Clostridium difficile Spore-Macrophage Interactions: Spore Survival

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 Findings: In 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 similar to 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/Significance: These 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

Landscapes and bacterial signatures of mucosa-associated intestinal microbiota in Chilean and Spanish patients with inflammatory bowel disease

Inflammatory bowel diseases (IBDs), which include ulcerative colitis (UC) and Crohn’s disease (CD), cause chronic inflammation of the gut, affecting millions of people worldwide. IBDs have been frequently associated with an alteration of the gut microbiota, termed dysbiosis, which is generally characterized by an increase in abundance of Proteobacteria such as Escherichia coli, and a decrease in abundance of Firmicutes such as Faecalibacterium prausnitzii (an indicator of a healthy colonic microbiota). The mechanisms behind the development of IBDs and dysbiosis are incompletely understood. Using samples from colonic biopsies, we studied the mucosa-associated intestinal microbiota in Chilean and Spanish patients with IBD. In agreement with previous studies, microbiome comparison between IBD patients and non-IBD controls indicated that dysbiosis in these patients is characterized by an increase of pro-inflammatory bacteria (mostly Proteobacteria) and a decrease of commensal beneficial bacteria (mostly Firmicutes). Notably, bacteria typically residing on the mucosa of healthy individuals were mostly obligate anaerobes, whereas in the inflamed mucosa an increase of facultative anaerobe and aerobic bacteria was observed. We also identify potential co-occurring and mutually exclusive interactions between bacteria associated with the healthy and inflamed mucosa, which appear to be determined by the oxygen availability and the type of respiration. Finally, we identified a panel of bacterial biomarkers that allow the discrimination between eubiosis from dysbiosis with a high diagnostic performance (96% accurately), which could be used for the development of non-invasive diagnostic methods. Thus, this study is a step forward towards understanding the landscapes and alterations of mucosa-associated intestinal microbiota in patients with IBDs.This study was supported by Fondo Nacional De Desarrollo Científico y Tecnológico FONDECYT grant 1161161 to R. Vidal, CONICYT-PCHA/2014-21140975 fellowship to N. Chamorro, FONDECYT 1120577 and 1170648 to Hermoso MA and the Spanish Ministry of Economy projects CLG2015 66686-C3-1-P to Rosselló-Mora R., as well as funds from the European Regional Development Fund (FEDER) and NSF Dimensions in Biodiversity grant OCE-1342694. Support was also provided by a Millennium Science Initiative grant from the Ministry of Economy, Development and Tourism to Paredes-Sabja D

Induction of a Specific Humoral Immune Response by Nasal Delivery of Bcla2ctd of Clostridioides difficile

Clostridioides difficile, formerly known as Clostridium difficile, is a spore-forming bacterium considered as the most common cause of nosocomial infections in developed countries. The spore of C. difficile is involved in the transmission of the pathogen and in its first interaction with the host; therefore, a therapeutic approach able to control C. difficile spores would improve the clearance of the infection. The C-terminal (CTD) end of BclA2, a spore surface protein of C. difficile responsible of the interaction with the host intestinal cells, was selected as a putative mucosal antigen. The BclA2 fragment, BclA2CTD, was purified and used to nasally immunize mice both as a free protein and after adsorption to the spore of Bacillus subtilis, a well-established mucosal delivery vehicle. While the adsorption to spores increased the in vitro stability of BclA2CTD, in vivo both free and spore-adsorbed BclA2CTD were able to induce a similar, specific humoral immune response in a murine model. Although in the experimental conditions utilized the immune response was not protective, the induction of specific IgG indicates that free or spore-bound BclA2CTD could act as a putative mucosal antigen targeting C. difficile spores. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.Indexación:Scopu

The Clostridium difficile Exosporium Cysteine (CdeC)-Rich Protein Is Required for Exosporium Morphogenesis and Coat Assembly

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 ΔcdeC isogenic knockout mutant derivative of C. difficile strain R20291, we demonstrated that (i) the distinctive nap layer is largely missing in ΔcdeC spores; (ii) CdeC is localized in the exosporium-like layer and is accessible to IgGs; (iii) Δ 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, Δ 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.Keywords: Spore surface, Subtilis, Infection, Germination, Culture, Gene, Caco-2, Bacterial spores, Identification, Bacillus anthracis exosporium

Epidemic Clostridium difficile Ribotype 027 in Chile

The increased severity of Clostridium difficile infection is primarily attributed to the appearance of an epidemic strain characterized as PCR ribotype 027. The only report that identified epidemic C. difficile ribotype 027 in an American country outside of North America comes from Costa Rica, raising the possibility that strains 027 might also be present in other countries of Latin America. Several studies between 2001 and 2009 have been conducted in South American countries to detect the incidence of C. difficile infection in hospitalized patients, but they did not identify which C. difficile strains were causing these infections

Early career interview: Marjorie Pizarro-Guajardo, Universidad Andrés Bello

Marjorie Pizarro-Guajardo is a postdoctoral fellow at the Universidad Andrés Bello (Santiago, Chile), where she studies Clostridium difficile spores. She won the 2019 Future Science Future Star Award. Here she tells us about her career to date, and how she felt winning the award. </jats:p

Early career interview: Marjorie Pizarro-Guajardo, Universidad Andrés Bello

Marjorie Pizarro-Guajardo is a postdoctoral fellow at the Universidad Andrés Bello (Santiago, Chile), where she studies Clostridium difficile spores. She won the 2019 Future Science Future Star Award. Here she tells us about her career to date, and how she felt winning the award