Type III secretion system 1 genes in Vibrio parahaemolyticus are positively regulated by ExsA and negatively regulated by ExsD

Vibrio parahaemolyticus harbours two distinct type III secretion systems (T3SS1 and T3SS2). A subset of 10 T3SS1 genes are transcribed when V. parahaemolyticus is grown in tissue culture medium [Dulbecco's modified Eagle's medium (DMEM)], while transcription of these genes (except exsD) is minimal upon growth in Luria-Bertani-Salt (LB-S). Transcription of T3SS1 genes and cytotoxicity towards HeLa cells was prevented by deletion of exsA while complementation with exsA restored these traits. Overexpression of ExsA in the wild-type strain, NY-4, activated the transcription of T3SS1 genes when bacteria were grown in LB-S. Thus, ExsA is necessary and sufficient to induce the transcription of T3SS1 genes. Deletion of the exsD permitted the transcription of T3SS1 genes when bacteria were grown in the LB-S medium and complementation with the wild-type exsD gene-blocked transcription of T3SS1 genes. Overexpression of ExsD in NY-4 prevented the transcription of T3SS1 gene when bacteria were grown in DMEM. A gel mobility shift assay demonstrated that purified ExsA protein binds a novel motif in the upstream region of vp1668 and vp1687, indicating that ExsA interacts directly with the promoter sequences of T3SS1 genes. ExsA positively regulates the expression and secretion of Vp1656 while ExsD negatively regulates the expression and secretion of Vp1656.