Pathogenisis Regulation Mechanisms In Vibrio Alginolyticus And Pseodumonas Aeruginosa

Vibrio alginolyticus is one of the important epizootic pathogens causing high mortality outbreaks of vibriosis in sea animals in farmed mariculture throughout Europe and South Asia, resulting in important economic losses. The abilities to adhere to the host surface, form biofilm and produce extracellular products such as the exotoxin alkaline serine protease (Asp) and siderophore have been suggested to be critically involved in virulence in this bacterium.Bacterial pathogens mainly rely on the activity of proteins secreted by a variety of protein secretion systems for the nutrients utilization, pathogenesis and niche adaptation. The type VI secretion system (T6SS) is a newly-defined, conserved bacterial protein translocation system and is precisely regulated in Gram-negative bacterial pathogens. We show that two sets of T6SS gene clusters (T6SSVA1and T6SSVA2) reside in the genomes of V. alginolyticus, encoding T6SS hallmark proteins such as Hcp, VgrG, and IcmF as well as serine-threonine kinase/phosphatase PpkA and PppA. A wide range of T6SS-related bacterial processes have been described while their underlying mechanisms are not clear. In this study, expression of a haemolysin co-regulated protein (Hcpl), which is one of the hallmarks of T6SS, was found to be strictly regulated in V. alginolyticus. The expression of Hcpl was growth phase-dependent and the production of Hcpl reached a maximum in the exponential phase. The expression of Hcpl was positively and negatively regulated by quorum sensing (QS) regulators LuxO and LuxR, respectively. In addition, we observed that Hcpl expression required the alternative sigma factor RpoN and the enhancer binding protein (EBP) VasH, which is encoded in T6SSVA1gene cluster. Moreover, LuxR, RpoN, and VasH could positively regulate the expression of other T6SS genes. Taken together, it was demonstrated that the expression of T6SS in V. alginolyticus was under the regulation of quorum sensing and alternative sigma factor.In this study, we identified a connection between the T6SS secretion/quorum sensing and various3’,5’-cyclic diguanylic acid (c-di-GMP) associated virulence phenotypes in V. alginolyticus. In-frame deletion mutants were made in the two T6SS gene loci to show their influences on motility, biofilm formation, cell aggregation, and exotoxin Asp production. The deletion of ppkA1and pppA resulted in an obvious alteration in the expression and secretion of Hcp1, signifying their essential roles in regulation of T6SS. PppA/PpkAl controlled pleiotropic phenotypes through the modulation of intracellular levels of second messenger c-di-GMP. Moreover, PppA/PpkAl also regulated the expression of Asp through manipulation of the expression of the quorum sensing regulator LuxR, which positively regulates the extracellular protease expression. Whole genome transcriptional profiling of ppkAl and pppA deletion strains revealed that the expression levels of over100genes were significantly co-regulated. These results demonstrate new roles of T6SS mediated by PppA/PpkAl in controlling pleiotropic phenotypes and contribute to our understanding of the conserved T6SS.In the gastrointestinal (GI) tract, complex communication occurs between the colonizing bacterial species and the cells of GI tract. This communication includes crosstalk between pathogens and the host. The opportunistic pathogen Pseudomonas aeruginosa is a well known wound and lung pathogen whose virulence is related to its abilities to monitor host chemical signals and to form communities of cells attached to surfaces known as biofilms. Here the breakdown product of ATP, adenosine, which is released into the GI tract by stressed eucaryotic cells, was found to play an important role in regulating the pathogenicity of P. aeruginosa. The addition of adenosine repressed P. aeruginosa biofilm formation and abolished bacterial swarming by preventing production of rhamnolipids. Since the adenosine metabolite, inosine, did not affect biofilm formation and a mutant unable to metabolize adenosine behaved like the wild-type strain, adenosine metabolism could be believed to be unrelated to the reduction of pathogenicity. Adenosine also reduces production of the virulence factors pyocyanin, elastase, EPS, siderophores, and the Pseudomonas quinolone signal which led to reduced virulence with Caenorhabditis elegans. To determine the mechanism by which adenosine reduces the virulence of P. aeruginosa, a whole-transcriptome analysis was conducted, and it was revealed that adenosine addition repressed genes similar to an iron-replete condition. DNA binding assays showed that adenosine repressed the iron-related genes through it increased the affinity of Fur for both the pvdS and fagA iron-related promoters. Therefore, adenosine decreases P. aeruginosa pathogenicity as an interkingdom signal by repressing genes related to iron acquisition. Significantly, this study provides a novel and nontoxic way (adenosine addition) to inhibit biofilm formation and virulence as well as an insight into how one of the most important virulence regulators is controlled.