AhaP, A Quorum Quenching Acylase from Psychrobacter sp. M9-54-1 That Attenuates Pseudomonas aeruginosa and Vibrio coralliilyticus Virulence

marine drugs Article AhaP, A Quorum Quenching Acylase from Psychrobacter sp. M9-54-1 That Attenuates Pseudomonas aeruginosa and Vibrio coralliilyticus Virulence JoséCarlos Reina 1, Manuel Romero 2, Rafael Salto 3, Miguel Cámara 2and Inmaculada Llamas 1,4,*   Citation: Reina, J.C.; Romero, M.; Salto, R.; Cámara, M.; Llamas, I. AhaP, A Quorum Quenching Acylase from Psychrobacter sp. M9-54-1 That Attenuates Pseudomonas aeruginosa and Vibrio coralliilyticus Virulence. Mar. Drugs 2021,19, 16. https:// doi.org/10.3390/md19010016 Received: 7 December 2020 Accepted: 27 December 2020 Published: 1 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Microbiology, Faculty of Pharmacy, Campus Universitario Cartuja s/n, University of Granada, 18071 Granada, Spain; [email protected] 2 National Biofilms Innovation Centre, Biodiscovery Institute and School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UK; [email protected] (M.R.); [email protected] (M.C.) 3 Department of Biochemistry, Faculty of Pharmacy, Campus Universitario Cartuja s/n, University of Granada, 18071 Granada, Spain; [email protected] 4 Biomedical Research Center (CIBM), Institute of Biotechnology, University of Granada, 18100 Granada, Spain *Correspondence: [email protected] Abstract: Although Psychrobacter strain M9-54-1 had been previously isolated from the microbiota of holothurians and shown to degrade quorum sensing (QS) signal molecules C6 and C10-homoserine lactone (HSL), little was known about the gene responsible for this activity. In this study, we determined the whole genome sequence of this strain and found that the full 16S rRNA sequence shares 99.78–99.66% identity with Psychrobacter pulmonis CECT 5989 T and P. faecalis ISO-46 T . M954-1, evaluated using the agar well diffusion assay method, showed high quorum quenching (QQ) activity against a wide range of synthetic N-acylhomoserine lactone (AHLs) at 4, 15, and 28 ◦ C. Highperformance liquid chromatography-mass-spectrometry (HPLC-MS) confirmed that QQ activity was due to an AHL-acylase. The gene encoding for QQ activity in strain M9-54-1 was identified from its genome sequence whose gene product was named AhaP. Purified AhaP degraded substituted and unsubstituted AHLs from C4to C14-HSL. Furthermore, heterologous expression of ahaP in the opportunistic pathogen Pseudomonas aeruginosa PAO1 reduced the expression of the QS-controlled gene lecA, encoding for a cytotoxic galactophilic lectin and swarming motility protein. Strain M9-54-1 also reduced brine shrimp mortality caused by Vibrio coralliilyticus VibC-Oc-193, showing potential as a biocontrol agent in aquaculture. Keywords: quorum quenching; acylase; Psychrobacter; marine habitat 1. Introduction The term quorum sensing (QS), which refers to a well-known population densitydependent gene expression mechanism, was introduced for the first time by Fuqua et al. in 1994 [ 1 ]. QS enables bacteria to communicate with each other through the production, release and detection of signal molecules, also known as autoinducers. After reaching a threshold concentration in the surrounding medium, these autoinducers coordinate the expression of multiple genes, including those coding for antibiotic and exoenzyme production, as well as for biofilm formation [ 2 – 4 ]. Some of the most studied autoinducers produced by Gram-negative bacteria are N-acylhomoserine lactones (AHLs). The canonical AHL molecule contains a homoserine lactone ring linked to an acyl chain which can present different levels of saturation and substitution [5–7]. AHL-mediated QS systems control virulence gene expression in multiple pathogens, including marine, agricultural and human pathogens [ 8 – 13 ]. Hence, interference with QS systems has been proposed as a novel strategy to prevent or attenuate these infections [ 14 ]. This interference can be achieved using molecules that block the interaction of AHLs with their cognate signal receptors without affecting signal integrity. These molecules are Mar. Drugs 2021,19, 16. https://doi.org/10.3390/md19010016 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021,19, 16 2 of 18 generally named QS inhibitors (QSIs). Alternatively, QS signaling can be disrupted by enzymatic inactivation of the signal molecules, a mechanism known as quorum quenching (QQ), which is one of the most studied approaches used to interfere with