1b) Analysis of the production and secretion profile of the VepA

1b). Analysis of the production and secretion profile of the VepA protein in each complement strain revealed that complementation with exsA or vp1701 increased the amount of VepA protein, whereas complementation with exsD

or vp1702 suppressed VepA protein production (Fig. 1c). The production and secretion profiles of the VepA protein in the vp1701 gene deletion and complementation strains were similar to those of the exsC deletion mutant of P. aeruginosa, indicating that VP1701 is orthologous to ExsC. That there was no homologue of the P. aeruginosa exsE gene in the V. parahaemolyticus T3SS1 see more region and VP1702 exerted a negative regulatory effect on the production of T3SS1-related proteins prompted us to examine the possibility that VP1702 is a functional equivalent of P. aeruginosa ExsE. As T3SS-dependent secretion is characteristic of ExsE, we then determined whether VP1702 is a specific substrate for T3SS1 using immunoblotting (Fig. 1d). As expected, VP1702 was not detected in the supernatants of the nonfunctional T3SS1 mutant strain (ΔvscN1). In contrast, the nonfunctional T3SS2 mutant strain AZD3965 research buy (ΔvscN2) secreted VP1702 protein in the supernatants, indicating that VP1702 is specifically secreted by T3SS1. These results indicate that VP1702 is a functional equivalent of ExsE and T3SS1 gene expression is regulated by the ExsACDE regulatory

cascade, similar to the regulation in P. aeruginosa. It is well known that extracellular calcium concentration is a potent signal for the induction of T3SS expression in P. aeruginosa. This type of transcriptional regulation is intimately coupled with type III secretory activity: transcription is repressed when the secretion channel is closed (high Ca2+ level) and is derepressed when the secretion channel is open (low Ca2+ Carbohydrate level). Therefore, the effect of extracellular calcium concentration on the production of T3SS1-related proteins (VscC1 and VepA) was examined using

immunoblotting. These proteins were detected in the bacterial pellet and the supernatant in the absence of calcium (inducing conditions), whereas the production of these proteins was repressed by the addition of CaCl2 (noninducing conditions) (Fig. 2a). We next determined the effect of the exs gene deletions on low-calcium-dependent production of VepA using immunoblotting (Fig. 2b). The ΔexsA and the ΔexsC strains did not express or secrete VepA, even under inducing conditions. In contrast, deletion of exsD or vp1702 resulted in derepression of VepA in the bacterial pellet. Although the production of VepA in the bacterial pellet was clearly induced in the ΔexsD and Δvp1702 strains, even under noninducing conditions, secretion still depended on the removal of extracellular calcium. These results suggest that VP1701 (ExsC of V. parahaemolyticus) functions as an anti-anti-activator for T3SS1 and that vp1702 is a functionally equivalent protein of P. aeruginosa ExsE.

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