SummaryChromobacterium violaceum is a Gram-negative bacterium that causes fatal septicaemia in humans and animals. C. violaceum ATCC 12472 possesses genes associated with two distinct type III secretion systems (T3SSs). One of these systems is encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a), another is encoded by Chromobacterium pathogenicity island 2 (Cpi-2). Here we show that C. violaceum causes fulminant hepatitis in a mouse infection model, and Cpi-1/-1a-encoded T3SS is required for its virulence. In addition, using C. violaceum strains with defined mutations in the genes that encode the Cpi-1/-1a or Cpi-2 locus in combination with cultured mammalian cell lines, we found that C. violaceum is able to induce cytotoxicity in a Cpi-1/-1a-dependent manner. Characterization of Chromobacterium-induced cytotoxicity revealed that cell lysis by C. violaceum infection involves the formation of pore structures on the host cell membrane, as demonstrated by protection by cytotoxicity in the presence of osmoprotectants. Finally, we demonstrated that CipB, a Cpi-1/-1a effector, is implicated in translocator-mediated pore formation and the ability of CipB to form a pore is essential for Chromobacterium-induced cytotoxicity. These results strongly suggest that Cpi-1/-1a-encoded T3SS is a virulence determinant that causes fatal infection by the induction of cell death in hepatocytes.

SummaryThe type III secretion system (T3SS) encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a) is critical for Chromobacterium violaceum pathogenesis. T3SS-dependent virulence is commonly characterized by type III effector virulence function, but the full repertoire of the effector proteins of Cpi-1/-1a T3SS is unknown. In this study, we showed that expression of Cpi-1/-1a T3SS is controlled by the master regulator CilA. We used transcriptional profiling with DNA microarrays to define CilA regulon and identified genes encoding T3SS effectors whose translocation into host cells was dependent on Cpi-1/-1a T3SS. From these effectors, we found that CopE (CV0296) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases in its C-terminal portion. The N-terminal portions (1-81 amino acids) of CopE and a CivB as a putative chaperone were required for its translocation. CopE specifically activates Rac1 and Cdc42 followed by the induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades human epithelial HeLa cells in a Cpi-1/-1a-encoded T3SS-and CopE-dependent manner. Finally, C. violaceum strains lacking copE and expressing a CopE-G168V deficient in GEF activity were attenuated for virulence in mice, suggesting that CopE contributes to the virulence of this pathogen.