QS-mediated regulatory mechanisms in order to control bacterial infections [15]. There are three main types of AHL-QQ enzymes: lactonases, which open the lactone ring; acylases, which cleave the AHL amide bond; and oxidoreductases, which modify the fatty acid chain whose recognition is impeded by the signal receptor [ 16 , 17 ]. Enzymatic degradation of AHLs has been reported in a wide range of microorganisms, including AHLand non-AHL-producing bacteria [ 18 ], as well as in mammalian cells [ 19 ], suggesting that QS inhibitory processes play an important role in different environments, whose actual ultimate physiological function, however, remains unexplained [20]. QQ enzymes have been shown to be effective in preventing infections caused by AHL-producing bacterial pathogens in konjac [ 21 ], potato tubers [ 22 – 24 ], tobacco and cauliflower [ 25 ], and shrimps [ 10 , 26 , 27 ]. QQ enzymes have also been shown to reduce the virulence of the human opportunistic pathogen Pseudomonas aeruginosa in several infection models [ 28 , 29 ]. With regard to the potential use of different QQ enzyme types in the fight against infections, it has been proposed that acylases, whose AHL-degrading activity is irreversible, could be more effective than lactonases, whose degradation of AHLs can be reverted in acidic environments [30,31]. The marine environment is a prolific and valuable source of numerous bioactive compounds [ 32 ], including quorum sensing inhibitor (QSI) and QQ enzymes [ 33 – 37 ]. Our laboratory recently examined marine invertebrates such as sea anemones and holothurians whose microbiota are a source of antimicrobial molecules [ 38 ]. From the same collection of bacteria, several active AHL-degrading bacteria from Stenotrophomonas maltophilia isolates [ 24 ] and a QSI-producing strain of Vibrio alginolyticus [ 39 ] have been identified and characterized. In this study, we analyzed the AHL-degrading capacity of strain M9-54-1 which was isolated from a holothurian and identified as belonging to the genus Psychrobacter. The QQ activity of M9-54-1 was tested against a wide range of synthetic AHLs, as well as crude extracts from AHL-producing aquacultural and human pathogens. We identified the gene responsible for this activity and also purified and characterized the enzyme encoded by this gene. The ability of this QQ enzyme to interfere with the QS systems of the pathogens Pseudomonas aeruginosa PAO1 and Vibrio coralliilyticus VibC-Oc-193 was also evaluated. 2. Results 2.1. Psychrobacter sp. M9-54-1 Shows Broad AHL-Degrading Activity Strain M9-54-1, belonging to Psychrobacter sp., had previously been isolated from the microbiota of Holothuria spp. [ 38 ] and selected for its ability to degrade C6-homoserine lactone (HSL) and C10-HSL. The QS interference mechanism has been reported to involve enzymatic inactivation of AHLs but not the production of QSI compounds [ 24 ]. In the present study, in order to evaluate M9-54-1 AHL-degradation activity in more detail, a wide range of AHLs, including both synthetic and crude AHL extracts, was tested using agar well diffusion assays (Figure 1). Reactions with synthetic AHLs (C4-, C6, C8-, C10-, 3-OH-C10-, C12-, and 3-O-C12-HSL) were carried out at 4, 15, and 28 ◦ C; the remaining AHL signal activity was measured as the diameter of colored halos developed by Agrobacterium tumefaciens biosensor strain NTL4 (pZLR4). Strain M9-54-1 showed activity against all AHLs at most temperatures, with or without substituted groups (Figure 1). Given that alkaline pH has been shown to drive AHL lactonolysis [ 31 ], pH was measured in all cultures to ensure that it was not the cause of AHL inactivation. Since all the AHLs analyzed were completely degraded at 28 ◦ C, the QQ activity of Psychrobacter sp. M9-54-1 was tested at this temperature against AHL crude extracts from pathogenic Vibrio spp. and Pseudomonas aeruginosa. AHLs produced by aquaculture-related pathogens V. owensii VibC-Oc-106 (Figure 1), V. mediterranei VibC-Oc-097, and V. coralliilyticus VibC-Oc-193 (data not shown) were found to be completely degraded by M9-54-1. In the case of AHLs produced by Mar. Drugs 2021,19, 16 3 of 18 the human pathogen P. aeruginosa PAO1, incubation with M9-54-1 resulted in extensive AHL degradation. Mar. Drugs 2021, 19, x FOR PEER REVIEW 3 of 20 1 was tested at this temperature against AHL crude extracts from pathogenic Vibrio spp. and Pseudomonas aeruginosa. AHLs produced by aquaculture-related pathogens V. owensii VibC-Oc-106 (Figure 1), V. mediterranei VibC-Oc-097, and V. coralliilyticus VibC-Oc-193 (data not shown) were found to be completely degraded by M9-54-1. In the case of AHLs produced by the human pathogen P. aeruginosa PAO1, incubation with M9-54-1 resulted in extensive AHL degradation. Figure 1. AHL-degradation activity of Psychrobacter sp. M9-54-1 explored using the agar plate diffusion assay method. (A) Degradation activity against synthetic AHLs, at different temperatures, expressed as percentage of halo diameter. Marine broth (MB) was used as negative control. (B) Detection of AHLs using biosensor strain Agrobacterium tumefaciens NTL4 (pZLR4): V. owensii VibC-Oc-106 crude extract (1), M9-54-1 degradation of V. owensii VibC-Oc-106 crude extract (2), P. aeruginosa PAO1 crude extract (3) and M9-54-1 degradation of a crude extract of P. aeruginosa PAO1 (4). 2.2. Whole-Genome Analysis of Psychrobacter sp. M9-54-1 Shows Potential for a New Species With its partial 16S rRNA gene homology (99.21% identity) but only 43.7% genome sequence completeness, strain M9-54-1 had previously been identified as Psychrobacter faecalis. In this study, the genomic DNA of Psychrobacter sp. M9-54-1 was extracted and sequenced. Draft genome assembly led to 3.2 Kb in 69 contigs, with an average coverage of 230 X and G+C content of 43.4 mol%. The genome was deposited in the NCBI genome database under accession number JADGFW000000000. Having obtained the whole genome sequence, we were able to identify and analyze the complete 16S rRNA gene sequence of strain M9-54-1, which shares 99.78% identity with P. pulmonis CECT 5989T and 99.66% identity with P. faecalis ISO-46T. The in silico DNA-DNA hybridization (DDH) and the average nucleotide identity (ANI) between strains M9-54-1 and P. faecalis SHUES1 (NZ_LXQA00000000.1), whose genome was available, were determined. The in silico DDH value was 60.4% and the ANI values based on ANIb and ANIm were 94.74 and 95.21%, respectively. Both DDH and ANI results were lower than the proposed cut-off values to describe a novel species (DDH, 70% and ANI, 95–96%), suggesting that strain M9-54-1 is a new species of the genus Psychrobacter. Nevertheless, no definitive conclusion can be drawn until the complete genome of P. pulmonis is published and compared with that of M9-54-1. 2.3. Psychrobacter sp. M9-54-1 AHL-Degrading Activity Is Cell-Associated and not Due to Lactonolysis To determine whether the QQ activity of strain M9-54-1 is due to an AHL lactonase, an acidification assay was performed using C10-HSL, an AHL that was completely degraded by this strain. The concentration of AHLs was not restored in the acidified and Figure 1. AHL-degradation activity of Psychrobacter sp. M9-54-1 explored using the agar plate diffusion assay method. ( A ) Degradation activity against synthetic AHLs, at different temperatures, expressed as percentage of halo diameter. Marine broth (MB) was used as negative control. ( B ) Detection of AHLs using biosensor strain Agrobacterium tumefaciens NTL4 (pZLR4): V. owensii VibC-Oc-106 crude extract (1), M9-54-1 degradation of V. owensii VibC-Oc-106 crude extract (2), P. aeruginosa PAO1 crude extract (3) and M9-54-1 degradation of a crude extract of P. aeruginosa PAO1 (4). 2.2. Whole-Genome Analysis of Psychrobacter sp. M9-54-1 Shows Potential for a New Species With its partial 16S rRNA gene homology (99.21% identity) but only 43.7% genome sequence completeness, strain M9-54-1 had previously been identified as Psychrobacter faecalis. In this study, the genomic DNA of Psychrobacter sp. M9-54-1 was extracted and sequenced. Draft genome assembly led to 3.2 Kb in 69 contigs, with an average coverage of 230 X and G+C content of 43.4 mol%. The genome was deposited in the NCBI genome database under accession number JADGFW000000000. Having obtained the whole genome sequence, we were able to identify and analyze the complete 16S rRNA gene sequence of strain M9-54-1, which shares 99.78% identity with P. pulmonis CECT 5989 T and 99.66% identity with P. faecalis ISO-46 T . The in silico DNA-DNA hybridization (DDH) and the average nucleotide identity (ANI) between strains M9-54-1 and P. faecalis SHUES1 (NZ_LXQA00000000.1), whose genome was available, were determined. The in silico DDH value was 60.4% and the ANI values based on ANIb and ANIm were 94.74 and 95.21%, respectively. Both DDH and ANI results were lower than the proposed cut-off values to describe a novel species (DDH, 70% and ANI, 95–96%), suggesting that strain M9-54-1 is a new species of the genus Psychrobacter. Nevertheless, no definitive conclusion can be drawn until the complete genome of P. pulmonis is published and compared with that of M9-54-1. 