tigecycline, (c) oxacillin, and (d) vancomycin, respect to untreated-control-USA300. Volcano Plots display both high statistical significance, p -value < 0.05 ( y -axis), and large magnitude fold changes, antibiotic vs control ( x -axis). The dashed black-line shows where p -value =0.05 expressed as -log t-test p-value (log10 p-value) = 1.30103, with points above the line p < 0.05 and pints below the line having p > 0.05. The filled square plot is such that points having a fold-changes less/more than 4x (4 < x < 0.25) expressed as ratio antibiotic / control and according to t-test difference [antibiotic – control]. The filled diamond plot is such that points having statistical significance. Aph3'A: aminoglycoside 3'-phosphotransferase; Aur: zinc metalloproteinase aureolysin; BlaZ: beta-lactamase; ClpP: ATPdependent Clp protease proteolytic subunit; Eap: extracellular adherence protein; EsaA: virulence protein; EzrA: septation ring formation regulator; Ffh: signal recognition particle protein; FtsZ: cell division protein; HemG: protoporphyrinogen oxidase; Hld: delta-haemolysin; Hly: alfa-haemolysin; IsaB: immunodominant antigen B; RpsA: 30S ribosomal protein S1; LukF-PV: Panton-Valentine leukocidin subunit F; LukS-PV: Panton-Valentine leukocidin subunit S; MecA: pencillin binding protein 2a (PBP 2a); PheT: phenylalanine--tRNA ligase beta subunit; ProRS: Proline-tRNA ligase; PurD: phosphoribosylamine-glycine ligase; PurL: phosphoribosylformylglycinamidine synthase 2; RplC: 50S ribosomal protein L3; RplE: 50S ribosomal protein L5; RpoB: DNA-directed RNA polymerase subunit beta; RpoC: DNA-directed RNA polymerase subunit beta; RpsE: 30S ribosomal protein S5; RpsJ: 30S ribosomal protein S10; SarA: Staphylococcal accessory regulator A; SdrE: serine-aspartate repeat-containing protein E; Seq: enterotoxin Q; SodA: superoxide dismutase A; SplC: serine protease C; SplE: serine protease E; SplF: serine protease F; Ssb: single-stranded DNA-binding protein; SspB: C47 family staphopain B; SspC: I57 family staphostatin B; TsaD: tRNA N6-adenosine threonylcarbamoyltransferase. Statistical significance proteins. Underline protein name. Manuscript: Results and Discussion 103 t-test difference linezolid [10e-1] -lg t-test pvalue. Linezolid -1.8 -1.2 -0.6 0 0.6 1 1.6 (a) Hld SdrE RplC SplE Seq Aur MecA RpsE -lg t-test pvalue. Tigecycline t-test difference tigecycline [10e-1] -2.6 -2 -1.6 -1.2 -0.6 0 0.6 1 1.6 (b) RplC Ffh ProRS TsaD ClpP PurL SplC SplE Aur Hly Aph3`A RpsA RNA methyltransferase PurD SspBIsaB LukS-PV BlaZ Manuscript: Results and Discussion 104 Mechanism of action -lg t-test pvalue. Vancomycin t-test difference vancomycin [10e-1] -1.8 -1.2 -0.6 0 0.6 1 (d) HemG Hld MecA -lg t-test pvalue. Oxacillin t-test difference oxacillin [10e-1] -1.8 -1.2 -0.6 0 0.6 1 1.6 (c) EsaA PheT IsaB RpsJ SspC RplE BlaZ LukF-PV Eap SplF PurD Fur family transcription regualtor FtsZ Ssb RpoB SodA ClpP EzrA RpoC Manuscript: Results and Discussion 105 Mechanism of action The protein synthesis inhibitors, linezolid and tigecycline, showed a significant increase in the expression of RplC (50S L3) (Figure 3a – b, Supplementary Table S5) assembly initiator protein involved in the initiation first step, which has also been implicated in resistance to linezolid.253 Tigecycline further showed a significant increase in the expression of other proteins involved in protein synthesis, such as proline-tRNA-ligase ProS and RNA-methyl-transferase (Figure 3b, Supplementary Table S5). This more significant increase with tigecycline was concurrent with the lower expression of ribosomal proteins, e.g., RplP (Table 2). The cell wall inhibitors, oxacillin