Shuyi Chen

Simple approach for ultrasensitive electrochemical immunoassay of Clostridium difficile toxin B detection

Clostridium difficile toxin B (Tcd B), as one of the primary contributing factors to the pathogenesis of C. difficile-associated diseases, has raised serious public concerns due to its virulence, spore-forming ability and persistence with major types of infectious diarrhea diseases, and been used as a potential biomarker in clinical diagnoses. Thus, a simple method for the determination of Tcd B was developed based on a sandwich-type electrochemical immunosensor. Greatly enhanced sensitivity was achieved based on fabricating the immunosensor by layer-by-layer coating carbon nanotubes (MWCNTs), Prussian blue (PB), Chitosan (CS), Glutaraldehyde (GA) composite on the working electrode as well as using graphene oxide (GO) as a nanocarrier in a multienzyme amplification strategy. In comparison with conventional methods, the proposed immunoassay exhibited high sensitivity and selectivity for the detection of Tcd B, providing a better linear response range from 0.003 to 320 ng/mL and a lower limit of detection (LOD) of 0.7 pg/mL (S/N=3) under optimal experimental conditions. The immunosensor exhibited convenience, low cost, rapidity, good specificity, acceptable stability and reproducibility. Moreover, satisfactory results were obtained for the determination of Tcd B in real human stool samples, indicating that the developed immunoassay has the potential to find application in clinical detection of Tcd B and other tumor markers as an alternative approach.

The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins

Clostridium difficile (C. difficile) is the main cause of antibiotic-associated diarrhea prevailing in hospital settings. In the past decade, the morbidity and mortality of C. difficile infection (CDI) has increased significantly due to the emergence of hypervirulent strains. Toxin A (TcdA) and toxin B (TcdB), the two exotoxins of C. difficile, are the major virulence factors of CDI. The common mode of action of TcdA and TcdB is elicited by specific glucosylation of Rho-GTPase proteins in the host cytosol using UDP-glucose as a co-substrate, resulting in the inactivation of Rho proteins. Rho proteins are the key members in many biological processes and signaling pathways, inactivation of which leads to cytopathic and cytotoxic effects and immune responses of the host cells. It is supposed that Rho GTPases play an important role in the toxicity of C. difficile toxins. This review focuses on recent progresses in the understanding of functional consequences of Rho GTPases glucosylation induced by C. difficile toxins and the role of Rho GTPases in the toxicity of TcdA and TcdB.

Recombinant Clostridium difficile toxin B induces endoplasmic reticulum stress in mouse colonal carcinoma cells

Clostridium difficile is the main cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans and animals. Its pathogenicity is primarily linked to the secretion of two exotoxins (TcdA and TcdB). Although great progress in the toxic mechanism of TcdA and TcdB has been achieved, there are many conflicting reports about the apoptotic mechanism. More importantly, apoptotic endoplasmic reticulum (ER) stress has been reported in cells treated with Shiga toxins-another kind of cytotoxins that can cause diarrhea and colitis. Herein we checked whether TcdB can induce ER stress. The results showed that recombinant TcdB (rTcdB) activated molecular markers of unfolded protein response, suggesting that rTcdB induced ER stress in CT26 cells. However, rTcdB did not induce the up-regulation of C/EBP homologous protein (CHOP), a classic mediator of apoptotic ER stress, but it activated the precursor of cysteine aspartic acid-specific protease 12 (caspase-12), a controversial mediator of apoptotic ER stress. Besides, glucosyltransferase activity-deficient mutant recombinant TcdB induced ER stress, though it has no cytotoxic or cytopathic effect on CT26 cells. Altogether, these data demonstrated that ER stress induced by rTcdB is glucosyltransferase-independent, indicating that ER stress induced by rTcdB is non-apoptotic. This work also offers us a new insight into the molecular mechanism of CHOP protein expression regulation and the role of CHOP expression in ER stress.