2.3. Psychrobacter sp. M9-54-1 AHL-Degrading Activity Is Cell-Associated and Not Due to Lactonolysis To determine whether the QQ activity of strain M9-54-1 is due to an AHL lactonase, an acidification assay was performed using C10-HSL, an AHL that was completely degraded by this strain. The concentration of AHLs was not restored in the acidified and filtered supernatant when they were tested by agar well diffusion assay (Figure 2A). To confirm this result, the AHL concentration was also analyzed by HPLC-MS (Figure 2B). The QQ activity of strain M9-54-1 was observed to be 100%, with no recovery in AHL concentration being detected following incubation under acidic conditions. These findings suggested that the mechanism by which autoinducers are degraded was not caused by an AHL lactonase. Mar. Drugs 2021,19, 16 4 of 18 Mar. Drugs 2021, 19, x FOR PEER REVIEW 4 of 20 filtered supernatant when they were tested by agar well diffusion assay (Figure 2A). To confirm this result, the AHL concentration was also analyzed by HPLC-MS (Figure 2B). The QQ activity of strain M9-54-1 was observed to be 100%, with no recovery in AHL concentration being detected following incubation under acidic conditions. These findings suggested that the mechanism by which autoinducers are degraded was not caused by an AHL lactonase. The cellular localization of the enzyme was also evaluated by testing C10-HSL-degrading activity in supernatants and crude cellular extracts (CCEs) from M9-54-1 cultures using the agar well diffusion assay method. QQ activity was detected in CCEs, whilst no AHL degradation activity was detected in filtered supernatants, indicating that the enzyme is not secreted (data not shown). Figure 2. Detection of the remaining C10-HSL activity by agar well diffusion assay using C. violaceum VIR07 (A) and HPLC-MS (B) before and after Psychrobacter sp. M9-54-1 supernatant acidification to determine the quorum-quenching enzymatic mechanism present in this bacterium. Marine broth (MB) was used as negative control. HPLC values are referred to as area under the curve (AUC). 2.4. Genome Analysis of Strain M9-54-1 Reveals that the AhaP Gene Is a Potential Acylase with AHL-Degrading Activity To identify the gene responsible for QQ activity in Psychrobacter sp. strain M9-54-1, a BLASTp search was carried out against a database of predicted proteins from the genome of this strain, using a group of 28 well-known QQ enzymes as queries (Supplementary Table S1). Two proteins (Nos. 2034 and 322), presented in Table 1, were found to have an e-value of under 1e-100 with different QQ enzymes, a threshold high enough to suggest that the hit could constitute a QQ enzyme. The automatic annotation of the two proteins was then confirmed by RAST. Protein no. 2034 had been automatically annotated as acyl-homoserine lactone acylase PvdQ, while protein no. 322 was annotated as a hypothetical protein. The corresponding genes 2034 (2.46 kb) and 322 (1.77 kb) were cloned into vector pGEX-4T-2, resulting in the production of pGEX-2034 and pGEX-322, respectively, and expressed in E. coli BL21 (DE3). Since only the expression of gene 2034 demonstrated an ability to degrade C10-HSL, as determined by the agar well diffusion assay method, gene 322 was discarded (data not shown). Protein No. 322 had homology with BpiB05, a QQ enzyme from an uncultured bacterium, and little is known about the key aminoacids for Figure 2. Detection of the remaining C10-HSL activity by agar well diffusion assay using C. violaceum VIR07 ( A ) and HPLC-MS ( B ) before and after Psychrobacter sp. M9-54-1 supernatant acidification to determine the quorum-quenching enzymatic mechanism present in this bacterium. Marine broth (MB) was used as negative control. HPLC values are referred to as area under the curve (AUC). The cellular localization of the enzyme was also evaluated by testing C10-HSLdegrading activity in supernatants and crude cellular extracts (CCEs) from M9-54-1 cultures using the agar well diffusion assay method. QQ activity was detected in CCEs, whilst no AHL degradation activity was detected in filtered supernatants, indicating that the enzyme is not secreted (data not shown). 