Salmonella biases epithelial differentiation in murine enteroids through Wnt and Notch signaling, which may contribute to diarrheal pathogenesis

Abstract

Salmonella enterica serovar Typhimurium is one of the most burdensome foodborne diarrheal pathogens worldwide. However, despite its prevalence, the mechanism by which Salmonella causes diarrhea is not entirely known. We have shown in mice that Salmonella infection decreases expression of SLC26A3 (Down‐Regulated in Adenoma; DRA), a chloride/bicarbonate exchanger. In order to understand the mechanism of this effect, we employed an enteroid model from murine colon. Enteroid‐derived monolayers (EDM) that recapitulate both the absorptive and secretory cell lineages of the native intestinal epithelium (enterocytes vs. enteroendocrine, goblet, and Paneth cells, respectively) were infected apically with Salmonella and relevant protein expression was studied using qRT‐PCR and western blot. Infection significantly reduced expression of DRA (40%, p = 0.0124) and Hes1 (45%, p = 0.0004) while upregulating expression of ATOH1 (3‐fold, p = 0.0292) and mucin 2 (Muc2) (2‐fold, p = 0.0134). Hes1 is a Notch pathway downstream signaling molecule and thus a precursor to the absorptive epithelial lineage that expresses DRA. ATOH1, on the other hand, is a Wnt pathway downstream signaling molecule and a precursor to secretory lineages, including goblet cells that express Muc2. Infection of EDMs also reduced levels of the Notch intracellular domain. The involvement of Notch was further investigated by inhibiting Notch signaling using a γ‐secretase inhibitor, which reproduced the downregulation in Hes1 and DRA and upregulation in ATOH1 and Muc2 as seen with infection. Because the balance of Wnt and Notch signaling determines relative intestinal epithelial differentiation, our findings suggest that the diarrheal pathogenesis of Salmonella may reflect Notch inhibition and an accompanying shift in epithelial differentiation from absorptive to secretory cell types, a decreased capacity for absorption, and thus the accumulation of diarrheal fluid.