and vancomycin, did not show significant statistical differences in the proteins expression involved in cell wall processes (Figure 3c - d, Supplementary Table S5). Nevertheless, oxacillin decreased the expression of FtsZ and its negative regulator EzrA (Figure 3c, Supplementary Table S5), predicted to be involved in cell division, and proteins such as DltD or MurF (Table 2), involved in cell wall biogenesis / degradation, as well as the organelle-organization crucial in chromosome dynamics Smc,254 were not observed (Table 2). This was expected considering that linezolid and tigecycline have effect on proteins synthesis, and oxacillin on cell wall synthesis. Virulence We observed that approximately 1 % of differentially expressed proteins in the control with a N -LFQ intensity more than 0.5, were not observed with linezolid or tigecycline treatments (Table 2), and the statistical analysis showed a decreased differential expression of virulence, especially with linezolid and tigecycline (Figures 3a - b respectively, Supplementary Table S5). In samples treated with linezolid, serine proteases SplC and Ear protein were not observed, while another serine protease SplE, enterotoxin Seq and haemolysin Hld were significantly less expressed. In samples treated with tigecycline, Hld, Ear protein, virulence proteins EsaA and EsxA, and serine protease SplA were not observed, while SplC and SplE were significantly less expressed. The main Manuscript: Results and Discussion 106 virulence factors Hla/Hly and LukS-PV (PVL)255 also showed a lower expression with tigecycline, besides other minor virulence factors (Aur, IsaB, SspB). In samples treated with oxacillin, only EsaA and SspC significantly, and minor virulence factors (IsaB, SspB) showed a lower expression (Figure 3c, Supplementary Table S5). By contrast, an highest expression of enterotoxin Sek was observed in samples treated with tigecycline and oxacillin (Table 3). In addition, in samples treated with oxacillin, the main virulence factor of S. aureus USA300 LukF-PV (PVL)30 was significantly more expressed (Figure 3c, Supplementary Table S5), likewise leukocidin LukDE ( N -LFQ intensity < 0.20) and SCIN (Table 3) were more highly expressed. In summary, the protein synthesis inhibitors, linezolid and tigecycline, were superior inhibitors of virulence proteins, and the uniques with a effect on serine proteases, toxins and haemolysins, which play a relevant role in the pathogenesis of S. aureus . Response to stress Biofilm formation. We observed that most of proteins predicted to be involved, direct or indirect mode, in biofilm formation were highly expressed under antibiotics conditions. In fact, FnbA was highly expressed in response to linezolid and tigecycline (Table 3); FnbB, coagulase Coa (Table 3) and SdrD ( N -LFQ intensity = 0.30) in response to tigecycline; ClfA was highly expressed in response to tigecycline and oxacillin (Table 3), and Eap showed a higher expression with oxacillin (Figure 3c, Supplementary Table S5). Unexpectedly, one of virulence factor for S. aureus USA300 also involved in biofilm formation, PsmA1, was more highly expressed only in response to oxacillin (Table 3). In contrast, linezolid was the unique antibiotic that significantly decreased the SdrE expression (Figure 3a, Supplementary Table S5). Therefore, the increment in the number of proteins involved in response to stress by biofilm formation was superior for tigecycline and oxacillin, according to increased of the antibiotic pressure carried out for these antibiotics. Manuscript: Results and Discussion 107 Mechanism of resistance. The proteins involved in