IDENTIFICATION OF GENES THAT REGULATE TYPE-III SECRETION SYSTEM I OF VIBRIO PARAHAEMOLYTICUS IN RESPONSE TO HOST-CELL CONTACT

Dan Erwin

Vibrio parahaemolyticus pandemic serotype O3:K6 causes acute gastroenteritis, wound infections and septicemia in humans. This organism encodes two type-III secretion systems (T3SS1 and T3SS2) of which host-cell cytotoxicity has been attributed to T3SS1. The purpose of this study is to identify factors responsible for regulating T3SS1 induction in response to host-cell contact. Transcription of T3SS1 genes is positively regulated by ExsA which is presumptively regulated by the ExsACDE pathway, similar to Pseudomonas aeruginosa. Herein we deleted the putative exsE from V. parahaemolyticus and found constitutive expression of the T3SS1 as expected. More importantly, however, in a cell culture model the ΔexsE strain was unable to induce cytotoxicity or autophagy. This is markedly different from P. aeruginosa where exsE deficiency has no effect on host-cell cytolysis. Swarming and cytoadhesion were reduced for the deletion mutant and could be recovered along with T3SS1-induced HeLa-cell cytotoxicity by in cis expression of exsE in the ΔexsE strain. Loss of adhesion and swarming motility was associated with the loss of flagella biogenesis in ΔexsE. Mouse mortality was unaffected by the deletion of exsE as compared to a wild-type control suggesting that additional adhesins are important for intoxication in vivo. ExsA synthesis, and the subsequent T3SS1-dependent virulence phenotype, was found to function independently of the ExsACDE pathway both in vitro and in vivo. To identify genetic factors required for regulation of ExsA synthesis in response to host-cell contact, a transposon mutant library was constructed and screened for loss of cytotoxicity using a HeLa-cell culture model. Of the isolates screened, nearly half were identified as mutations in known T3SS1 genes confirming that the assay was functioning as expected. The remaining insertional mutations were found in genes encoded on both chromosome 1 and chromosome 2. Based on these data we conclude that ExsE contributes to the negative regulation of T3SS1 through the ExsACDE pathway. In addition, the unexpected loss of cytoadhesion indicates that there is probable cross-talk between the flagella systems and T3SS1. Positive regulation of ExsA in response to host-cell contact was confirmed and several candidate genes for an ExsACDE-independent induction pathway were identified by transposon mutagenesis.

IDENTIFICATION OF GENES THAT REGULATE TYPE-III SECRETION SYSTEM I OF VIBRIO PARAHAEMOLYTICUS IN RESPONSE TO HOST-CELL CONTACT

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