A plasmid-encoded T3SS underlies the virulence of enteropathogenic Providencia alcalifaciens strains isolated from a large foodborne outbreak in Japan.

Providencia alcalifaciens is a gut commensal bacterium and also an emerging enteric pathogen associated with sporadic infections and outbreak cases in humans. The most notable outbreak caused by this bacterium occurred in 1996 in Fukui Prefecture, Japan, affecting 270 individuals. However, the pathogenic mechanisms responsible for this outbreak remain unknown. In this study, we identified the key virulence determinants of the Fukui outbreak strains through genomic and functional analyses. These strains uniquely carry a ~162 kb large plasmid encoding a type III secretion system (T3SS) closely homologous to the Salmonella SPI-1 T3SS. We also show that the plasmid-encoded T3SS (T3SSp) constitutes a functional secretion system and is essential for the pathogenicity of the Fukui outbreak strain, including the invasion of cultured epithelial cells and the induction of diarrhea in a rabbit model. Secretome analysis identified effectors secreted in a T3SSp-dependent manner, among which PipA-sharing limited sequence similarity with SipA, a SPI-1 T3SS effector-plays a crucial role in inducing diarrhea. Ectopic expression of PipA in HeLa cells caused focal accumulation of F-actin, indicating its cytoskeleton-modulating activity. Comparative genomics with other Providencia species revealed the dissemination of the large plasmid, with structural variations among enteropathogenic strains of P. alcalifaciens and Providencia rustigianii associated with clinical cases in humans and animals. Thus, our findings underscore the molecular basis of P. alcalifaciens pathogenicity in the Fukui outbreak and highlight the significance of the large plasmids encoding T3SS in driving pathogenic evolution among Providencia species.