the mechanism of resistance to βlactam antibiotics were higher expressed as response to oxacillin pressure as expected, though also as response to linezolid and vancomycin pressure. Oxacillin and linezolid increased the expression of BlaZ (Figure 3a - c, Supplementary Table S5), linezolid and vancomycin increased the Pbp2a/MecA expression (Figure 3a - d, Supplementary Table S5), and with linezolid, even Ppb3 was highest expressed ( N -LFQ intensity = 0.24). Both proteins, MecA and Pbp3, are involved in the resistance to all β-lactam antibiotics, thus it might indicate that linezolid confer an increased resistance to this antibiotic family, at least with this sub-concentration. The resistance to glycopeptides was only increased with oxacillin by the higher expression of RpoB256 (Figure 3 c, Supplementary Table S5). The resistance to aminoglycosides was only reduced with tigecycline by the significantly lower expression of AphA (3´)III (Figure 3b, Supplementary Table S5). Endopeptidases Clp family. This heterogeneous Clp family, highly conserved among eubacteria, has a dual role, as it both eliminates stress-damaged proteins, as well as ensures the timely degradation of major stress regulators.86 In our study, endopeptidases Clp were differentially expressed according to antibiotic and subinhibitory concentration, especially ClpP. Endopeptidase ClpP was significantly more expressed with tigecycline and more expressed with oxacillin (Figure 3b – c, Supplementary Table S5) whereas in samples treated with linezolid and vancomycin (Supplementary Table S5) was not changed, likely according with the cell density and antibiotic pressure. Hence, we support that Clp endopeptidases play a key role in regulatory network in response to stress and pathogenesis, and furthermore, ClpP could also have a key role in the S. aureus USA300 regulation, by interacting with regulatory systems, for the coordinated response against antibiotic pressure. Manuscript: Results and Discussion 114 proteins highly expressed and 96 proteins not expressed. Among the highly expressed proteins under antibiotic conditions (Table 4), approximately 40 % have been documented to be related with virulence or response to stress, including colonization and biofilm formation. For example, PstS has been involved in a reversible adaptation to antibiotic stress to survive in the presence of β-lactams,264 and by extension, in our study to other antibiotics. In addition, IsdA protein and ClfB are involved in the cell adhesion and biofilm formation,265 and are overexpressed under restricted environments of iron266 and calcium265 respectively. Hence, the common bacterial response against antibiotic pressure was the higher increase of response to stress and biofilmformation,265,267 according to the literature. The differentially expressed proteins in control not observed under antibiotic conditions, close to 80 % and only 3 % had N -LFQ intensities less than 0.20 and more than 0.5, respectively (data not shown). The haemolysin HlgA was the unique virulence protein highly expressed in control ( N -LFQ intensity = 14.32) that was not observed with linezolid, tigecycline and oxacillin. Aforementioned AgrC (control N -LFQ intensity = 0.20) was not observed with tigecycline, oxacillin and vancomycin treatments, probably owing to lower cell density (CFU / ml) observed due to bactericidal activity of these antibiotics (Supplementary Table S2). Another EssB/C system (control N -LFQ intensities = 0.23 / 0.17) involved in the establishment of infection in the host and dispensable for laboratory growth,268 were not detected with any treatment, as expected. Manuscript: