Friday, April 4, 2025
4/4/2025
Mechanisms of persistence of Clostridioides difficile in the intestinal mucosa - PROJECT SUMMARY/ABSTRACT Clostridioides difficile is a Gram-positive, spore forming and strictly anaerobic and pathogenic bacterium that is the leading cause of antibiotic-associated diarrhea worldwide. C. difficile infections (CDI) are highly contagious, difficult to treat and prevent recurrence of the infection. There are several critical gaps in our current knowledge of the molecular mechanisms of persistence of C. difficile spores during CDI-treatment that lead to recurrence of the infection. Our long-term goals are to elucidate how C. difficile spores interact with the intestinal mucosa to facilitate spore-persistence and how these interactions lead to disease recurrence. Our previous studies revealed that C. difficile spores internalized and adhered in intestinal epithelial cells act as a reservoir for the recurrence of the infection. These results lead to our central hypothesis: that C. difficile spores combine intracellular and adherence mechanisms to persist in the intestinal mucosa during infection and treatment, to drive disease recurrence. Towards this hypothesis, we found that C. difficile spores internalize into intestinal epithelial cells (IECs). During vancomycin treatment of C. difficile-infected mice, we observed in ileal, cecum, and colonic samples, significant persistence of adhered and intracellular C. difficile spores. We observed that C. difficile spores interact, in a concentration dependent manner, with the extracellular matrix proteins fibronectin (Fn) and vitronectin (Vn), and uses them as molecular bridges to gain entry into IECs in an a5b1 and avb1 integrin- dependent manner. The spore surface protein, BclA3, is essential for spore entry into IECs via the Fn/Vn-integrin pathway. BclA3 contributes to disease recurrence, apparently by enhancing spore adherence to the intestinal mucosa. Administration of the cholesterol-sequestering agent, nystatin, strongly attenuated spore entry into IECs in an ileal and colonic loop mouse model, while administration of nystatin during vancomycin treatment of C. difficile-infected mice diminished disease recurrence in mice. Our results also show that E-cadherin interacts with C. difficile spores and plays a role in spore-adherence to IECs, and that E-cadherin-mediated spore- adherence to IECs is enhanced by toxin-mediated damage, which may also play a role in recurrence of CDI. Guided by our strong preliminary data, we propose to pursue three Specific Aims to characterize the mechanism underlying the interactions between C. difficile spores and the intestinal mucosa that contribute to recurrence of the infection: (1) Dissect the mechanism of internalization of C. difficile spores into intestinal epithelial cells; (2) Determine the role of adhered and intracellular spores in C. difficile persistence and disease recurrence; (3) Investigate the impact of toxins in E-cadherin-mediated C. difficile persistence and infection recurrence. Completion of these aims may expose novel targets for decolonization, therapeutics, and vaccine strategies to combat this pathogen.
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