Entry of spores into intestinal epithelial cells contributes to recurrence of Clostridioides difficile infection

Abstract

Clostridioides difficile spores produced during infection are important for the recurrence of the disease. Here, we show that C. difficile spores gain entry into the intestinal mucosa via pathways dependent on host fibronectin-α5β1 and vitronectin-αvβ1. The exosporium protein BclA3, on the spore surface, is required for both entry pathways. Deletion of the bclA3 gene in C. difficile, or pharmacological inhibition of endocytosis using nystatin, leads to reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model. Our findings indicate that C. difficile spore entry into the intestinal barrier can contribute to spore persistence and infection recurrence, and suggest potential avenues for new therapies.

A ligated intestinal loop mouse model protocol to study the interactions of Clostridioides difficile spores with the intestinal mucosa during aging

“…Also, that C. difficile spore entry is increased in 2year-old mice. This finding, coupled with our recent observations that C. difficile spore entry is associated with R-CDI rates [20], may explain the increased R-CDI rates observed in elderly patients and in animal models [5,6,37].…”

“…Additionally, in human biopsies of older adults have been observed an increased intestinal permeability by a reduced transepithelial electric resistance compared to young humans [40] being those changes in the permeability to ions and not for macromolecules [41]. Recently we demonstrated that C. difficile spores gain access into the intestinal epithelial cells via pathways dependent on fibronectin-α5β1 and vitronectin-αvβ1 [20]. Although fibronectin, vitronectin, and integrins α5, αv, and β1 are mainly located in the basolateral membrane [42,43] we have shown that fibronectin and vitronectin are luminally accessible into the colonic mucosa of healthy young mice [20].…”

“…During CDI, C. difficile forms metabolically dormant spores that are essential for R-CDI [19]. Accordingly, with this observation, we developed a surgical procedure of intestinal ligated loop, which allowed us to demonstrate that C. difficile spores are able to adhere and internalize into the intestinal mucosa contributing to disease recurrence [20]. However, whether aging affects the adherence and internalization of C. difficile spores to the intestinal mucosa remains unclear.…”

“…Add 70-90µL of 1:1,000 chicken primary antibody anti-C. difficile spore IgY batch 7246 (AvesLab, USA) and 1:150 phalloidin Alexa-Fluor 568 (A12380 Invitrogen, USA); in PBS-3% BSA overnight at 4° C. This antibody does not immunoreacted with epitopes of vegetative cells neither with murine microbiota [20,23]. After adding the antibody solution, check that there are no bubbles in the tube and that the tissue is completely submerged.…”

Whole genome sequencing evidences higher rates of relapse in Clostridioides difficile infection caused by PCR ribotype 106

“…However, the fact that we did not observe isogenic strains among the majority of patients (differing between 4 and 43 SNPs) makes this theory less likely, since it would be expected to find more isogenic strains among the different patients if they had been reinfected with persistent spores of the same strain present in the environment. In these sense, a new important study has recently demonstrated a novel mechanism employed by C. difficile spores to gain entry into the intestinal mucosa via pathways dependent on host fibronectin-α5β1 and vitronectin-αvβ1 where the exosporium protein BclA3, on the spore surface, is required for both entry pathways contributing to the recurrence of disease [27].…”