Regulation of type III secretion system 1 gene expression in Vibrio parahaemolyticus is dependent on interactions between ExsA, ExsC and ExsD

Vibrio parahaemolyticus ExsA is the transcriptional regulator for type III secretion system 1 (T3SS 1) while ExsD blocks T3SS 1 expression. Herein we show that deletion of exsC from V. parahaemolyticus blocked synthesis of T3SS 1-dependent proteins under inducing conditions (contact with HeLa cells), while in trans complementation of the Delta exsC strain with wild-type exsC restored protein synthesis. Under non-inducing conditions (Luria broth plus salt), in trans expression of exsC in a wild-type strain resulted in synthesis and secretion of T3SS 1-dependent proteins. Deletion of exsC does not affect the synthesis of ExsA while expression of T3SS 1 genes is independent of ExsC in the absence of ExsD. Co-expression of recombinant proteins with different antigenic tags demonstrated that ExsC binds ExsD and that the N-terminal amino acids of ExsC (positions 7 to 12) are required for binding. Co-expression and purification of antigentically tagged ExsA and ExsD demonstrated that ExsD directly binds ExsA and presumably prevents ExsA from binding promoter regions of T3SS 1 genes. Collectively these data demonstrate that ExsD binds ExsA to block expression of T3SS 1 genes, while ExsC binds ExsD to permit expression of T3SS 1 genes. ExsA, ExsC and ExsD from V. parahaemolyticus appear to be functional orthologues of their Pseudomonas aeruginosa counterparts.