A Clostridium difficile gene encoding flagellin The GenBank accession numbers for the sequences reported in this paper are AF065259 (strain 79-685) and AF077341 (strain VPI 10463).

Tasteyre, Albert; Barc, Marie-Claude; Karjalainen, T.; Dodson, Paul; Hyde, S.; Bourlioux, P.; Borriello, Peter

doi:10.1099/00221287-146-4-957

Cited by 53 publications

(37 citation statements)

References 56 publications

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“…However, not all C. difficile-associated disease is caused by toxigenic strains of C. difficile (5,8,14,46,47). Thus, other virulence factors, such as adhesins, are likely important in the pathogenesis of C. difficile-associated disease.…”

Section: Discussionmentioning

confidence: 99%

Manganese Binds to Clostridium difficile Fbp68 and Is Essential for Fibronectin Binding

Lin¹,

Kuo²,

Koleci³

et al. 2011

Journal of Biological Chemistry

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Clostridium difficile is an etiological agent of pseudomembranous colitis and antibiotic-associated diarrhea. Adhesion is the crucial first step in bacterial infection. Thus, in addition to toxins, the importance of colonization factors in C. difficileassociated disease is recognized. In this study, we identified Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects people and multiple animal species. Colonization of the gastrointestinal tract may be asymptomatic or cause a variety of disorders, including mild diarrhea, pseudomembranous colitis, and antibiotic-associated diarrhea (1). Recent outbreaks in North America and Europe document the seriousness of C. difficile infection (2). C. difficile toxins, including toxin A, toxin B, and a recently identified toxin, binary ADP-ribosyltransferase toxin C. difficile transferase, are thought to be the primary virulence factors that mediate C. difficile-associated disease (3). Because C. difficile colonizes gastrointestinal tissues and enterocyte-like Caco-2 cells (4, 5), colonization factors are also recognized as important virulence factors of C. difficile. A variety of colonization factors has been identified, including the following: capsule (6); proteolytic enzymes (7, 8); S-layer proteins P36 and P47 (9); adhesins such as SLPA, CCAP6, Fbp68, and 12-kDa protein (4, 9 -14); flagellins such as FliC and FliD (15, 16); and GroEL chaperones (17,18).Adhesion is the first step in bacterial infection, and several adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) 2 contribute to this step (19). MSCRAMMs located on bacterial surfaces mediate adhesion by binding to various extracellular matrices including fibronectin (Fn), laminin, collagen, elastin, and proteoglycan on host surfaces (19). Loss of some of these genes may attenuate virulence, indicating that MSCRAMMs are pivotal mediators of bacterial disease (20). Although a number of MSCRAMMs have been investigated in other bacterial pathogens, only a few clostridial adhesins, such as Fbp68, have been characterized, and the colonization mechanisms of C. difficile are still poorly understood (11).Manganese-binding proteins are important players in bacterial physiology by participating in cation homeostasis (21), carbon metabolism to promote nutrient acquisition (22), signal transduction (23), resistance to oxidative stress (24), and nutrient-deprived stress (25). In addition, the interaction of some bacterial adhesins and ECM components can be modulated by metal ions such as calcium (26,27). To date, the ability of manganese to mediate bacterial adhesion has not been reported.Previously, Fbp68 on the surface of C. difficile was shown to serve as an adhesin by binding to Fn, fibrinogen, and vitronectin (11). Interestingly, antibody to Fbp68 can be detected in sera from patients with C. difficile-associated disease, indicating that Fbp68 can induce a host immune response during C. difficile infection (28). Structural analysis of Fbp68 indicates t...

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Section: Discussionmentioning

confidence: 99%

Manganese Binds to Clostridium difficile Fbp68 and Is Essential for Fibronectin Binding

Lin¹,

Kuo²,

Koleci³

et al. 2011

Journal of Biological Chemistry

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“…releasing suitable substrates from available protein sources for metabolism and which could be involved in mucus penetration (Poilane et al, 1998 ;Seddon & Borriello, 1992) ; (iii) adhesins, which are involved in mucus and cell association (Borriello et al, 1988a ;Eveillard et al, 1993 ;Karjalainen et al, 1994) ; (iv) fimbriae, the role of which is obscure (Borriello et al, 1988a) ; and (v) flagella, for which no role in C. difficile colonization has yet been assigned (Tasteyre et al, 2000).…”

