Coordinated regulation of pathogenic phenotypes and hostresponse by the Vibrio cholerae type three secretionsystem

Vibrio cholerae strain AM-19226, which causes sporadic cholera-like disease, encodes a horizontally-acquired type three secretion system (T3SS) as its major virulence mechanism. Disease mediated by strain AM-19226 in animal models requires a functional T3SS, which translocates bacterial effectors directly into the host cell cytoplasm. Putative open reading frames within the AM-19226 T3SS genomic island were screened for their ability to inhibit growth when expressed in Saccharomyces cerevisiae, and were further screened in yeast strains deleted for components of MAPK pathways to potentially identify interacting partners, and also tested for T3SS-dependent translocation into HeLa cells. Eleven translocated proteins with varying levels of translocation were identified. Previous studies showed that AM-19226 T3SS transcriptional regulators, VttRA and VttRB, are required for bile-dependent expression of T3SS structural genes in vitro. To better understand the scope of genes that are potentially regulated by VttRA and VttRB, deep RNA sequencing was performed on wild type AM-19226 and derivatives deleted for vttRA and vttRB grown in bile. Comparative transcriptome analysis revealed genes encoded outside the T3SS genomic island, such as those involved in biofilm formation, motility, and type six secretion as potentially regulated by VttRA/B. In addition, T3SS effectors also appeared to be positively regulated by VttRA/B. Infant mouse competition assays showed that strains individually deleted for effectors, Vops H, A, M, I, and W, exhibited a 100-1000 fold defect in colonization whereas others had ≈ 10-fold defect (VopG) or no defect (VopZ). Because colonization can alter host cell signaling pathways and AM-19226-mediated disease has an inflammatory component, we evaluated the chemokine profile of Caco2-BBE cells during AM-19226 infection. A standard ELISA detected increased levels of IL-8 during Caco2-BBE/AM-19226 co-culture that required a functional T3SS and bile. Collective data suggest that colonization by strain AM-19226 is mediated by 7 translocated proteins, 5 of which are encoded within the T3SS structural gene operons, and VttRA and VttRB are global transcriptional regulators that directly or indirectly coordinate T3SS gene expression with diverse phenotypes such as motility and biofilm formation during different stages of infection.