2.4. Genome Analysis of Strain M9-54-1 Reveals that the AhaP Gene Is a Potential Acylase with AHL-Degrading Activity To identify the gene responsible for QQ activity in Psychrobacter sp. strain M9-54-1, a BLASTp search was carried out against a database of predicted proteins from the genome of this strain, using a group of 28 well-known QQ enzymes as queries (Supplementary Table S1). Two proteins (Nos. 2034 and 322), presented in Table 1, were found to have an e-value of under 1 × 10 −100 with different QQ enzymes, a threshold high enough to suggest that the hit could constitute a QQ enzyme. Table 1. Homologues to quorum quenching putative proteins identified in the genome of Psychrobacter sp. M9-54-1. Homologues Identity (%) Positives (%) e-Value Protein No. 2034 AaC Ralstonia sp. XJ12B (AAO41113.1) 37 54 3.00 ×10−171 AaC Shewanella sp. MIB015 (BAF94155.1) 32 47 1.00 ×10−112 AhlM Streptomyces sp. M664 (AAT68473.1) 35 52 1.00 ×10−131 HacA Pseudomonas psyringae pv. syringae B728a (YP_235052.1) 35 53 4.00 ×10−152 PvdQ Pseudomonas aeruginosa PAO1 NP_251075.1 36 53 1.00 ×10−162 Protein No. 322 Uncultured bacterium BpiB05 Bio8 (ABU51109) 40 59 3.00 ×10−159 The automatic annotation of the two proteins was then confirmed by RAST. Protein no. 2034 had been automatically annotated as acyl-homoserine lactone acylase PvdQ, while protein no. 322 was annotated as a hypothetical protein. The corresponding genes 2034 (2.46 kb) and 322 (1.77 kb) were cloned into vector pGEX-4T-2, resulting in the production of pGEX-2034 and pGEX-322, respectively, and expressed in E. coli BL21 (DE3). Since only the expression of gene 2034 demonstrated an Mar. Drugs 2021,19, 16 5 of 18 ability to degrade C10-HSL, as determined by the agar well diffusion assay method, gene 322 was discarded (data not shown). Protein No. 322 had homology with BpiB05, a QQ enzyme from an uncultured bacterium, and little is known about the key aminoacids for its activity, which may explain why no QQ activity was found in Protein No. 322. Thus, the protein encoded by gene 2034 was selected for further study and named a cylh omoserine lactone acylase from Psychrobacter sp. (AhaP). The sequence of the predicted protein encoded by gene 2034 was compared with that of well-known QQ acylases with demonstrated AHL-degrading capacity using the neighbour-joining algorithm. The phylogenetic tree revealed that the enzyme AhaP from Psychrobacter sp. M9-54-1 shows high clustering similarity to other AHL acylases (Figure 3A). In fact, the predicted amino acid sequence of AhaP shared 36% identity with the amino acid sequence of PvdQ from P. aeruginosa PAO1 (NP_251075.1). As expected, the protein structure of AhaP, which was predicted using Phyre2 software, shared high similarity to that of the known acylase PvdQ from PAO1 (Figure 3B). The alignment between AhaP and PvdQ is shown in Supplementary Figure S1. Mar. Drugs 2021, 19, x FOR PEER REVIEW 5 of 20 its activity, which may explain why no QQ activity was found in Protein No. 322. Thus, the protein encoded by gene 2034 was selected for further study and named acyl-homoserine lactone acylase from Psychrobacter sp. (AhaP). Table 1. Homologues to quorum quenching putative proteins identified in the genome of Psychrobacter sp. M9-54-1. Homologues Identity (%) Positives (%) e-Value Protein No. 2034 AaC Ralstonia sp. XJ12B (AAO41113.1) 37 54 3.00 × 10−171 AaC Shewanella sp. MIB015 (BAF94155.1) 32 47 1.00 × 10−112 AhlM Streptomyces sp. M664 (AAT68473.1) 35 52 1.00 × 10−131 HacA Pseudomonas psyringae pv. syringae B728a (YP_235052.1) 35 53 4.00 × 10−152 PvdQ Pseudomonas aeruginosa PAO1 NP_251075.1 36 53 1.00 × 10−162 Protein No. 322 Uncultured bacterium BpiB05 Bio8 (ABU51109) 40 59 3.00 × 10−159 The sequence of the predicted protein encoded by gene 2034 was compared with that of well-known QQ acylases with demonstrated AHL-degrading capacity using the neighbour-joining algorithm. The phylogenetic tree revealed that the enzyme AhaP from Psychrobacter sp. M9-54-1 shows high clustering similarity to other AHL acylases (Figure 3A). In fact, the predicted amino acid sequence of AhaP shared 36% identity with the amino acid sequence of PvdQ from P. aeruginosa PAO1 (NP_251075.1). As expected, the protein structure of AhaP, which was predicted using Phyre2 software, shared high similarity to that of the known acylase PvdQ from PAO1 (Figure 3B). The alignment between AhaP and PvdQ is shown in Supplementary Figure S1. Figure 3. (A). Phylogenetic analysis of the acylase AhaP from Pyschrobacter sp. M9-54-1 and other known acylases using the neighbor-joining method with 1000 bootstrap replications. (B). Comparison of the predicted structures of the acylases AhaP from Psychrobacter sp. M9-54-1 and PvdQ from Pseudomonas aeruginosa PAO1 using the Phyre2 program. Figure 3. ( A ). Phylogenetic analysis of the acylase AhaP from Pyschrobacter sp. M9-54-1 and other known acylases using the neighbor-joining method with 1000 bootstrap replications. ( B ). Comparison of the predicted structures of the acylases AhaP from Psychrobacter sp. M9-54-1 and PvdQ from Pseudomonas aeruginosa PAO1 using the Phyre2 program. 