Section: Abbreviationsmentioning

confidence: 99%

GroEL (Hsp60) of Clostridium difficile is involved in cell adherence

et al. 2001

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Previous results have demonstrated that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses ; this study focussed on whether the GroEL heat shock protein is implicated in this process. The 1940 bp groESL operon of C. difficile was isolated by PCR. The 1623 bp groEL gene is highly conserved between various C. difficile isolates as determined by RFLP-PCR and DNA sequencing, and the operon is present in one copy on the bacterial chromosome. The 58 kDa GroEL protein was expressed in Escherichia coli in fusion with glutathione S-transferase and the fusion protein was purified from IPTG-induced bacterial lysates by affinity chromatography on glutathione-Sepharose. A polyclonal, monospecific antiserum was obtained for GroEL which established by immunoelectron microscopy, indirect immunofluorescence and immunoblot analysis that GroEL is released extracellularly after heat shock and can be surface associated. Cell fractionation experiments suggest that GroEL is predominantly cytoplasmic and membrane bound. GroEL-specific antibodies as well as the purified protein partially inhibited C. difficile cell attachment and expression of the protein was induced by cell contact, suggesting a role for GroEL in cell adherence.

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“…The fliC, fliD and cwp84 genes were PCR-amplified from an ermB, Clin r , ribotype 001, North American pulsotype 2 C. difficile Calgary Laboratory Services clinical isolate # 2007 provided by Dr Thomas Louie (Department of Medicine, University of Calgary). The PCR primers were designed as described previously (Savariau-Lacomme et al, 2003;Tasteyre et al, 2000Tasteyre et al, , 2001b but lacking the 59 restriction sites. Next, the PCR products were cloned into pQE-30 UA (Qiagen) following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Therapeutic potential of egg yolk antibodies for treating Clostridium difficile infection

Mulvey

Dingle

Fang³

et al. 2011

Journal of Medical Microbiology

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Herein we present evidence for the therapeutic potential of colonization factor (CF)-specific egg yolk antibodies (IgY) for potentially treating acute and recurring Clostridium difficile infection (CDI) in humans. The study involved cloning, expressing as 6¾His-tagged proteins in Escherichia coli, and Ni-affinity purifying three previously identified CFs (FliC, FliD and Cwp84) from C. difficile. The recombinant CF antigens were then used to immunize Leghorn chickens and CF-specific IgY antibodies were prepared from their eggs. The specificity and titre of the resulting C. difficile CF-specific IgY antibodies were assessed by ELISA and Western immunoblotting techniques. The antibodies were also screened for their ability to inhibit C. difficile adherence to human colon-derived T84 cells, and, based on these findings, one of them (FliD-specific IgY) was evaluated for its potential to prevent C. difficile-mediated morbidity and mortality in Syrian hamsters. The results revealed that purified FliD-specific IgY significantly protected hamsters from C. difficile strain 630 infection relative to control animals treated with carbonate buffer alone or IgY produced from unimmunized chicken eggs. The results suggest that egg yolk preparations obtained from chickens immunized with recombinant C. difficile CFs may represent another safe and cost-effective treatment option in humans suffering from acute or recurring CDI. INTRODUCTIONConventional antibiotics alter both the numbers and complexity of the normal gastrointestinal (GI) microflora, thereby reducing the ability of these protective organisms to competitively exclude opportunists from colonizing the GI tract. This disruption may render hospitalized subjects susceptible to colonization by C. difficile (Job & Jacobs, 1997). Once established in the colon, C. difficile expresses two very large exotoxins, toxin A (TcdA) and toxin B (TcdB), which are primarily responsible for the clinical symptoms of C. difficile infection (CDI) (Voth & Ballard, 2005). These range from diarrhoea to pseudomembranous colitis, toxic megacolon and multi-system organ failure, which may be fatal.The accepted approach to treating CDI is to replace the antibiotic responsible for initiating the condition with one, typically metronidazole, to which C. difficile is sensitive. In about 80 % of cases, this approach leads to complete recovery without further complications. In the remaining cases, however, recurring CDI (rCDI) can occur, due, presumably, to a failure of the normal GI microflora to become re-established after the metronidazole is discontinued. This leaves the colon of these individuals susceptible to C. difficile relapse or reinfection. Multiple recurrences are not unusual, resulting in a significant reduction in the quality of life in these individuals.In response to the increasing risk of CDI to human health, much research has focused on new and improved treatment regimens, particularly in subjects suffering from rCDI. These studies have concentrated on novel toxin neutralizing agents, an...

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A Clostridium difficile gene encoding flagellin The GenBank accession numbers for the sequences reported in this paper are AF065259 (strain 79-685) and AF077341 (strain VPI 10463).

Cited by 53 publications

References 56 publications

Manganese Binds to Clostridium difficile Fbp68 and Is Essential for Fibronectin Binding

Manganese Binds to Clostridium difficile Fbp68 and Is Essential for Fibronectin Binding

GroEL (Hsp60) of Clostridium difficile is involved in cell adherence

Therapeutic potential of egg yolk antibodies for treating Clostridium difficile infection

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