Identification of an Essential Region for Translocation of Clostridium difficile Toxin B

Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the major virulence factors involved in C. difficile-associated diarrhea and pseudomembranous colitis. TcdA and TcdB both contain at least four distinct domains: the glucosyltransferase domain, cysteine protease domain, receptor binding domain, and translocation domain. Few studies have investigated the translocation domain and its mechanism of action. Recently, it was demonstrated that a segment of 97 amino acids (AA 1756–1852, designated D97) within the translocation domain of TcdB is essential for the in vitro and in vivo toxicity of TcdB. However, the mechanism by which D97 regulates the action of TcdB in host cells and the important amino acids within this region are unknown. In this study, we discovered that a smaller fragment, amino acids 1756–1780, located in the N-terminus of the D97 fragment, is essential for translocation of the effector glucosyltransferase domain into the host cytosol. A sequence of 25AA within D97 is predicted to form an alpha helical structure and is the critical part of D97. The deletion mutant TcdB∆1756–1780 showed similar glucosyltransferase and cysteine protease activity, cellular binding, and pore formation to wild type TcdB, but it failed to induce the glucosylation of Rho GTPase Rac1 of host cells. Moreover, we found that TcdB∆1756–1780 was rapidly degraded in the endosome of target cells, and therefore its intact glucosyltransferase domain was unable to translocate efficiently into host cytosol. Our finding provides an insight into the molecular mechanisms of action of TcdB in the intoxication of host cells.

Rapid detection of Clostridium difficile toxins and laboratory diagnosis of Clostridium difficile infections

BackgroundClostridium difficile is an anaerobic, spore-forming and Gram-positive bacillus. It is the major cause of antibiotic-associated diarrhea prevailing in hospital settings. The morbidity and mortality of C. difficile infection (CDI) has increased significantly due to the emergence of hypervirulent strains. Because of the poor clinical different between CDI and other causes of hospital-acquired diarrhea, laboratory test for C. difficile is an important intervention for diagnosis of CDI.ObjectiveLaboratory tests for CDI can broadly detect either the organisms or its toxins. Currently, several laboratory tests are used for diagnosis of CDI, including toxigenic culture, glutamate dehydrogenase detection, nucleic acid amplification testing, cell cytotoxicity assay, and enzyme immunoassay towards toxin A and/or B. This review focuses on the rapid testing of C. difficile toxins and currently available methods for diagnosis of CDI, giving an overview of the role that the toxins rapid detecting plays in clinical diagnosis of CDI.

Novel Cysteine Desulfidase CdsB Involved in Releasing Cysteine Repression of Toxin Synthesis in Clostridium difficile

Clostridium difficile, a major cause of nosocomial diarrhea and pseudomembranous colitis, still poses serious health-care challenges. The expression of its two main virulence factors, TcdA and TcdB, is reportedly repressed by cysteine, but molecular mechanism remains unclear. The cysteine desulfidase CdsB affects the virulence and infection progresses of some bacteria. The C. difficile strain 630 genome encodes a homolog of CdsB, and in the present study, we analyzed its role in C. difficile 630Δerm by constructing an isogenic ClosTron-based cdsB mutant. When C. difficile was cultured in TY broth supplemented with cysteine, the cdsB gene was rapidly induced during the exponential growth phase. The inactivation of cdsB not only affected the resistance of C. difficile to cysteine, but also altered the expression levels of intracellular cysteine-degrading enzymes and the production of hydrogen sulfide. This suggests that C. difficile CdsB is a major inducible cysteine-degrading enzyme. The inactivation of the cdsB gene in C. difficile also removed the cysteine-dependent repression of toxin production, but failed to remove the Na2S-dependent repression, which supports that the cysteine-dependent repression of toxin production is probably attributable to the accumulation of cysteine by-products. We also mapped a δ54 (SigL)-dependent promoter upstream from the cdsB gene, and cdsB expression was not induced in response to cysteine in the cdsR::ermB or sigL::ermB strain. Using a reporter gene fusion analysis, we identified the necessary promoter sequence for cysteine-dependent cdsB expression. Taken together, these results indicate that CdsB is a key inducible cysteine desulfidase in C. difficile which is regulated by δ54 and CdsR in response to cysteine and that cysteine-dependent regulation of toxin production is closely associated with cysteine degradation.

