Harnessing the Glucosyltransferase Activities of Clostridium difficile for Functional Studies of Toxins A and B

Abstract

The incidence of Clostridium difficile infection (CDI) has been increasing within the last decade. Pathogenic strains of C. difficile produce toxin A and/or toxin B, which are important virulence factors in the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect either the bacterium or the toxins. We have developed an assay (Cdifftox activity assay) to detect C. difficile toxin A and B activities that is quantitative and cost-efficient and utilizes a substrate that is stereochemically similar to the native substrate of the toxins (UDP-glucose). To characterize toxin activity, toxins A and B were purified from culture supernatants by ammonium sulfate precipitation and chromatography through DEAE-Sepharose and gel filtration columns. The activities of the final fractions were quantitated using the Cdifftox activity assay and compared to the results of a toxin A-and B-specific enzyme-linked immunosorbent assay (ELISA). The affinity for the substrate was >4-fold higher for toxin B than for toxin A. Moreover, the rate of cleavage of the substrate was 4.3-fold higher for toxin B than for toxin A. The optimum temperature for both toxins ranged from 35 to 40°C at pH 8. Culture supernatants from clinical isolates obtained from the stools of patients suspected to be suffering from CDI were tested using the Cdifftox activity assay, and the results were compared to those of ELISA and PCR amplification of the toxin genes. Our results demonstrate that this new assay is comparable to the current commercial ELISA for detecting the toxins in the samples tested and has the added advantage of quantitating toxin activity.

Toxin Synthesis by Clostridium difficile Is Regulated through Quorum Signaling

“…1A ). To examine the regulation of toxin production, we first developed a rapid and sensitive assay (the Cdifftox activity assay) to detect toxin A and B activity ( 33 ). This activity assay enabled the detection of toxin synthesis by C. difficile strain 630 at 16 h of growth in culture, compared to >32 h for analysis by enzyme-linked immunosorbent assay (ELISA) ( Fig.…”

Large clostridial glycosylating toxins modifying small GTPases

“…The LCGTs also cleave p-nitrophenyl-β-d-glucopyranoside (PNPG), a chromogenic substrate with stereochemical characteristics similar to UDP-glucose. This reaction may be exploited to detect LCGTs from culture supernatants [56]. …”

“…Phospho-S71-Rac1/Cdc42 are protected against glucosylation, thus reducing the effects of TcdB and TcsL [136,137]. Furthermore, taurocholate (TCA) has been shown to protect Caco-2 colonic epithelial cells from the damaging effects of TcdB, which has been attributed to inhibition of the glycosyltransferase activity of the toxins by TCA [56,138]. Cysteine protease activity has further been identified as a new target for the development of toxin-inhibiting compounds, as the autocatalytic cleavage is specifically inhibited using either dipeptide and tripeptide TcdB-CPD inhibitors or NO donors, resulting in transnitrosylation of the catalytic cysteine [103][104][105][106][139][140][141].…”

Multiple factors contribute to bimodal toxin gene expression in Clostridioides (Clostridium) difficile

“…Disease is mediated primarily through two exotoxins known as TcdA and TcdB (Lyerly et al, 1982;Lyerly et al, 1985;Triadafilopoulos et al, 1987;Voth and Ballard, 2005). Both toxins are glucosyltransferases that glucosylate host proteins, particularly the Rho family of GTPases (Schirmer and Aktories, 2004;Gerhard et al, 2008;Darkoh et al, 2011;Carter et al, 2012;Shen, 2012;Chandrasekaran and Lacy, 2017). This leads to collapse of the actin cytoskeleton and loss of tight junctions resulting in gastrointestinal distress (Moore et al, 1990;Stubbe et al, 2000;Feltis et al, 2000;Nusrat et al, 2001;Gerhard et al, 2008;Shen, 2012).…”