Results and Discussion 115 Table 4. Differentially expressed proteins only under 0.5MICs of all or three antibiotics. Normalized LFQ intensities, not transformed by log2, represented as the mean among the three replicates. (see excel file SI003 for more information). Num Ids Protein Name ANTIBIOTIC Biological Category Group KEGG_PATHWAY Linezolid Tigecyclin Oxacillin Vancomycin 0.5 μg/ml 0.25 μg/ml 16 μg/ml 1 μg/ml 338 3-oxoacyl-[acyl-carrier-protein] synthase 3 (EC 2.3.1.180) FabH 0.24 0.28 0.16 0.26 1 Fatty acid biosynthesis 728 Nicotinamidase (EC 3.5.1.19) 0.09 0.20 0.19 1 132 tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG (Glucose-inhibited division protein A) 0.21 0.67 0.54 0.20 3 1013 Chaperone protein DnaJ 0.11 0.36 0.21 0.15 3 1351 DNA topoisomerase 1 (EC 5.99.1.2) TopA 0.20 0.15 0.15 3 808 D-lactate dehydrogenase (EC 1.1.1.28) Ddh 2.58 0.58 0.18 4 Pyruvate metabolism 919 Phosphate-binding protein PstS (PBP);Phosphate ABC superfamily ATP binding cassette transporter, binding protein 0.64 2.51 0.29 5 ABC transporters. Two-component system 303 Transferrin receptor. Iron (Fe+3) ABC superfamily ATP binding cassette transporter, binding protein 0.10 0.29 0.26 5 ABC transporters 1163 Clumping factor B ClfB 1.20 3.50 0.43 6 560 Iron-regulated surface determinant protein A (Fur-regulated protein A) (Staphylococcal transferrin-binding protein A) IsdA 0.57 0.99 1.82 7 1173 Putative surface anchored protein. LPXTG family cell wall surface anchor protein SasF 1.63 1.25 0.54 743 Staphylokinase Sak 3.66 4.74 1.59 1182 Uncharacterized protein (1) 2.62 2.02 1.02 (1) Putative immunity protein/bacteriocin. Only identified in supernatants. Manuscript: Conclusions 117 Conclusions S. aureus has a marked ability to adapt to different environments by modulation its central metabolism according to changing conditions, in vitro and in vivo . The adaptive biological response to able to survive, is led by regulatory systems, such as the quorumsensing. Nevertheless, to understand the differential protein expression as response to antibiotic pressure of S. aureus USA300 strain, it would be necessary a specific regulatory network. We think that, the Clp protein family could contribute to antibiotic adaptation, and probably can interplay pathogenicity and response to antibiotic pressure. This response of S. aureus USA300 strain, to antibiotic pressure of subinhibitory concentrations, was led towards greater expression of proteins involved in biofilm formation, especially with tigecycline and oxacillin treatments. The higher dose were superior and showed an effect on the proteins involved in each mechanism of action. The protein synthesis inhibitors, linezolid and tigecycline, inhibited the expression of several virulence factors involved in pathogenesis, such as serine proteases and haemolysins. The 0.5 MIC of linezolid further significantly decreased the expression of enterotoxin Seq and sialoprotein SdrE, which can be an advantage for clinical treatment of CA-MRSA infections such as osteomyelitis, bacteremia and even endocarditis. Tigecycline was superior against S. aureus USA300 using both subinhibitory concentrations, and showed a decreased expression of the main virulence factor of S. aureus Hla, as well as of PVL. Tigecycline was also considered to show a bactericidal activity. The sub-concentrations of oxacillin were efficient against S. aureus USA300, though the higher expression of virulence factor involved in the pathogenesis such as PSMs and Luk-F (PVL), more than the possible selection of homotypic-resistant MRSA, would reject its clinical used. The vancomycin had not effect over the