Virulence Regulation By Two-Component Systems In Edwardsiella Tarda

Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro-and extra-intestinal infections in humans. Outbreaks of E. tarda-associated edwardsiellosis in various piscine species increasingly occur throughout the world and cause enormous losses to marine and freshwater aquaculture industries. A few of virulence factors have been characterized in E. tarda, including biofilm formation, hemolysin, type III secretion system (T3SS), and type VI secretion system (T6SS). To date, however, the genetic factors that govern them and other determinants of pathogenicity in E. tarda remain largely unknown. With the availability of the complete genome sequence of E. tarda EIB202, systematic and large-scale studies of the genes responsible for environment adaptation and virulence of E. tarda have become feasible.The ability to adapt to and thrive in diverse environments outside and inside of its hosts prompts us to investigate the roles of the two-component signal transduction systems (TCSs) in E. tarda. Through bioinformatic analysis, we identified63putative TCS proteins encoded by E. tarda EIB202, including30histine kinases and33response regulators. We successfully constructed deletion mutations in each of the response regulator genes, suggesting that none of the TCSs are essential for cell viability in E. tarda. The mutants were further examined for roles in biofilm formation, antibiotic resistance, stress response, expression and secretion of proteins involved in either the T3SS or T6SS, as well as virulence. Through these assays, we identified four regulators of growth, four regulators of biofilm development, two regulators of antibiotic resistance, and four regulators involved in stress responses. It was found that two regulators, EsrB and PhoP, are essential for the pathogenicity of E. tarda and further demonstrated that these two regulators have codependent and independent contributions to E. tarda virulence. Mutation of EsrB resulted in the complete loss of both the T3SS and T6SS proteins, while PhoP partially regulated the expression of T3SS and T6SS genes through EsrB, and was essential for resistance to structurally different cationic antimicrobial peptides (CAMPs).To investigate the mechanism underlying CAMP resistance in E. tarda, the function of UDP-glucose dehydrogenase (Ugd) was characterized. Ugd is an enzyme that converts UDP-glucose into UDP-glucuronic acid. Transcriptional fusion analysis revealed that CAMP induced the expression of ugd through PhoP. The lipopolysaccharide (LPS) produced by Δugd consisted of a truncated core oligosaccharide (OS) with no O-antigen attached. The mutant also exhibited enhanced autoaggregation and biofilm formation and reduced hemolytic activity. The ugd mutant was extremely sensitive to CAMPs, presumably because of alterations in LPS structure. Using different infection models it was found that Augd was impaired in survival within macrophages and displayed significantly attenuated virulence and an impaired ability to persist within the host. Moreover, vaccination of turbot (Scophthalmus maximus) with Augd by intraperitoneal injection elicited significant protection against the wild-type E. tarda strain, suggesting that Augd may be promising as a potential vaccine candidate against edwardsiellosis.As PhoQ/PhoP TCS plays important roles in both virulence and stress tolerance in E. tarda, we compared the proteomes of the wild-type and phop mutant strains to define components of the PhoP regulon. Overall,18proteins whose expression levels exhibited a2-fold or greater change were identified, and13of these proteins were found to require the presence of PhoP for full expression, while5were expressed at a higher level in the phoP mutant background. Identified proteins were representing diverse functional categories including energy production, amino acid metabolism, and oxidative stress defense. Quantitative real-time PCR (qRT-PCR) analysis of the mRNA levels for the identified proteins confirmed the proteomics data. Interestingly, P subunit of the F1F0ATP synthase (AtpD) playing an important role in growth and virulence of E. tarda, was listed as one of the proteins whose expression was greatly dependent on PhoP. The F1F0ATP synthase was encoded in a gene cluster (atpIBEFHAGDC) and the9genes were transcribed as an operon. qRT-PCR revealed that transcription of the9ATP synthase genes was positively regulated by PhoP. Electrophoretic mobility shift assay (EMSA) indicated that PhoP exerted this effect through direct binding to the promoter of atpl. The results provide new insights into the PhoP regulon and unravel a novel role for PhoP in the regulation of the F1F0ATP synthase.