Edwardsiella tarda is a Gram-negative enteric bacterium affecting both animals and humans. Recently, a type III secretion system (TTSS) was found in Ed. tarda. Such systems are generally used by bacterial pathogens to deliver virulence factors into host cells to subvert normal cell functions. Genome-walking was performed from the eseB and esrB genes (homologues of Salmonella sseB and ssrB, respectively) identified in previous studies, to determine the sequences of the TTSS. Thirty-five ORFs were identified which encode the TTSS apparatus, chaperones, effectors and regulators. Mutants affected in genes representing each category were generated and found to have decreased survival and growth in fish phagocytes. LD 50 values of the mutants were increased by at least 10-fold in comparison to those of the wild-type strain. The adherence and invasion rates of the esrA and esrB mutants were enhanced while those of the other mutants remained similar to the wild-type. The eseC and eseD mutants showed slight autoaggregation in Dulbecco's Modified Eagle Medium, whereas the rest of the mutants failed to autoaggregate. Regulation of the TTSS was found to involve the two-component regulatory system esrA-esrB. This study showed that the TTSS is important for Ed. tarda pathogenesis. An understanding of this system will provide greater insight into the virulence mechanisms of this bacterial pathogen.

Edwardsiella tarda is a gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastroand extraintestinal infections in humans. A type III secretion system (TTSS) and a putative secretion system (EVP) have been found to play important roles in E. tarda pathogenesis. Our previous studies suggested that the TTSS and EVP gene clusters were regulated by a two-component system of EsrA-EsrB. In the present study, we characterized another regulator, EsrC, which showed significant sequence similarity to the AraC family of transcriptional regulators. Mutants with in-frame deletions of esrC increased the 50% lethal doses in blue gourami fish, reduced extracellular protein production, and failed to aggregate. Complementation of esrC restored these three phenotypes. Two-dimensional gel electrophoresis showed that EsrC regulated the expression of secreted proteins encoded by the TTSS (such as EseB and EseD) and EVP (EvpC) gene clusters. The expression of esrC required a functional two-component system of EsrA-EsrB. EsrC in turn regulated the expression of selected genes encoded in TTSS (such as the transcriptional unit of orf29and orf30, but not esaC) and genes encoded in the EVP gene cluster. The present study sheds light on the regulation of these two key virulence-associated secretion systems and provides greater insight into the pathogenic mechanisms of this bacterium.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries. Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions. We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants. Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function. Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins. Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69. Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown. These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens.Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens (7). EHEC strains, especially those of serotype O157:H7, which produce Shiga-like toxin (Stx), are a common cause of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome, while EPEC is the most common bacterial cause of infant diarrhea (29). Both have been implicated in food-borne outbreaks in many countries and cause diarrhea by colonizing the intestinal mucosa (29, 30). EHEC and EPEC strains are distinguished from other pathogenic E. coli strains by their ability to produce a characteristic histopathological feature known as attaching and effacing (AE) lesions on the mucosa (12, 29). AE lesions are characterized by the destruction of the microvilli and the induction of actin-based pedestal formation underneath the eukaryotic membrane at the site of attachment (6). EHEC and EPEC secrete many extracellular proteins (ECPs), and the type III secretion system (TTSS) is a major secretion apparatus for secreting virulence factors which interact directly with the host (20, 25). The TTSS is located within a chromosomal pathogenicity island designated the locus of enterocyte effacement (LEE) which is necessary for the formation of AE lesions (18,19). The LEE-encoded regulator (Ler) activates most of the genes within the LEE region and is centr...