2.5. Purified AhaP Degrades AHLs with Different Substitutions and a Wide Range of Acyl Chain Lengths To facilitate the purification of AhaP, the ahaP gene was cloned into vector pET-24b (+) with a C-terminal 6xHis tag and expressed in E. coli NiCo21. The use of this construct and host should improve the solubility and increase the yield. The purified protein, which integrity was analyzed by SDS-PAGE (data not shown), was used in a QQ assay with a wide range of synthetic AHLs (C4-, O-C4-, OH-C4-, C6-, O-C6-, OH-C6-, C8-, O-C8-, OH-C8-, C10-, O-C10-, OH-C10-, C12-, O-C12-, OH-C12-, C14-, O-C14-, and OH-C14-HSL). For each AHL, the remaining signal activity was detected by using the bioluminescent biosensor strains E. coli JM109 (pSB536) for C4-HSL and its derivatives E. coli JM109 (pSB401) for C6-, C8-, and C10-HSL and their derivatives, as well as E. coli JM109 (pSB1142) for C12and C14-HSL and their derivatives. The results obtained demonstrated the presence of AhaP QQ activity against all the AHLs tested except for O-C4-, OH-C4-, and C14-HSL (Figure 4). Mar. Drugs 2021,19, 16 6 of 18 Mar. Drugs 2021, 19, x FOR PEER REVIEW 7 of 20 Figure 4. Quorum quenching activity of the purified enzyme AhaP against synthetic AHLs (C4HSL (A), OH-C4-HSL (B), O-C4-HSL (C), C6-HSL (D), OH-C6-HSL (E), O-C6-HSL (F), C8-HSL (G), OH-C8-HSL (H), O-C8-HSL (I), C10-HSL (J), OH-C10-HSL (K), O-C10-HSL (L), C12-HSL (M), OH-C12-HSL (N), O-C12-HSL (O), C14-HSL (P), OH-C14-HSL (Q), O-C14-HSL (R). Autoinducer activity was detected with the biosensor strains E. coli (pSB536) for C4-HSL and its derivatives; E. coli (pSB401) for C6-, C8and C10-HSL and their derivatives; and E. coli (pSB1142) for C12and C14-HSL and their derivatives. Values are expressed as the area under the curve (AUC) of relative light units/OD600. Different letters above the bars indicate that the values are significantly different (p < 0.05). Figure 4. Quorum quenching activity of the purified enzyme AhaP against synthetic AHLs (C4-HSL ( A ), OH-C4-HSL ( B ), O-C4-HSL ( C ), C6-HSL ( D ), OH-C6-HSL ( E ), O-C6-HSL ( F ), C8-HSL ( G ), OH-C8-HSL ( H ), O-C8-HSL ( I ), C10-HSL ( J ), OH-C10-HSL ( K ), O-C10-HSL ( L ), C12-HSL ( M ), OH-C12-HSL ( N ), O-C12-HSL ( O ), C14-HSL ( P ), OH-C14-HSL ( Q ), O-C14-HSL ( R ). Autoinducer activity was detected with the biosensor strains E. coli (pSB536) for C4-HSL and its derivatives; E. coli (pSB401) for C6-, C8and C10-HSL and their derivatives; and E. coli (pSB1142) for C12and C14-HSL and their derivatives. Values are expressed as the area under the curve (AUC) of relative light units/OD 600 . Different letters above the bars indicate that the values are significantly different (p< 0.05). Mar. Drugs 2021,19, 16 7 of 18 2.6. Heterologous Expression of ahaP in Pseudomonas aeruginosa Attenuates QS-Controlled Virulence Traits The ahaP gene was cloned into the broad host-range plasmid pME6000 and expressed in Pseudomonas aeruginosa PAO1 to evaluate its effect, if any, on phenotypes regulated by AHL-based QS systems. To this end, swarming motility of PAO1 overexpressing ahaP (pME6000::ahaP) was tested and compared to that of the same strain carrying the empty plasmid (pME6000) and the PAO1 wild type strain. As shown in Figure 5A, the expression of ahaP significantly reduced swarming motility in this opportunistic pathogen. Moreover, the pME6000::ahaP construct and empty plasmid were transferred to the biosensor strain PAO1 lecA::lux in which the expression of lectin gene lecA was evaluated. The strain’s bioluminescence output without plasmid pME6000 was also assessed as control. The luminescence produced by the reporter strain was significantly reduced (p< 0.05) when the incubation was carried out in the presence of exogenous C4and C6-HSL which increase the biosensor response (Figure 5B). These results indicate that AhaP enzymatic activity reduces the expression of these two virulence factors in the human pathogen P. aeruginosa PAO1. Mar. Drugs 2021, 19, x FOR PEER REVIEW 