The Basic Studies On Quorum Quenching As A Novel Tool To Fight Against Bacterial Infections In Aquaculture

Quorum sensing (QS) is a mechanism for bacteria to communicate, regulate geneexpression and synchronize social behaviors such as biofilm formation,bioluminescence and secretion of virulence factors. Nowadays, QS, especially theAHL (N-acyl-homoserine lactone)-based QS, has been recognized to play animportant role in the expression of virulence factors in many pathogens includingvibrios. The emergence of antibiotic-resistant pathogens poses a global threat andexhorts an urgent need to develop alternative therapeutic strategies. Since QS is notessential for the growth of bacteria, quenching QS (quorum quenching, QQ) in thesepathogens would disarm virulence rather than kill the bacteria, which may greatlyweaken the selective pressure imposed on the pathogens and attenuate the evolutionof resistance to QQ drugs. All of these traits make QS an ideal target for antivirulencetherapy. Although various QQ agents have been identified, most of them are terrestrial.Therefore, in this thesis, a high-throughput method was developed for identifying QQbacteria from marine candidates. The QQ agents of Muricauda olearia Th120werefurther characterized. Moreover, genomic analysis of M. olearia suggested that strainTh120might possess a complex pathway for iron metabolism. All of these resultscould hence provide us with new agents for anti-virulence strategies to control diseasecaused by QS dependent pathogens and/or QS dependent host-microbial interactions.In this thesis, a sensitive and high-throughput method named “the A136liquidX-gal assay” was developed for identifying QQ bacteria. This assay is based on theprinciple of bio-measurement of the residual AHLs after reaction, and β-galactosidaseactivities can be accurately quantified by absorbance measurements followed bynormalization to the cell density of the biosensor. Additionally, the PIPES buffer wasapplied for successfully removing false positives caused by alkaline hydrolysis of AHLs. This method allows simultaneous measurement of large numbers of sampleswithin a minimum of hands-on time, requiring merely standard equipment available inany research laboratory. This assay was successfully applied in identifying QQbacteria among366candidate strains and25QQ strains belonging to14species wereobtained. It was the first report of QQ activity in the genera Flaviramulus, Muricauda,Salinimonas, Thalassomonas, Marivita, Novosphingobium, Colwellia andRhodobacter. Moreover,12out of14species (except the species of Olleyamarilimosa and Tenacibaculum discolor) were reported to bear QQ activities for thefirst time. Further studies showed that three of them possessing AHL-lactonaseactivities and also indicate that a high diversity of AHL-degrading activities existingin these QQ strains. The supplementation of O. marilimosa T168, Pseudoalteromonasprydzensis Th125and M. olearia Th120into water significantly attenuatedAeromonas hydrophila infection in zebrafish, which indicates their potential forfurther application in aquaculture.QQ bacteria M. olearia Th120showed an extremely high AHL-degradingactivity. Strain Th120possesses small molecular QS inhibitors and QQ enzymes. Fiveproteins with diverse AHL-degrading activities were identified by in-gel QQ activityassay, mass spectrometry analysis and whole genome sequencing technique. Two ofthem are AHL lactonase and acylase, and named as MomL (Muricauda oleariamarine lactonase) and MomA (M. olearia marine acylase), respectively. RecombinantMomL and MomA were overexpressed in E. coli and showed relatively highAHL-degrading activities. The sequence analysis of MomL and MomA with knownQQ enzymes show that they are most closely related with Bacillus sp.240B1AiiA(28.85%identity) and Pseudomonas aeruginosa PAO1HacB (37.92%identity) in thededuced amino acid sequences, respectively. MomL is the only secretory AHLlactoanase belonging to metallo-β-lactamase superfamily at present. RecombinantMomL showed a broad substrate specificity and a preference for substrates withlong-chain AHLs or3-oxo-AHLs, and further displayed a moderate heat-resistance. ICP-MS (inductively coupled plasma mass spectrometry) analysis indicates thatrecombinant MomL may have a high affinity with magnesium but can barely combinecobalt. Combination with beryllium may promote its AHL-degrading activity. Theseresults indicate that MomL may have a distinct metal-combining propertie. In addition,site-directed mutagenesis showed that His117, His119, Asp121, His122, His189and His214are necessary for MomL activity. BLAST sequence-similarity searchingin GenBank databases showed that MomL homologues mainly exist in several marineFlavobacteriaceae bacteria. Therefore, MomL may represent a novel marine AHLlactonase. Furthermore, MomL can greatly decrease the protease and pyocyaninproduction of P. aeruginosa PAO1, which indicates its potential for the application inaquaculture to control bacterial disease.The genome of M. olearia Th120was sequenced using the Illumina HiSeq2000,and a total of3,730protein encoding genes were predicated. Analysis of the relativeenrichment of COG classification revealed that strain Th120possesses a strikinglyhigh abundance of genes involved in carbohydrate transport and metabolism,transcription, signal transduction mechanisms and inorganic ion transport andmetabolism, especially in iron uptake, transport, storage and metabolism. The genemomL is located in the middle of suf operon whose products composing an importantFe-S cluster biosynthesis machinery SUF. The leaving group thio effect of AHLlactonase and the inhibitory effect of Fe2+on MomL activity indicate that MomL mayplay a further regulatory role in Fe-S cluster biosynthesis and iron metabolismpathway in strain Th120. The bioinformatics analysis of whole genome data showedthat strain Th120may possess an N-acyl-amino acid hydrolase which may beresponsible for the further degradation of a ring-opened AHL yielding a homoserineand the corresponding fatty acid, both of which are contained in the integratedbiosynthesis of amino acids pathway of strain Th120. Except strain Th120, O.marilimosa T168, Tenacibaculum soleae strains and T. discolor T84are also belong tothe family Flavobacteriaceae. The characters of AHL-degrading activity of T. discolor T84showed similar with strain Th120, and strain T168and T133showedlactonase-like activities. Since the MomL homologues might be widespread in themarine Flavobacteriaceae bacteria, bioinformatics analysis based on the genomes ofseveral representative marine Flavobacteriaceae bacteria available in NCBI databaseindicates the existence of MomL homologues in O. marilimosa T168, T. discolor T84and T. soleae strains while MomA homologues only in T. discolor and T. soleae.In a summary, a sensitive and high-throughput method for identifying QQbacteria was developed and was successfully applied in identifying novel QQ bacteriabelonging to14species. Five proteins with diverse AHL-degrading activities wereidentified in M. olearia Th120and QQ enzyme MomL may represent a novel marineAHL lactonase. Whole genomic analysis revealed a strikingly high abundance ofgenes involved in iron metabolism in Th120and MomL may play a further regulatoryrole in Fe-S cluster biosynthesis.