| Edwardsiella piscicida is a kind of broad-host intestinal pathogenic bacterium that infects a variety of freshwater and seawater fish,which causes significant economic losses to the aquaculture industries.Understanding of its pathogenic mechanism is essential for the development of prevention and treatment of edwardsiellosis.At present,several virulence factors related to E.piscicida pathogenic process have been identified,including type III secretion system（T3SS）,type VI secretion system（T6SS）,chondroitinase,hemolysin and flagella,etc.However,most of these virulence factors are prevalent in other pathogens and may play similar roles in E.piscicida.By applying transposon insertion sequencing（TIS）analysis,a large number of genes essential for infection of EIB202 have been screened out.Among them are some genes annotated as function unknown genes.These virulence-associated factors or regulatory genes were first discovered in E.piscicida,which might be of great significance on the understanding of its pathogenesis.Thus these virulence-associated factors or regulators were identified and their functions were further analyzed in this study.By the pattern analysis of conditional essential loci（PACE）,89 candidate live attenuated vaccine（LAV）targets were previously screened out in our laboratory.In this study,the conserved hypothetical protein ETAE0023 was selected for further characterization.EnrR belongs to the DcrB protein family and is related to phage adsorption,but no research has been reported about its relationship to the virulence in pathogens.Its influence on bacterial virulence was verified in the in vivo competition index experiment,and it was found that the deletion mutant of ETAE0023 decreased bacterial viability in fish.Compared with wild type strain and other live attenuated strains,ΔETAE0023 could be fast cleared out in fish,but still effectively stimulated the immune response of fish,especially the expression of immunoglobulin IgM.In clinical trials,the immune protection to fish elicited byΔETAE0023 reached to 73.7%.In addition,ETAE0023 did not affect the expression of T3SS-and T6SS-related proteins,although it played important role in the adhesion of E.piscicida to zebrafish cell line ZF4.All the results indicated that ETAE0023 is a promising LAV target of E.piscicida.But how it is associated to the in vivo virulence remains to be elucidated.Among the genes essential for infection in vivo,mutations in ftsH,hflK,and hflC could greatly reduce the survival ability of EIB202 in vivo.The three genes encodes membrane protease FtsH and its two modulators HflK and HflC,respectively,and all of them function together on degrading membrane proteins.Previous reports have shown that ftsH is an essential gene for growth in a variety of intestinal pathogens,while hflK and hflC have not been found to affect the virulence of bacteria,which indicates that they may have unique functions in E.piscicida.In E.piscicida,the functions of ftsH and its modulators were analyzed in this study.First,it was confirmed in in vivo competition experiment that all of the deletion mutation strains of these genes in E.piscicida could significantly reduce the colonization ability in vivo,and the cell invasion experiment showed that they could affect the adaptability of E.piscicida in the intracellular environment of fish cells.The LpxC could be degraded by FtsH,but the level of LpxC had no influence on the normal growth of E.piscicida,which is significantly different from other Gram-negative bacteria.In vitro environmental stress tolerance analysis showed that FtsH and its modulators affected the tolerance towards low nutrition,high osmotic pressure and mitomycin,and the the high osmotic pressure tolerance was probably regulated by the cellular accumulation of YfgM,the substrate of FtsH.This study revealed the unique functions of FtsH and its modulators in E.piscicida pathogenesis.Genomic islands（GIs）acquired by horizontal transfer play crucial roles in bacterial genome evolution.T3SS,T6SS and other genomic islands that confer pathogenecity towards hosts are the source of pathogen virulence evolution.We focused on a putative horizontally acquired protein ETAE0051（Edwardsiella nucleoic-associated regulator R,EnrR）,which regulates the expression of GIs.The enrR is widely distributed in a variety of bacteria,and its deletion mutant strain ΔenrR displayed the decreased expressions of T3SS-and T6SS-related proteins,which significantly influenced the virulence of E.piscicida.Comprehensive analysis of the ChIP-seq and RNA-seq showed that EnrR could bind low G+C content regions in E.piscicida genome,and regulate the expression of T3SS-and T6SS-related proteins by cro in GI-12.The binding ability of EnrR to DNA was found to be nonspecific and dependent on length as revealed by various DNA binding analysis techniques.Furthermore,EnrR was verified to be a novel nucleoid associated protein（NAP）.Expression of EnrR in different bacteria showed that EnrR could also regulate the lysogeny-lytic switch of prophages in Escherichia coli and enhance the virulence of Salmonella Typhimurium by activating the expression of SPIs（Salmonella pathogenicity islands）.The crystal structure of EnrR showed that the 47th arginine residue（R47）was the key animo acid for DNA binding ability.This study unraveled a universal NAP that regulates bacterial GIs and virulence.To summarize,this project identified and characterized 3 pivotal in vivo virulence-associated regulators in E.piscicida.The results will facilitate the understanding of the bacterial pathogenesis as well as the development of novel vaccines against the infections of Edwardsiella bacteria. |