8 of 20 Figure 5. Evaluation of ahaP expression in P. aeruginosa PAO1. (A). Swarming motility assay of PAO1 wild type and strains containing the pME6000::ahaP construct and empty plasmid pME6000. (B). Detection of luminescence production in the biosensor strains PAO1 lecA::lux and PAO1 lecA::lux containing the pME6000::ahaP construct and empty plasmid pME6000 in the presence of C4and C6-HSL. Different letters above the bars indicate that the values are significantly different (p < 0.05). Figure 5. Evaluation of ahaP expression in P. aeruginosa PAO1. ( A ). Swarming motility assay of PAO1 wild type and strains containing the pME6000::ahaP construct and empty plasmid pME6000. ( B ). Detection of luminescence production in the biosensor strains PAO1 lecA::lux and PAO1 lecA::lux containing the pME6000::ahaP construct and empty plasmid pME6000 in the presence of C4and C6-HSL. Different letters above the bars indicate that the values are significantly different (p< 0.05). 2.7. Psychrobacter sp. M9-54-1 Attenuates the Virulence of Vibrio Coralliilyticus VibC-Oc-193 Both In Vitro and In Vivo Given that Psychrobacter sp. M9-54-1 abolished AHL-mediated signal activity present in crude extracts from three Vibrio spp., the V. coralliilyticus strain VibC-Oc-193 was selected to test the effect of AHL degradation by M9-54-1 on virulence factors produced by this aquaculture pathogen. Under the conditions tested, the AHLs produced by V. coralliilyticus VibC-Oc-193 (Figure 6A) were degraded in the presence of the M9-54-1 strain. In the Mar. Drugs 2021,19, 16 8 of 18 phenotypic analyses carried out, only the production of gelatinase in V. coralliilyticus VibC-Oc-193 was drastically reduced (Figure 6B). Mar. Drugs 2021, 19, x FOR PEER REVIEW 8 of 20 Figure 5. Evaluation of ahaP expression in P. aeruginosa PAO1. (A). Swarming motility assay of PAO1 wild type and strains containing the pME6000::ahaP construct and empty plasmid pME6000. (B). Detection of luminescence production in the biosensor strains PAO1 lecA::lux and PAO1 lecA::lux containing the pME6000::ahaP construct and empty plasmid pME6000 in the presence of C4and C6-HSL. Different letters above the bars indicate that the values are significantly different (p < 0.05). Figure 6. Production of AHLs ( A ) and gelatinase ( B ) by V. coralliilyticus VibC-Oc-193 (1) and the co-culture of Psychrobacter M9-54-1 and V. coralliilyticus VibC-Oc-193 (2). ( C ). Survival rate of Artemia salina nauplii after 48 h and 72 h of incubation with the different cultures. Different letters indicate significant differences (p< 0.05). To evaluate its potential use to control bacterial infections, we tested the QQ activity of M9-54-1 in a disease model of Artemia salina brine shrimp after 48 and 72 h infection with V. coralliilyticus VibC-Oc-193. Statistically significant disease attenuation was observed after 72 h of infection, resulting in an increase of 17.5% in the survival rate of Artemia salina incubated with M9-54-1 as compared to the infected Artemia salina without the Psychrobacter strain (Figure 6C). To confirm that the reduction in virulence recorded was due to QS interference and not to a growth inhibitory effect on VibC-Oc-193, colony forming units from M9-54-1 were assessed. No differences in VibC-Oc-193 viability were observed from either culture (data not shown), suggesting that the decrease in virulence observed is likely associated with AHL degradation by M9-54-1. 3. Discussion During the last decade, antimicrobial resistance (AMR) has become a serious global threat to human and animal health. International organizations such as FAO and WHO are working closely on implementing a global plan to minimize the threat of AMR which includes the development of novel strategies to fight bacterial infection diseases (www.fao. org). One novel and promising approach in which the research community is interested is interference with quorum sensing which controls virulence gene expression in numerous pathogens. The majority of studies have focused on enzymatic AHL degradation, with only a few devoted to non-enzymatic inhibitory mechanisms [14]. The marine environment has proven to be an excellent source of novel enzymes and compounds that interfere with QS systems [ 11 , 40 , 41 ]. In an attempt to discover novel QS-inhibiting compounds, we previously explored the marine symbiotic bacteria of invertebrates from which we selected AHL-degrading [ 24 ] and QSI-producing bacteria [ 39 ]. Thus, strain M9-54-1, with its