Molecular characterization of the surface layer proteins from <i>Clostridium difficile</i>

Calabi, Emanuela; Ward, Steven; Wren, Brendan W.; Paxton, Thanai; Panico, Maria; Morris, Howard R.; Dell, Anne; Dougan, Gordon; Fairweather, Neil F.

doi:10.1046/j.1365-2958.2001.02461.x

Cited by 174 publications

(212 citation statements)

References 49 publications

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“…However, C. difficile adherence to epithelial cells in vitro has been reported to be mediated by several colonization factors, including the low-and high-molecular-mass Slayer proteins which are derived by post-translational cleavage of the S-layer precursor protein, SlpA (Calabi et al, 2001). The high-molecular-mass S-layer protein is largely responsible for C. difficile binding to both human GI tissues and to components of the extracellular matrix (Calabi et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells

Dingle

Mulvey

Humphries

et al. 2010

Journal of Medical Microbiology

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Herein we describe a real-time quantitative PCR assay for evaluating the adherence of Clostridium difficile to differentiated human intestinal Caco-2 cells. Our investigations demonstrated that the method, employing the C. difficile-specific triose-phosphate isomerase gene, is as reliable but less time-consuming than counting c.f.u. We conclude that the method will be useful for evaluating the role of host cell adherence in the pathogenesis of C. difficile infection. INTRODUCTIONClostridium difficile is a Gram-positive anaerobic microorganism that is the most important cause of antibioticassociated diarrhoea, now generally referred to as C. difficile infection (CDI), in humans. To cause clinical illness, C. difficile first penetrates the mucus layer covering the epithelial surface of the gastrointestinal (GI) tract; a strategy that is most likely facilitated by flagella and proteases (Deneve et al., 2009;Tasteyre et al., 2001). Once established in the GI tract, C. difficile expresses two large exotoxins, toxin A (TcdA) and toxin B (TcdB) (Jank & Aktories, 2008;Voth & Ballard, 2005). Delivery of TcdA and TcdB to the cytoplasmic compartment of epithelial cells then leads to glucosylation of Rho family GTPases, resulting in disorganization of the host cell cytoskeleton (Deneve et al., 2009). This results in the loosening of tight junctions between adjacent intestinal epithelial cells ultimately leading to inflammation and diarrhoea.Although TcdA and TcdB represent primary virulence factors, whether adherence to host epithelial cells is also important in the C. difficile pathogenic strategy is less clear. However, C. difficile adherence to epithelial cells in vitro has been reported to be mediated by several colonization factors, including the low-and high-molecular-mass Slayer proteins which are derived by post-translational cleavage of the S-layer precursor protein, SlpA (Calabi et al., 2001). The high-molecular-mass S-layer protein is largely responsible for C. difficile binding to both human GI tissues and to components of the extracellular matrix (Calabi et al., 2002). In addition, Cwp84, a putative colonization factor and cysteine protease, has recently been implicated in a process whereby SlpA is cleaved into lowand high-molecular-mass counterparts (Janoir et al., 2007;Kirby et al., 2009). Several other cell-surface colonization factors including Cwp66, Fbp68 and GroEL, may also all play a role in C. difficile adherence to intestinal epithelial cells (Hennequin et al., , 2003Waligora et al., 2001). Moreover, the flagellar proteins FliC (flagellin) and FliD (flagellar cap protein) bind to axenic mouse mucus possibly facilitating the initial penetration of the intestinal mucus layer (Tasteyre et al., 2001). CDI patients are known to produce serum antibodies to one or more of these cellsurface proteins during infection, emphasizing their potential role in pathogenesis and generating a protective host immune response (Pechine et al., 2005;Wright et al., 2008).Several in vitro C. difficile adherence assays have be...