Carbon storage regulator CsrA plays important roles in multiple virulence-associated processes of Clostridium difficile

The carbon storage regulator CsrA is a global regulator that controls multiple virulence-associated processes including host cell invasion, virulence secretion, quorum sensing, biofilm formation, and motility in many pathogenic bacteria. However, the roles of CsrA in Clostridium difficile still remain unclear. In this study, a C. difficile strain overexpressing csrA was constructed to investigate its effects on multiple virulence associated processes. Overexpression of csrA resulted in flagella defect and poor motility in C. difficile 630Δerm, suggesting that CsrA involves in the regulation of flagellum synthesis. The levels of toxin production were increased in the C. difficile 630Δerm overexpressing of csrA. Moreover, csrA overexpression enhanced the adherence ability to Caco-2 cells and solvent production of C. difficile 630Δerm. Altogether, CsrA of C. difficile participates in multiple virulence processes including toxin production, motility, and adherence, and in the regulation of carbon metabolism. These results enhance our understanding of the regulatory functions of CsrA and reveal that CsrA is an important regulator in C. difficile contributing to virulence regulation.

High-mobility group box 1 protein contributes to the immunogenicity of rTcdB-treated CT26 cells

Clostridium difficile TcdB is a key virulence factor that causes C. difficile-associated diseases. Our previous studies have shown that recombinant full-length TcdB (rTcdB) induces cell death in CT26 cells, and rTcdB-treated CT26 cells with high immunogenicity could stimulate dendritic cell (DC) activation and T cell activation in vitro. The rTcdB-treated CT26 cells also induce antitumor immunity in mice and protect mice from CT26 cells. High-mobility group box 1 protein (HMGB1) is a non-histone nuclear protein, which has various biological functions within the nucleus and also acts as an extracellular signal molecule involving in inflammatory diseases, cancers or autoimmune diseases. In this study, HMGB1 was found to be released from the rTcdB-treated CT26 cells. HMGB1 knockdown by using specific siRNA weakened the capacity of the BMDCs loaded with the rTcdB-treated CT26 cells to prime T cells in vitro and in vivo. The released HMGB1 from CT26 cells could interact with the receptor TLR4, which is closely related to DC activation and immune responses. The knockdown of HMGB1 also affected the phagocytosis of the rTcdB-treated CT26 cells by DCs in vitro. Furthermore, HMGB1 weakened the antitumor immunity of the rTcdB-treated CT26 cells, which protects mice from rechallenge of the live CT26 cells. Taken together, these results suggest that HMGB1 plays an important role on the immunogenicity of the rTcdB-treated dying CT26 cells.

Salubrinal protects against Clostridium difficile toxin B-induced CT26 cell death.

Clostridium difficile (C. difficile) is considered to be the major cause of the antibiotic-associated diarrhea and pseudomembranous colitis in animals and humans. The prevalence of C. difficile infections (CDI) has been increasing since 2000. Two exotoxins of C. difficile, Toxin A (TcdA) and Toxin B (TcdB), are the main virulence factors of CDI, which can induce glucosylation of Rho GTPases in host cytosol, leading to cell morphological changes, cell apoptosis, and cell death. The mechanism of TcdB-induced cell death has been investigated for decades, but it is still not completely understood. It has been reported that TcdB induces endoplasmic reticulum stress via PERK-eIF2α signaling pathway in CT26 cell line (BALB/C mouse colon tumor cells). In this study, we found that salubrinal, a selective inhibitor of eIF2α dephosphorylation, efficiently protects CT26 cell line against TcdB-induced cell death and tried to explore the mechanism underlying in this protective effect. Our results demonstrated that salubrinal protects CT26 cells from TcdB-mediated cytotoxic and cytopathic effect, inhibits apoptosis and death of the toxin-exposed cells via caspase-9-dependent pathway, eIF2α signaling pathway, and autophagy. These findings will be helpful for the development of CDI therapies.