virulence factors and was the less efficient. Hence, this study provides for the proteomic changes in a CAMRSA USA300 strain and an insight in cellular response to different sub-concentrations of several antibiotics. All these findings might ultimately be useful for further study in a clinical-setting. Manuscript: Supplementary Information 119 Supplementary data Quantitative Proteomic Analysis of Community-Acquired Methicillin-Resistant Staphylococcus aureus USA300 in Response to Subinhibitory Concentrations of Antibiotics Torres-Sangiao, Eva (1,2), Kucharova, Veronika (1), de Souza, Gustavo Antonio (3), Bou, Germán (2) García Riestra, Carlos (4) and Wiker, Harald G (1)* (1) The Gade Research Group for Infection and Immunity, Faculty of Medicine & Dentistry at University of Bergen (Norway), (2) Clinical Microbiology Lab., University Hospital Complex of A Coruña – INIBIC (Spain), (3) Proteomics Core Facility, Centre for Immune Regulation and Dept. of Immunology, University of Oslo and Oslo University Hospital, Norway, (4) Department of Microbiology, University Hospital Complex of Santiago de Compostela-IDIS-University of Santiago de Compostela Supplementary Information Manuscript: Supplementary Information 121 Table S1 . Minimal Inhibitory Concentration (MICs) of antibiotics tested against USA 300 . Figure S1. Schematic diagram of sample preparation (A) culture and (B) digestion , for proteomics analysis by LFQ. Table S2. CFU/ml and amount of protein (means) of subMICs tested against USA 300 . Cultures were incubated until optical density OD600 reached at 2 + 0.1 (stationary phase) and the aliquots were plated on blood-agar. The proteins extracts were previously concentrated and 20 μg of protein were loaded and fractionated by SDS-PAGE. Table S3. Pathway enrichment study by DAVID with a score more than 2.5. Annotation coverage of 45%. Upor downregulation of metabolic pathways was defined by score enrichment values from DAVID web for each pathway cluster using the score value for the antibiotic condition minus the score value of the control (antibiotic – control). Differences between + 1 was considered not significant. Table S4. Biological Process distribution of identified proteins (absolute values). Definition of Biological Category group in the left. The GO annotations coverage was 75%. Figure S2 . Stress Profile Scatter Plot.. Profile scatter-plot for differentially expressed proteins involved in pathogenesis and response to stress identified for 0.5MICs. LFQ intensities normalized without transforming, represented as the mean among the three (treated samples) and six biological replicates (control). n= number of identified proteins, LFQ i = LFQ intensities means. Figure S3. Plots correlation by Pearson coefficient R. MultiScatter Plot showing the correlation between LFQ intensities means of identifiend proteins for each subinhibitory concentration of every antibiotic, plotted against LFQ intensities means of identifiend proteins for the control, and their corresponding Pearson correlation R, for (a) pellet cells, and (b) supernatant cells. BLUE = proteins from pellet; BLACK = shared proteins between both fractions; RED = proteins from supernatant; GREEN = proteins are not within control. P = pellet; S = supernatant; Ctrl = CONTROL; LNZ= linezolid; TIG= tigecycline; OXA= oxacilline; VAN= vancomycin. Figure S4. HeatMap and detail of the most relevant differences between treated-USA300 and control-USA300 . The HeatMap was generated by Perseus software, it shows the LFQ intensities means of the untreated-control USA300 and the treated USA300 (columns), and the proteins names (rows) clustered