Role of OspC1 and OspD3 (ShET2) in interleukin-8 secretion during Shigella flexneri infection

Shigella flexneri causes millions of diarrheal infections and hundreds of thousands of deaths each year, mostly in children in developing countries. Shigella uniquely evolved to adapt and utilize host variables to its advantage despite the harsh gastrointestinal environment. Our previous research has demonstrated that Shigella resists the bactericidal nature of bile salts, found mostly in the small intestine, and utilizes the compound as a signal to regulate virulence for subsequent colonic infection. The ospC1 and ospD3 genes, which are encoded on the virulence plasmid and known to affect neutrophil migration and interleukin-8 (IL-8) secretion during infection, are upregulated in the presence of bile salts. The goal of this study was to examine the effects of bile salts pre-exposure on subsequent colonic cell infection and IL-8 secretion. We first confirmed the expression of ospC1 and ospD3 was induced in bile salts. Next, overnight colonic HT-29 epithelial cell infections and IL-8 secretion analyses were performed using wild-type bacteria, a virulence-plasmid cured strain (BS103) as a negative infection control, and mutants ΔospC1, ΔospD3, and ΔospC1+ΔospD3. Single mutants retained wild-type levels of infection; but surprisingly, the double mutant ΔospC1+ΔospD3 had less bacterial recovery after infection that was exacerbated following bile salts pre-exposure. For IL-8 secretion in the absence of bile salts, ΔospD3 had significantly higher levels, indicating the OspD3 protein suppresses IL-8 secretion. However, following bile salts pre-exposure, ΔospC1 displayed higher IL-8 secretion, suggesting the OspC1 protein is more important to suppress IL-8 secretion. Future analyses will continue to explore these findings and evaluate subsequent effects on neutrophil migration. Our data provide support that Shigella exposure to appropriate in vivo-like conditions is essential to understand infection dynamics in the colonic epithelium.2026-10-3