Pathogenic diversification of the gut commensal <i>Providencia alcalifaciens</i> via acquisition of a second type III secretion system

<i>Providencia alcalifaciens</i> is a Gram-negative bacterium found in various water and land environments and organisms, including insects and mammals. Some <i>P. alcalifaciens</i> strains encode gene homologs of virulence factors found in pathogenic Enterobacterales members, such as <i>Salmonella enterica</i> serovar Typhimurium and <i>Shigella flexneri</i>. Whether these genes are pathogenic determinants in <i>P. alcalifaciens</i> is not known. In this study, we investigated <i>P. alcalifaciens</i>-host interactions at the cellular level, focusing on the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS_1b is widespread in <i>Providencia</i> spp. and encoded on the chromosome. A large plasmid that is present in a subset of <i>P. alcalifaciens</i> strains, primarily isolated from diarrheal patients, encodes for T3SS_1a. We show that <i>P. alcalifaciens</i> 205/92 is internalized into eukaryotic cells, lyses its internalization vacuole, and proliferates in the cytosol. This triggers caspase-4-dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS_1a in entry, vacuole lysis, and cytosolic proliferation is host cell type-specific, playing a more prominent role in intestinal epithelial cells than in macrophages or insect cells. In a bovine ligated intestinal loop model, <i>P. alcalifaciens</i> colonizes the intestinal mucosa and induces mild epithelial damage with negligible fluid accumulation in a T3SS_1a- and T3SS_1b-independent manner. However, T3SS_1b was required for the rapid killing of <i>Drosophila melanogaster</i>. We propose that the acquisition of two T3SS has allowed <i>P. alcalifaciens</i> to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.