Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa

Mora-Uribe, Paola; Miranda-Cárdenas, Camila; Castro-Córdova, Pablo; Gil, Fernando; Calderón, Iván L.; Fuentes, Juan A.; Rodas, Paula I.; Banawas, Saeed; Sarker, Mahfuzur R.; Paredes-Sabja, Daniel

doi:10.3389/fcimb.2016.00099

Cited by 54 publications

(61 citation statements)

References 35 publications

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“…The success of carbohydrate-based subunit vaccines for treatment of other important bacterial infections [32][33][34] was an important factor which led to our study of the glycan structures produced by both vegetative cells and spores of C. difficile as potential subunit vaccine candidates [12,35]. Although spores are a metabolically dormant form of C. difficile, which persist in the gut during antibiotic treatment, recent studies have indicated that the exosporangial layer may be involved in host cell interactions and as such may present a good therapeutic target [19,36]. Hair-like projections similar to those found on B. anthracis spore surface have been identified on C. difficile spores.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine

et al. 2020

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The BclA3 glycoprotein is a major component of the exosporangial layer of Clostridium difficile spores and in this study we demonstrate that this glycoprotein is a major spore surface associated antigen. Here, we confirm the role of SgtA glycosyltransferase (SgtA GT) in BclA3 glycosylation and recapitulate this process by expressing and purifying SgtA GT fused to MalE, the maltose binding protein from Escherichia coli. In vitro assays using the recombinant enzyme and BclA3 synthetic peptides demonstrated that SgtA GT was responsible for the addition of β-O-linked GlcNAc to threonine residues of each synthetic peptide. These peptide sequences were selected from the central, collagen repeat region of the BclA3 protein. Following optimization of SgtA GT activity, we generated sufficient glycopeptide (10 mg) to allow conjugation to KLH (keyhole limpet hemocyanin) protein. Glycosylated and unglycosylated versions of these conjugates were then used as antigens to immunize rabbits and mice. Immune responses to each of the conjugates were examined by Enzyme Linked Immunosorbent Assay ELISA. Additionally, the BclA3 conjugated peptide and glycopeptide were used as antigens in an ELISA assay with serum raised against formalin-killed spores. Only the glycopeptide was recognized by anti-spore polyclonal immune serum demonstrating that the glycan moiety is a predominant spore-associated surface antigen. To determine whether antibodies to these peptides could modify persistence of spores within the gut, animals immunized intranasally with either the KLH-glycopeptide or KLH-peptide conjugate in the presence of cholera toxin, were challenged with R20291 spores. Although specific antibodies were raised to both antigens, immunization did not provide any protection against acute or recurrent disease.

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Section: Discussionmentioning

confidence: 99%

In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine

et al. 2020

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“…The BclA family of collagen-like glycoproteins are homogeneously distributed in the exosporium of C. difficile spores. Two members of the family, BclA2 and BclA3, have been identified as responsible of the formation of hair-like projections on the spore surface of the hypervirulent strain R20291 [15]. Both BclA2 and BclA3 are involved in the interaction with intestinal epithelial cells [15] and, therefore, are potential targets of new anti-CDI treatments.…”

Section: Introductionmentioning

confidence: 99%

“…Two members of the family, BclA2 and BclA3, have been identified as responsible of the formation of hair-like projections on the spore surface of the hypervirulent strain R20291 [15]. Both BclA2 and BclA3 are involved in the interaction with intestinal epithelial cells [15] and, therefore, are potential targets of new anti-CDI treatments. BclA2 protein of C. difficile R20291 is a 47.8 kDa protein, organized into three domains: (i) an N-terminal domain (NTD) anchored to the exosporium; (ii) a collagen-like domain; (iii) and a C-terminal domain (CTD) exposed to the exterior [16].…”

Section: Introductionmentioning

confidence: 99%

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

Maia

Reyes-Ramírez

Pizarro-Guajardo

et al. 2020

IJMS

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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.

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“…One factor that may contribute to C. difficile virulence is an isolate's ability to adhere to intestinal epithelium. Adherence of C. difficile spores to epithelium is dependent on the characteristics of exosporium, and the composition of this outmost layer can vary between strains [40][41][42]. Recently, two cysteine-rich proteins, cdeC and cdeM, were shown to influence the ability of C. difficile spores to adhere to intestinal epithelium [40].…”

Section: Discussionmentioning

confidence: 99%

Epidemic Ribotypes Of Clostridium (Now Clostridioides) Difficile Are Likely To Be More Virulent Than Non-epidemic Ribotypes In Animal Models

Vitucci

Pulse

Tabor-Simecka

et al. 2019

Preprint

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Background. Clostridium difficile infections have become more frequently diagnosed and associated with greater disease severity, which has resulted in an increase burden on the healthcare system. These increases are attributed to the increased prevalence of hypervirulent strains encompassing select ribotypes. These epidemic ribotypes were characterized as hypervirulent due to higher in vitro spore and toxin production, as well as increased incidence, severity and mortality within patients. However, it is unclear whether epidemic ribotypes are truly more virulent than non-epidemic ribotypes in vivo. Furthermore, there is conflicting evidence about the ability of a strain’s in vitro phenotype to be predictive of their in vivo virulence. The goals of the current studies were to determine if epidemic ribotypes are more virulent than other ribotypes in animal models, and whether the in vitro virulence phenotype of an isolate or ribotype predict in vivo virulence. Results. To determine if epidemic strains were truly more virulent than other non-epidemic strains, the in vivo virulence of thirteen C. difficile isolates (7 non-epidemic and 6 epidemic ribotype isolates) were determined in murine (C57BL/6 mice) and hamster (golden Syrian hamster) models of C. difficile infections. The isolates of epidemic ribotype of C. difficile were found to be more virulent in both the murine and hamster models than non-epidemic isolates. In particular, the group of epidemic ribotypes of C. difficile had lower LD50 values in hamsters. The increased severity of disease was associated with higher levels of Toxin A and Toxin B production found in fecal samples, but not numbers of organisms recovered. The isolates were further characterized for their in vitro virulence phenotypes, e.g. toxin production, growth rates, spore formation and adherence of spores to intestinal epithelial cell lines. Although there were higher levels of toxins produced and greater adherence for the group of epidemic ribotypes, the in vitro profiles of individual isolates were not always predictive of their in vivo virulence. Conclusions. Overall, the group of epidemic ribotypes of C. difficile were more virulent in vivo despite individual isolates having similar phenotypes to the non-epidemic isolates in vitro.

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Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa

Cited by 54 publications

References 35 publications

In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine

In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine

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

Epidemic Ribotypes Of Clostridium (Now Clostridioides) Difficile Are Likely To Be More Virulent Than Non-epidemic Ribotypes In Animal Models

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