Scale bar?=?50 m

Scale bar?=?50 m. toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to NECA Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium. NEW & NOTEWORTHY In this article, we developed a novel model of infection, also known as CDI, is a prevalent complication of long-term hospitalization. Risk factors for disease include antibiotic usage that alters gut microbiota, immunocompromised status, and advanced age (9, 18, 37, 160). CDI causes a range of clinical manifestations, including mild to severe watery diarrhea, pseudomembranous colitis, toxic megacolon, and death. The pathogenesis of infection involves ingestion of spores and germination of the spores in the presence of unconjugated primary bile acids, which are abundant in the small intestine, followed by vegetative cell colonization of the intestinal mucus layer NECA (45, 58, 75, 101, 135, 139). toxins are responsible for clinically significant CDI disease manifestations (5, 7, 15C17, 27, NECA 28, 32, 33, 40, 56, 59, 61, 69, 72, 73, 94, 98, 104, 106, 108, 111, 113). CDI is most frequently documented in the colon, prompting a substantial effort devoted to understanding pathogenesis in this intestinal segment (17, 18, 23, 27, 30, 44, 45, 66). Although CDI in the colon is well recognized, CDI in the small intestine, termed CDI-enteritis, has also been reported (1, 3, 4, 6, 8, 12C14, 22, 35, 43, 50, 51, 54, 55, 60, 62, 65, 71, 86C89, 92, 95, 97, 99, 100, 103, 105, 107, 109, 114C116, 121, 122, 127, 136C138, 142, 143, 145, 146, 148, Rabbit Polyclonal to ATP5S 152, 154C158, 161). These CDI cases include infection of the jejunum and ileum. CDI-enteritis frequently occurs in individuals with inflammatory bowel disease (IBD), a condition that is increasing in prevalence (6, 87, 103, 114). Although strains produce two major toxins, TcdA and TcdB, which belong to the family of large clostridial toxins (23). In addition, hypervirulent strains of can produce an additional binary toxin called CDT (57). These toxins enter epithelial cells by binding to one or more receptors present on the cell surface (2, 26, 30, 61, 68, 98, 112, 119, 124C126, 141, 149). Receptor binding is followed by endocytosis, which rapidly initiates multiple signaling events (48, 49, 66, 86). For TcdA and TcdB, toxin endocytosis leads to the inactivation of Rho GTPases (77, 78), which results in disassembly of actin stress fibers and collapse of the NECA actin cytoskeleton. This actin cytoskeleton collapse can be visualized as the rounding of cells, decreased cell volume, loss of cell-cell contacts, and formation of neurite-like NECA retraction fibers (33, 67, 130). For CDT, toxin endocytosis results in ADP-ribose caps in the actin protein, which prevents actin filament elongation, resulting in the collapse of the actin cytoskeleton. Thus, all toxins result in cell rounding. Because toxins are necessary for symptomatic infection, toxin activity has become a hallmark of the study of pathogenesis. Traditionally, cancer-derived cell lines, including HeLa, HT29, Caco-2, and T84 cells, or immortalized monkey fibroblast Vero cells have been used to examine toxin activity. However, multiple studies possess noted variants in the cytotoxic potencies from the same toxin across different cell lines. Another problem in learning CDI-enteritis originates from the cell type the original model systems.