following the instructions from Perseus..The row cluster was defined as < 20. The row color bar show the untreated-control USA300´s cluster. The used parameters were the euclidean distance linkage as mean, one preprocess with k-means and number the cluster 300 (parameters by default) to the imputed LFQ intensities and without the preprocess to no imputed LFQ intensities. LFQ intensities means calculate as the average among six (control) or three replicates (antibiotic). Ctrl= control; LNZ= linezolid; TIG= tigecycline; OXA= oxacilline; VAN= vancomycin. Bars of LFQ intensities and GREY = undetectable protein. Table S5. Statistical significance and fold change of differentially expressed Manuscript: Supplementary Information 122 proteins for 0.5 MICs . Proteins summarized to be associated with mechanism of action of antibiotics or pathogenesis Statistical analysis performed by Perseus v.1.4.1.3. Foldchange 2 (Antibiotic / Control) calculated as [(Antibiotic mean / Control mean)] for each protein individually, and 1 log t-test p -value = (log10 p -value)*(-1). absolute value = 1.30103. (See excel file SI004 for more information). Excel file SI_001. This file includes a list of the 1284 identified proteins and their LFQ intensities, as well as the Protein ID, total number of unique peptides, sequence coverage, posterior error probability (PEP) score from MaxQuant. Excel file SI_002. Proteins uniquely expressed in untreated USA300 but not observed in treated-control-USA300. LFQ intensities normalized without transforming, represented as the mean among the six biological replicates. Excel file SI_003. Proteins uniquely expressed in treated USA300 but not observed in untreated-control-USA300. LFQ intensities normalized without transforming, represented as the mean among the three biological replicates. Excel file SI_004. Statistical significance proteins . Statistical analysis performed by Perseus v.1.4.1.3 where log t-test p -value = (log10 p -value)*(-1), absolute value = 1.30103 and the foldchange as t -test difference (antibiotic – control). Manuscript: Supplementary Information 123 FIGURES Figure S1. Schematic diagram of sample preparation (A) culture and (B) digestion, for proteomics analysis by LFQ. TSB= Tripticase-soy broth. LNZ= linezolid; TIG= tigecycline; OXA= oxacilline; VAN= vancomycin. Manuscript: Supplementary Information 130 Table S3. Pathway enrichment study by DAVID with an annotation coverage of 45%. ANTIBIOTIC Pathway Cluster Linezolid Tigecycline Oxacillin Vancomycin 0.25 μg/ml 0.5 μg/ml 0.12 μg/ml 0.25 μg/ml 8 μg/ml 16 μg/ml 0.5 μg/ml 1 μg/ml Ribosome Purine metabolism Aminoacyl-tRNA biosynthesis Pyrimidine metabolism Glycolysis / Gluconeogenesis Citrate cycle (TCA cycle) Pyruvate metabolism / Propanoate metabolism Peptidoglycan biosynthesis Amino sugar and nucleotide sugar metabolism Oxidative Phosphorylation Fatty acid biosynthesis Nucleotide excision repair ND ND ND D-Alanine metabolism ND = Enrichment score No determined +3 +1.5 +1 -regulation / Down- -1 -1.5 -3 -6 Manuscript: Supplementary Information 131 Table S4. Biological Process distribution of identified proteins (absolute values). Biological Category group BIOLOGICAL PROCESS Control Linezolid Tigecycline Oxacilllin Vancomycin Total 0.25 μg/ml 0.5 μg/ml 0.12 μg/ml 0.25 μg/ml 8 μg/ml 16 μg/ml 0.5 μg/ml 1 μg/ml 1 METABOLISM CARBOHIDRATES 41 40 40 28 31 41 38 39 39 46 LIPID-FATTY AC MP 17 18 17 9 11 15 17 14 18 21 CATABOLIC MP 2 3 3 1 2 2 2 3 2 4 BIOSYNTHETIC MP 8 6 6 5 4 8 6 6 8 10 MP 31 28 28 14 13 31 23 26 31 41 PRIMARY MP 10 7 8 5 5 7 7 9 9 11 2 AMINO ACIDS & DERIVATIVES MP 44 42 39 18 24 38 31 36 48 56 PROTEINS MP 49 