Here, we have shown that BclA3 uses Fn and Vn, and their specific integrins to gain entry into IECs and that BclA3 is essential for spore adherence to the intestinal mucosa and contributes to the recurrence of the disease. The work presented here also shows that spore entry into IECs contributes to spore dormancy in the presence of primary bile salts (i.e., taurocholate) and that blocking in vivo spore entry during antibiotic treatment (vancomycin) leads to reduced R-CDI in mice. Deletion of the gene in spore entry into the intestinal barrier can contribute to spore persistence and infection recurrence, and suggest potential avenues for new therapies. is a strict anaerobic Gram-positive pathogenic bacterium that forms highly resistant spores that easily persist in the environment and contribute to the transmission of infections (CDI) through fecalCoral route1. Disruption of the gut microbiota by broad-spectrum antibiotics leads to an optimal environment for colonization and proliferation in the colon and disease manifestation. CDI currently leads hospital-acquired diarrhea associated to antibiotics in the United States and worldwide2. In the US alone, ~500,000 patients per year become infected with CDI, and mortality rates reach ~8% of total patients2. The annual cost of CDI to the health care system is estimated at ~US 4.8 billion2. Treatment of CDI usually involves antibiotic therapy, typically vancomycin or metronidazole and, most recently, fidaxomicin2, which, although resolves the infection in ~95% of the cases, leads to recurrence of CDI (R-CDI) in 15C30% of the individuals3C5. During infection, produces two major virulence factors, toxins TcdA and TcdB, responsible for the clinical manifestation of the disease, induce pro-inflammatory cytokines, disruption of tight junctions, detachment of intestinal epithelial cells (IECs), and loss of transepithelial barrier6. also initiates a sporulation pathway that leads to the production of new metabolically dormant spores in the hosts intestine1. In vivo, spore formation is essential for the recurrence of the disease7. Moreover, spore-based therapies that remove spores from the intestinal mucosa contribute to reducing the recurrence of the disease in animal models8. Recent in vivo studies in the laboratory strain 630 suggest that the spore-surface mucus-binding protein, peroxiredoxinCchitinase CotE, and the exosporium collagen-like BclA1 proteins are required for the colonization and infectivity in a mouse model of CDI9,10. However, the surface layer of 630 spores does not resemble that of clinically relevant strains, which exhibit hair-like projections in their spore NSD2 surface, structures that are absent in strain 6301,11,12. Notably, most clinically relevant sequenced isolates, including isolates of the epidemically relevant 027 ribotype, have a truncated due to a premature stop codon in the N-terminal domain13, resulting in the translation of a small polypeptide, which localizes to the spore surface10; thus limiting the breadth and depth of these results. spores exhibit high levels of adherence to IECs in vitro14,15, and that the hair-like projections of spores Gepotidacin come in close proximity with the microvilli of differentiated Caco-2 cells; furthermore, spores interact in a dose-dependent manner with fibronectin (Fn) and vitronectin (Vn)15, two extracellular matrix proteins used by several enteric pathogens to infect the host16,17. However, the mechanisms that underline how these interactions contribute to spore persistence in vivo and contribute to the recurrence of the disease remain unclear. Herein, we first demonstrate that spores gain entry into the intestinal epithelial barrier of mice and that spore entry into IECs requires serum molecules, specifically Fn and Vn, that are luminally accessible in the colonic mucosa. We also demonstrate that Gepotidacin the spore entry pathway is Fn-51 and Vn-v1 integrin-dependent. Next, we Gepotidacin show that the spore-surface collagen-like BclA3 protein is essential for spore entry into IECs through these pathways in vitro and for spore adherence to the intestinal mucosa. Our results also show that BclA3 contributes to the recurrence of the disease in mice. We also observed the therapeutic potential of blocking spore entry into the intestinal epithelial barrier, and how coadministration of nystatin with vancomycin reduces spore persistence and R-CDI in mice. Together, our results reveal a novel mechanism employed by spores that contributes to R-CDI, which involves gaining intracellular access into the intestinal barrier via BclA3-Fn-51 and BclA3-Vn-v1 specific, and that blocking spore entry contributes to reduced recurrence of the disease. Results spores internalize into the intestinal barrier in vivo To study the interaction of spores and the hosts intestinal barrier, we used a colonic/ileal.