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Section: Introductionmentioning

confidence: 99%

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells

Dingle

Mulvey

Humphries

et al. 2010

Journal of Medical Microbiology

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“…The virulence of C. difficile is mainly due to toxins A and B, although the role of potential colonization factors has been elucidated to a certain extent. The latter include the capsule (Davies & Borriello, 1990), proteolytic enzymes (Poilane et al, 1998;Seddon & Borriello, 1992), and the S-layer proteins P36 and P47 (Calabi et al, 2001(Calabi et al, , 2002Cerquetti et al, 2000;Karjalainen et al, 2001Karjalainen et al, , 2002. Our laboratory has identified several adhesins potentially involved in colonization: Cwp66 Waligora et al, 2001), the flagellin FliC (Tasteyre et al, 2000a(Tasteyre et al, , b, 2001b, the flagellar cap protein FliD (Tasteyre et al, 2001a, b), and a member of the Hsp60 family of chaperones GroEL (Hennequin et al, 2001a, b).…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of a fibronectin-binding protein from Clostridium difficile

et al. 2003

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A 68 kDa fibronectin-binding protein (Fbp68) from Clostridium difficile displaying significant homology to several established or putative Fbps from other bacteria was identified. The one-copy gene is highly conserved in C. difficile isolates. Fbp68 was expressed in Escherichia coli in fusion with glutathione S-transferase; the fusion protein and the native Fbp68 were purified. Immunoblot analysis and cell fractionation experiments revealed that Fbp68 is present on the surface of the bacteria. Far-immuno dot-blotting demonstrated that Fbp68 was capable of fixing fibronectin. Indirect immunofluorescence and ELISA were employed to demonstrate that C. difficile could bind both soluble and immobilized fibronectin. With competitive adherence inhibition assays it was shown that antibodies raised against Fbp68 partially inhibited attachment of C. difficile to fibronectin and Vero cells. Furthermore, Vero cells could fix purified membrane-immobilized Fbp68. Thus Fbp68 appears to be one of the several adhesins identified to date in C. difficile.

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“…The S-layer is composed of two proteins, the high molecular weight (HMW) 2 and low molecular weight (LMW) S-layer proteins (SLPs), derived by post-translational extracellular cleavage of the precursor SlpA (8). Following cleavage, the HMW and LMW SLPs form a high affinity heterodimer, the basic subunit of the assembled S-layer (9).…”

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confidence: 99%

Clostridium difficile Has Two Parallel and Essential Sec Secretion Systems

Fagan

Fairweather

2011

Journal of Biological Chemistry

221

326

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Protein translocation across the cytoplasmic membrane is an essential process in all bacteria. The Sec system, comprising at its core an ATPase, SecA, and a membrane channel, SecYEG, is responsible for the majority of this protein transport. Recently, a second parallel Sec system has been described in a number of Gram-positive species. This accessory Sec system is characterized by the presence of a second copy of the energizing ATPase, SecA2; where it has been studied, SecA2 is responsible for the translocation of a subset of Sec substrates. In common with many pathogenic Gram-positive species, Clostridium difficile possesses two copies of SecA. Here, we describe the first characterization of the C. difficile accessory Sec system and the identification of its major substrates. Using inducible antisense RNA expression and dominant-negative alleles of secA1 and secA2, we demonstrate that export of the S-layer proteins (SLPs) and an additional cell wall protein (CwpV) is dependent on SecA2. Accumulation of the cytoplasmic precursor of the SLPs SlpA and other cell wall proteins was observed in cells expressing dominant-negative secA1 or secA2 alleles, concomitant with a decrease in the levels of mature SLPs in the cell wall. Furthermore, expression of either dominant-negative allele or antisense RNA knockdown of SecA1 or SecA2 dramatically impaired growth, indicating that both Sec systems are essential in C. difficile.

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Molecular characterization of the surface layer proteins from Clostridium difficile

Cited by 174 publications

References 49 publications

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells

Identification and characterization of a fibronectin-binding protein from Clostridium difficile

Clostridium difficile Has Two Parallel and Essential Sec Secretion Systems

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