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spore 1.5 1 patch with 121
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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.
C. difficile spores exhibit high levels of adherence to IECs in vitro14,15, and that the hair-like projections of C. difficile spores come in close proximity with the microvilli of differentiated Caco-2 cells; furthermore, C. difficile 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 C. difficile spore persistence in vivo and contribute to the recurrence of the disease remain unclear.
Herein, we first demonstrate that C. difficile 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 the spore entry pathway is Fn-α5β1 and Vn-αvβ1 integrin-dependent. Next, we 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 C. difficile spores that contributes to R-CDI, which involves gaining intracellular access into the intestinal barrier via BclA3-Fn-α5β1 and BclA3-Vn-αvβ1 specific, and that blocking spore entry contributes to reduced recurrence of the disease.
C. difficile spores of epidemically relevant strains exhibit hair-like projections that are likely to be formed by the collagen-like exosporium proteins1,13. Fn and Vn have a gelatin/collagen-binding domain16,17, suggesting that these molecules might interact with C. difficile spores through these hair-like projections. Indeed, through TEM coupled with immunogold labeling of Fn and Vn, we observed that more than 50% of the spores were positive for Fn- or Vn-immunogold particles (Supplementary Fig. 13a, b); immunogold Fn- and Vn-specific particles were observed in proximity to the hair-like extensions of C. difficile R20291 spores (Fig. 5a, b), suggesting that these structures might be implicated in spore entry into IECs. Most epidemically relevant strains encode two collagen-like exosporium proteins, BclA2 and BclA31,13. During the sporulation of R20291 strain, bclA3 expression levels are 60-fold higher than those of bclA228. Consequently, we first hypothesized whether BclA3 was responsible for the formation of the hair-like extensions. Therefore, we constructed a single bclA3 mutant strain, in an epidemic R20291 background, by removing the entire gene through a pyrE-based allelic exchange system29 (Supplementary Fig. 14). Electron micrographs demonstrate that, as expected, wild-type R20291 (ΔpyrE/pyrE+) spores exhibited typical hair-like projections observed in previous reports1,11,12 (Fig. 5c). By contrast, the ΔbclA3 deletion mutant formed spores that lacked the hair-like projections (Fig. 5d) that were restored upon complementation of the ΔbclA3 mutant strain with a single wild-type copy of bclA3 in the pyrE locus (ΔbclA3/bclA3+; Fig. 5e), indicating that BclA3 is required for the formation of these projections on the surface of C. difficile spores.
During CDI, C. difficile spore formation is essential in the recurrence of the disease7, yet the underlying mechanisms that correlate C. difficile spore persistence and recurrence of the disease remain unclear. In this study, we unravel a novel and unexpected mechanism employed by C. difficile spores to interact with the intestinal mucosa that contributes to the recurrence of disease. Our results have identified host molecules, cellular receptors, and a spore-surface ligand involved in spore entry into IECs. Notably, intracellular spores remain dormant in the presence of germinant. Using nystatin, a pharmacological inhibitor of spore entry in combination with antibiotic treatment leads to a reduction in the recurrence of the disease in mice. Together, these observations open a new angle for therapeutic interventions of CDI to prevent the recurrence of the disease.
Our results identified host molecules and cellular receptors involved in the entry of C. difficile spores into IECs. The presence of Fn or Vn allows C. difficile spores to gain intracellular access to IECs, in an RGD-specific manner and through specific integrin receptors (i.e., α5β1 and αvβ1). These observations were confirmed by the in vivo inhibition of C. difficile spore entry in the presence of the RGD peptide, which inhibits specifically interactions between Fn-α5β1 and Vn-αvβ120,21. Although Fn and Vn are mainly located in the basal and basolateral membrane of IECs, contributing to cell polarity16,17, antibody staining of healthy ileum and colonic tissue demonstrates that Fn and Vn are luminally accessible in a significant fraction of the IECs. Most of these cells were positive for luminally accessible Ecad and suggests that these cell types include cell extrusion, cells next to extrusion sites, and epithelial folds that typically undergo adherent junction reorganization22,23,24. However, a small fraction of cells positive for luminally accessible Fn and Vn were negative for luminally accessible Ecad, suggesting a novel phenotype within cells at the intestinal epithelial barrier. We also confirmed previous observations in mice that identified GCs have luminally accessible Ecad22, suggesting that C. difficile spores might also target these cell types to gain entry into the epithelial barrier. M cells are an additional cell type that might contribute to C. difficile spore entry into the intestinal epithelial barrier includes since they express β1-integrin at the apical surface in contrast to its normal basolateral location in enterocytes22,37,38. The fact that in vivo spore entry was RGD-binding integrin-specific suggests that Fn and Vn are accessible and employed by C. difficile spores to gain entry into IECs, which is consistent with the presence of accessible Fn and Vn in ileal and colonic loops. It is noteworthy that while RGD-specific entry was observed in both ileal and colonic loops, nystatin was only able to reduce spore entry into the ileum, but not colonic mucosa. This suggests that caveolae-independent endocytosis of C. difficile spores might prime in the colonic epithelia. During CDI, C. difficile toxins disrupt adherent junctions, leading to progressive exposure of deep regions of the colonic epithelium as infection advances. One consequence of this cellular disorganization may alter the distribution of cell receptors that may lead to increased adherence and internalization of C. difficile spores into the intestinal mucosa. Together, these observations prompt further studies to address how epithelium remodeling contributes to persistence of C. difficile spores and recurrence of the disease.
Another major contribution of this work is the role of the spore-surface collagen-like BclA3 exosporium protein in C. difficile spore entry into IECs in a Fn-α5β1- and Vn-αvβ1-dependent manner. Our previous work shows that Fn and Vn bind in a dose-dependent manner to C. difficile spores15. By immunogold-electron microscopy, our results demonstrate that Fn and Vn bind to the hair-like projections of C. difficile spores. We also demonstrate that they are formed by the collagen-like exosporium glycoprotein BclA3. It is noteworthy that experiments with monolayers of Caco-2 cells and CHO cells expressing integrin subunits demonstrate that BclA3 is essential for spore entry in the presence of Fn and Vn in an integrin-dependent manner; results that contrast with BclA3 being essential for adherence to the intestinal mucosa, but not for spore entry into the intestinal barrier. Coupling these results with those of in vivo RGD-specific C. difficile spore entry into the intestinal barrier indicates that additional spore-surface proteins might play redundant roles during in vivo spore entry or some uncharacterized host factors may also contribute. Regardless of these incongruencies, we observed that BclA3 contributes to the recurrence of the disease in a mouse model, suggesting that BclA3-mediated spore adherence to the intestinal mucosa might contribute to spore persistence and recurrence of the disease. The differences in spore adherence to the colonic tissue after R-CDI observed in the medium colonic tissue of mice might relate to the absence of mucosal folds typically observed in the distal and proximal colon of mice39. 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 C. difficile spore adherence to the intestinal mucosa and contributes to the recurrence of the disease. 2ff7e9595c
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