45 43 28 33 43 45 38 44 56 3 NUCLEOTIDE, NUCLEOSIDE AND NUCLEIC ACID MP 56 52 56 30 36 57 46 44 63 71 DNA MP 17 16 17 11 11 22 14 12 21 27 REPLICATION 13 11 15 10 8 16 10 11 17 20 TRANSCRIPTION 25 21 22 14 10 23 22 20 27 37 TRANSLATION 80 78 82 64 70 77 69 77 79 84 4 OXIDO-REDUTION 26 21 27 12 15 22 19 18 28 33 GENERATION OF PRECUSOR METABOLITES &ENERGY 22 22 22 19 18 21 18 22 22 22 OTHERS (Vit, CoEnz, Cofactor & others) 23 22 20 10 15 30 23 20 25 32 5 TRANSPORT Metal IonTransport 12 12 16 7 7 10 8 11 9 17 Others 25 20 21 5 6 15 11 19 22 30 6 CELL-CELLULAR MP CELL ORGANIZATION & BIOGENESIS 32 29 32 21 19 29 25 26 28 33 CELL CYCLE, CELL ADHESION & CELL DIVISION 8 3 7 5 8 6 5 4 6 14 7 CELL DEATH, CELL KILLING & REPRODUCTION 11 10 10 8 8 12 11 10 12 14 SIGNAL TRANSDUCTION 8 7 7 3 3 7 7 6 10 10 CELLULAR PROCESS 6 6 6 1 1 5 3 3 6 6 CELLULAR HOMEOSTASIS 8 8 8 3 3 9 8 7 8 9 CELLULAR COMPONENT ORGANIZATION 1 1 1 1 1 1 0 1 1 1 RESPONSE TO STRESS 9 10 8 7 7 13 12 8 10 13 TO BIOTIC STIMULUS 0 0 0 0 0 1 1 0 0 1 PATHOGENESIS 8 8 8 6 9 10 12 8 8 12 - UNKNOWN 182 169 162 73 88 157 123 137 163 249 TOTAL 774 715 731 418 466 728 616 635 764 980 Manuscript: Supplementary Information 132 Table S5. Statistical significance and fold change of differentially expressed proteins for 0.5 MICs. Num Ids Protein Name ANTIBIOTIC Biological Category Group KEGG_PATHWAY linezolid tigecyclin oxacillin vancomycin (1) log ttest pvalue2 Foldchange3 (1) log ttest pvalue2 Foldchange3 (1) log ttest pvalue2 Foldchange3 (1) log ttest pvalue2 Foldchange3 (1) Statistically Significant Proteins 320 ATP-dependent Clp protease proteolytic subunit (EC 3.4.21.92) Endopeptidase ClpP 0.03 2.41 + 1.96 4.63 2.41 2.87 0.01 0.78 2 363 I57 family staphostatin B SspC NaN NaN NaN NaN + 2.13 0.16 0.15 0.98 2 1074 Serine protease SplE (EC 3.4.21.-) + 1.84 0.39 + 2.68 0.05 0.05 0.43 0.20 0.62 2 1076 Serine protease SplC (EC 3.4.21.-) NaN NaN + 2.61 0.01 1.18 0.06 0.05 0.76 2 1096 tRNA N6-adenosine threonylcarbamoyltransferase (EC 2.3.1.-) TsaD NaN NaN + 2.52 1.75 NaN NaN 0.89 1.41 2 1256 Signal recognition particle protein (Fifty-four homolog) Ffh 2.15 1.58 + 3.88 6.03 0.20 1.56 0.40 1.06 2 Protein export. Bacterial secretion system 206 Single-stranded DNA-binding protein Ssb 0.31 1.00 NaN NaN + 3.92 4.11 0.16 0.49 3 DNA replication. Mismatch repair. Homologous recombination 229 RNA methyltransferase, TrmH family, group 3 1.21 1.06 + 2.04 2.79 0.79 3.31 1.31 1.45 3 534 Phosphoribosylformylglycinamidine synthase 2 (EC 6.3.5.3) PurL 0.26 0.68 + 4.93 0.07 0.60 0.73 0.14 0.82 3 Purine metabolism 538 Phosphoribosylamine--glycine ligase (EC 6.3.4.13) PurD 0.02 0.87 + 3.34 0.12 1.84 0.39 0.02 0.97 3 Purine metabolism 564 Phenylalanine-tRNA ligase / tRNA synthetase beta subunit (EC 6.1.1.20) PheT 0.14 0.90 0.35 1.72 + 3.93 4.76 0.29 1.06 3 Aminoacyl-tRNA biosynthesis 632 Transcriptional regulator, Fur family NaN NaN NaN NaN + 1.93 0.26 0.20 1.04 3 901 Proline--tRNA ligase (EC 6.1.1.15) (Prolyl-tRNA synthetase) ProRS 1.30 1.23 + 4.03 3.91 0.35 1.66 0.69 2.21 3 Aminoacyl-tRNA biosynthesis 972 30S ribosomal protein S1 RpsA 0.64 0.54 + 3.23 0.36 1.42 0.34 0.28 0.66 3 Ribosome 1204 50S ribosomal protein L3 RplC + 3.15 10.99 + 4.09 26.97 0.48 11.77 0.13 1.40 3 Ribosome 2 Protoporphyrinogen oxidase (EC 1.3.3.4) HemG NaN NaN NaN NaN NaN NaN + 4.38 1.96 4 Porphyrin and chlorophyll metabolism 253 Ser-Asp rich fibrinogen/bone sialoprotein-binding protein SdrE + 3.18 0.04 0.50 0.62 0.11 0.54 0.55 0.33 6 Manuscript: Supplementary Information 133 504 Staphylococcal enterotoxin Q Seq + 1.10 1.03 0.25 1.84 0.08 0.88 0.07 1.39 7 622 Panton-Valentine leukocidin, LukF-PV 0.08 