high levels of AHL-degrading activity, was isolated from holothurians and selected for further study. In the present study, a cold-adapted QQ Mar. Drugs 2021,19, 16 9 of 18 enzyme named AhaP, which was identified and purified from strain M9-54-1, demonstrated its ability to degrade with high levels of activity substituted and unsubstituted AHLs. The strain M9-54-1 has been identified as belonging to the genus Psychrobacter based on the 16S rRNA gene sequence and whole genome sequencing. Its full-length 16S rRNA gene sequence similarity to P. faecalis and P. pulmonis was over 99.5%, indicating that this strain could belong to either of these species. However, a whole genome comparison between M9-54-1 and P. faecalis based on digital DNA-DNA hybridization and ANI values, suggests that M9-54-1 could be a new species of the genus Psychrobacter [ 42 ]. Since the whole genome sequence of P. pulmonis was not available, a whole-genome comparison with this species could not be performed. Interestingly, despite high similarities amongst their 16S rRNA gene sequences, these species differ in terms of phenotypic characteristics such as carbohydrate use [43]. Most bacteria of the genus Psychrobacter are psychrotolerant, with a growth temperature ranging from − 10 to 38 ◦ C, and halotolerant, showing growth in the presence of up to 6.5% (w/v) NaCl. Many of these bacteria are isolated from cold and high-salt environments such as Antarctic and seawater [ 44 ]. Psychrophile and psychrotolerant bacteria have become important vehicles for isolating novel high-activity enzymes at low temperatures, making them excellent energy-saving biocatalysts with a reduced environmental impact [ 45 ]. In addition, these enzymes are more thermostable than those originating from plants and animals [46]. Cold-adapted enzymes are considered to have potential biotechnological and industrial applications that require activity at mild temperatures and rapid heat inactivation rates, such as that found in molecular biology, medical research, food/feed processing, pharmaceuticals, detergents and cosmetics [ 45 , 47 – 49 ]. With regard to Psychrobacter, coldadapted enzymes, such as ribonucleases, proteases, esterases and lipases, have been found in some species of this genus [ 45 , 47 , 50 – 52 ]. Nevertheless, the production of compounds that interfere with quorum sensing (QS) in Psychrobacter strains has been the subject of a limited number of studies. For example, a marine sponge Psychrobacter sp. strain was found to degrade 3-O-C8-HSL and to be able to reduce swarming motility in Pseudomonas aeruginosa [ 53 ]. Another strain of Psychrobacter sp. isolated from a marine sponge has been reported to produce cyclic dipeptides that act as QS inhibitors via a non-enzymatic mechanism [ 54 ]. In our study, the experimental evidence presented, together with a phylogenetic analysis of the AhaP protein, suggest that the QQ enzyme produced by strain M9-54-1 belongs to a family of AHL acylases, showing high sequence and structural similarity to PvdQ from P. aeruginosa [ 55 ]. These findings reinforce the notion that the QS interference mechanism of strain M9-54-1 is not caused by the production of compounds that block the interaction of AHLs with their cognate signal receptors. Psychrobacter sp. strain M9-54-1 has shown high levels of activity against a broad range of synthetic AHLs as well as those produced by bacterial pathogens. Thus, the potential use of AhaP enzyme to combat bacterial infections was evaluated by expressing it in the human pathogen P. aeruginosa PAO1. A recurrent problem associated with infections caused by P. aeruginosa is the multiple antibiotic resistance displayed by clinical isolates of this bacterium [ 56 , 57 ], which greatly limits the treatment choices available. In this study, by using the strain P. aeruginosa PAO1 and the P. aeruginosa bioreporter PAO1 lecA::lux, we demonstrated that ahaP gene expression interferes with the production of the carbohydratebinding protein LecA which acts as an adhesin and a cytotoxin [ 58 ]. The reduction in lecA gene expression when PAO1 cultures were spiked with AHLs, as well as swarming motility inhibition, a well-studied QS-regulated phenotype in P. aeruginosa [ 59 ], suggest the potential use of AhaP as an anti-virulence treatment technique. Strain M9-54-1, which is a marine bacterium, was also tested as a potential biocontrol tool to combat infections in aquaculture. 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