Clostridium difficile is an important nosocomial pathogen that has become a major cause of antibiotic-associated diarrhea. There is a general consensus that C. difficile spores play an important role in C. difficile pathogenesis, contributing to infection, persistence, and transmission. Evidence has demonstrated that C. difficile spores have an outermost layer, termed the exosporium, that plays some role in adherence to intestinal epithelial cells. Recently, the protein encoded by CD1067 was shown to be present in trypsin-exosporium extracts of C. difficile 630 spores. In this study, we renamed the CD1067 protein Clostridium difficile exosporium cysteine-rich protein (CdeC) and characterized its role in the structure and properties of C. difficile spores. CdeC is expressed under sporulation conditions and localizes to the C. difficile spore. Through the construction of an ⌬cdeC isogenic knockout mutant derivative of C. difficile strain R20291, we demonstrated that (i) the distinctive nap layer is largely missing in ⌬cdeC spores; (ii) CdeC is localized in the exosporium-like layer and is accessible to IgGs; (iii) ⌬cdeC spores were more sensitive to lysozyme, ethanol, and heat treatment than wild-type spores; and (iv) despite the almost complete absence of the exosporium layer, ⌬cdeC spores adhered at higher levels than wild-type spores to intestinal epithelium cell lines (i.e., HT-29 and Caco-2 cells). Collectively, these results indicate that CdeC is essential for exosporium morphogenesis and the correct assembly of the spore coat of C. difficile.

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.

Background Clostridium difficile is the main cause of nosocomial infections including antibiotic associated diarrhea, pseudomembranous colitis and toxic megacolon. During the course of Clostridium difficile infections (CDI), C. difficile undergoes sporulation and releases spores to the colonic environment. The elevated relapse rates of CDI suggest that C. difficile spores has a mechanism(s) to efficiently persist in the host colonic environment.Methodology/Principal FindingsIn this work, we provide evidence that C. difficile spores are well suited to survive the host’s innate immune system. Electron microscopy results show that C. difficile spores are recognized by discrete patchy regions on the surface of macrophage Raw 264.7 cells, and phagocytosis was actin polymerization dependent. Fluorescence microscopy results show that >80% of Raw 264.7 cells had at least one C. difficile spore adhered, and that ∼60% of C. difficile spores were phagocytosed by Raw 264.7 cells. Strikingly, presence of complement decreased Raw 264.7 cells’ ability to phagocytose C. difficile spores. Due to the ability of C. difficile spores to remain dormant inside Raw 264.7 cells, they were able to survive up to 72 h of macrophage infection. Interestingly, transmission electron micrographs showed interactions between the surface proteins of C. difficile spores and the phagosome membrane of Raw 264.7 cells. In addition, infection of Raw 264.7 cells with C. difficile spores for 48 h produced significant Raw 264.7 cell death as demonstrated by trypan blue assay, and nuclei staining by ethidium homodimer-1.Conclusions/SignificanceThese results demonstrate that despite efficient recognition and phagocytosis of C. difficile spores by Raw 264.7 cells, spores remain dormant and are able to survive and produce cytotoxic effects on Raw 264.7 cells.

Impaired wound healing complicates a wide range of diseases and represents a major cost to healthcare systems. Here we describe the use of discarded wound dressings as a novel, cost effective, accessible, and non-invasive method of isolating viable human cells present at the site of skin wounds. By analyzing 133 discarded wound dressings from 51 patients with the inherited skin-blistering disease epidermolysis bullosa (EB), we show that large numbers of cells, often in excess of 100 million per day, continually infiltrate wound dressings. We show, that the method is able to differentiate chronic from acute wounds, identifying significant increases in granulocytes in chronic wounds, and we show that patients with the junctional form of EB have significantly more cells infiltrating their wounds compared with patients with recessive dystrophic EB. Finally, we identify subsets of granulocytes and T lymphocytes present in all wounds paving the way for single cell profiling of innate and adaptive immune cells with relevance to wound pathologies. In summary, our study delineates findings in EB that have potential relevance for all chronic wounds, and presents a method of cellular isolation that has wide reaching clinical application.

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Clostridioides difficile spores produced during infection are essential for the recurrence of the disease. However, how C. difficile spores persist in the intestinal mucosa to cause recurrent infection remains unknown. Here, we show that C. difficile spores gain entry into the intestinal mucosa via fibronectin-α5β1 and vitronectin-αvβ1 specific-pathways. The spore-surface exosporium BclA3 protein is essential for both spore-entry pathways into intestinal epithelial cells. Furthermore, C. difficile spores of a bclA3 isogenic mutant exhibited reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model of the disease. Inhibition of C. difficile spore-entry led to reduced spore-entry into the intestinal epithelial barrier and recurrence of C. difficile infection in vivo. These findings suggest that C. difficile spore-entry into the intestinal barrier is a novel mechanism of spore-persistence that can contribute to infection recurrence and have implications for the rational design of therapies.

In the Methods, the incorrect supplier and catalogue number for an antibody was listed. The sentence 'phalloidin Alexa-Fluor 568 (#ab176753 Abcam, USA)' should read 'phalloidin Alexa-Fluor 568 (#A12380 Thermo Fisher, USA)'.The Methods, in the section 'Colonic and ileal loop assay ', originally incorrectly read 'To evaluate the effect of nystatin or RGD peptide in C. difficile spore internalization, mice were treated with 17,000 UI kg −1 nystatin (n = 4) 24 h before the surgery. In the loop, as control, mice were treated with 0.9% NaCl (saline; n = 4) then, ligated loops were injected with 3 × 10 C. difficile R20291. In the case of RGD, ligated loops were injected with 250 nmol of RGD peptide (n = 4)'. The correct version replaces this text with 'To evaluate the effect of nystatin and RGD peptide in C. difficile spore internalization in vivo, 24 h prior to surgery, mice (n = 4) were treated with nystatin (17,000 IU kg −1 ) in 100 µL of DPBS by oral gavage; control mice (n = 8; 4 for control and 4 for RGD treatment) where treated with 100 µL of DPBS by oral gavage. On the day of surgery, ileal and colonic ligated loops of control mice (n = 4) were injected with 100 µL of DPBS containing 3 × 10 8 C. difficile R20291 spores; ileal and colonic ligated loops of nystatin-treated mice (n = 4) were injected with 100 µL of DPBS containing 3 × 10 8 C. difficile R20291 spores and 340 IU (17,000 IU kg −1 ) nystatin; ileal and colonic ligated loops of RGD-treated mice (n = 4) were injected with 100 µL of DPBS containing 3 × 10 8 C. difficile R20291 spores and 86.6 µg (250 nM) of RGD peptide. DPBS was used to resuspend nystatin and RGD peptides because it rendered higher solubility than saline solution (0.9% weight vol −1 NaCl)'.The Methods, in the section 'Quantification of C. difficile spores from feces and colon of mice', originally incorrectly read '1.5% weight vol −1 (BD, USA; TCCFA plates)'. The correct version replaces this text with '1.5% weight vol −1 agar (BD, USA) (TCCFA plates)'.