0.96 0.04 0.59 + 1.98 1.94 0.40 0.58 7 337 Possible extracellular adherence protein Eap 0.08 1.34 0.21 0.95 + 1.67 2.07 0.42 0.53 402 Virulence protein EsaA 0.30 0.73 NaN NaN + 3.75 0.08 0.60 0.66 1187 3'5'-aminoglycoside phosphotransferase (Aminoglycoside 3'- phosphotransferase) (EC 2.7.1.95) Aph(3')III 0.71 0.60 + 1.83 0.57 0.78 0.65 1.20 1.93 Not statistically Significant Proteins 1166 Zinc metalloproteinase aureolysin (EC 3.4.24.29) Aur 1.13 0.15 1.91 0.08 0.48 0.26 0.63 2.13 2 1184 Beta-lactamase BlaZ 1.15 4.44 0.82 1.47 2.52 2.76 0.91 1.14 2 beta-Lactam resistance. Two-component system 236 DNA-directed RNA polymerase subunit beta (RNAP subunit beta) (EC 2.7.7.6) RpoB 0.48 1.39 0.93 2.28 1.48 2.44 0.12 0.88 3 Purine metabolism. Pyrimidine metabolism. RNA polymerase 237 DNA-directed RNA polymerase subunit beta' (RNAP subunit beta') (EC 2.7.7.6) RpoC 0.36 1.48 0.72 1.83 1.14 2.22 0.24 1.03 3 Purine metabolism. Pyrimidine metabolism. RNA polymerase 452 Transcriptional regulator SarA (Staphylococcal accessory regulator A) 0.84 2.10 0.35 1.95 1.12 4.89 0.07 1.27 3 769 30S ribosomal protein S5 RpsE 0.77 2.88 0.19 3.40 0.04 2.04 0.20 1.19 3 Ribosome 1205 30S ribosomal protein S10 RpsJ 0.68 0.67 0.04 1.61 1.45 0.51 0.47 1.10 3 Ribosome 149 MecA (Pbp2a) (Pencillin binding protein 2a (PBP 2a, methicillin resistance determinant MecA, transpeptidase); (EC 3.4.16.4) 0.92 2.28 0.18 1.19 0.07 0.97 0.87 2.25 6 beta-Lactam resistance 364 Cysteine protease (EC 3.4.22.48);C47 family staphopain B SspB 0.31 0.54 1.90 0.05 0.71 0.10 0.25 0.24 7 574 Alpha-haemolysin Hly 0.56 0.42 2.20 0.07 0.45 0.22 0.16 1.17 7 623 Panton-Valentine leukocidin, LukS-PV 0.63 0.71 1.03 0.35 0.48 0.48 0.07 1.11 7 704 Septation ring formation regulator EzrA 0.98 1.26 0.43 0.95 1.58 0.50 0.00 0.89 7 993 Superoxide dismutase [Mn/Fe] 1 (EC 1.15.1.1) SodA 0.07 1.05 1.29 3.48 1.88 2.15 0.27 0.92 7 1252 Cell division protein FtsZ 1.55 1.61 0.71 1.31 1.24 0.66 0.10 0.93 7 1397 Delta-haemolysin Hld 1.03 0.15 NaN NaN 0.12 0.77 0.32 0.65 7 1167 Immunodominant staphylococcal antigen B IsaB 0.07 0.69 1.73 0.18 2.12 0.14 0.08 0.91 (1) Statistically significant proteins = + (2) log t-test p-value = (log10 p-value)*(-1), absolute value = 1.30103 (3) Fold-change (Antibiotic / Control) calculated as [(Antibiotic mean / Control mean)] for each protein individually. Values < 0.5 are down-regulated proteins and values > 2 are up-regulated proteins. Annexed: Identification and antimicrobial susceptibility of methicillinresistant Staphylococcus aureus (MRSA) clones. 143 Letters Manuscript: Supplementary Information 139 Free PMC article available : http://jcm.asm.org.pva.uib.no/content/46/12/4114 PMID:18945834 [PubMed - indexed for MEDLINE] PMCID: PMC2593277 Abstract not available. "Staphylococcus aureus strains with resistance to methicillin or oxacillin (MRSA) represent one of the main nosocomial pathogens at present. MRSA infections are clearly associated with higher mortality and economic cost than those caused by methicillinsusceptible S. aureus (1). In Spain, the prevalence of methicillin resistance among S. aureus isolates has increased since the 1990s and in some cases has now reached levels higher than 30% (2). A total of 301 S. aureus strains were evaluated (51 mecA negative and 250 mecApositive as determined by PCR). S. aureus ATCC 29213 was used as a negative control. Molecular typing of the X region of the spa gene was done with the 250mecA-positive isolates (4), and these were then grouped into a spa clonal complex (BURST.Ridom StaphType software), with 4 types more prevalent than the others (t02, 23.6%; t18, 22%; t67, 17.6%; and t12, 16.3%)."