The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis

Abstract

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.

Structural basis for selective modification of Rho and Ras GTPases by Clostridioides difficile toxin B

“…However, TcdB becomes inaccessible to antibodies after entering the host cells. We found that mutations that selectively disrupt GTPases binding drastically reduced the cytopathic toxicity of TcdB holotoxin, which is consistent with prior studies showing that a glucosyltransferase-deficient TcdB mutant was atoxic in vivo ( 50 ). Therefore, the GTD is an ideal molecular target for therapeutic interventions once the toxin is inside the cells, which will directly target the root cause of disease symptoms and cellular damage.…”

Murine Intrarectal Instillation of Purified Recombinant Clostridioides difficile Toxins Enables Mechanistic Studies of Pathogenesis

“…On the other hand, the DxD TcdB has been used in vitro and ex vivo to demonstrate the glucosyltransferase-independent mechanism for TcdB-induced necrotic cell death (17)(18)(19). It is unclear how 50 mg of DxD TcdB failed to produce significant epithelial injury in the present model, but a recently published report shows isogenic spores lacking glucosyltransferase activity have minimal, if any, colonic inflammation in C. difficile infected mouse and hamster models (41). This could be the result of species-specific differences.…”

Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection

“…However, these systems may have lacked the sensitivity of the C. difficile infection model, in which toxins are constantly produced and interact with the host epithelium over a course of several days. Recently, GT activity of TcdB was shown to be necessary to cause CDI, and there were no observed GTindependent effects in the mouse model of infection using defined GTX mutants in the C. difficile 630 strain [16]. This study had two limitations: the first being that the C. difficile 630 strain produces few CDI symptoms in the mouse model of infection when compared to epidemic C. difficile strains, and it also does not cause epithelial damage [16][17][18].…”

“…The relevance of GT-independent cell death during infection has been unclear since it is unknown if cells encounter the concentration of TcdB required to stimulate necrotic cell death. A recent study concluded that GT activity was necessary to cause CDI in mouse and hamster models of infection, but this does not preclude an additional role for GT-independent events as the infection progresses [16]. The study also involved GT-defective C. difficile mutants in the 630 strain, which is genetically tractable, but causes only mild disease symptoms in animal models [17].…”

“…The study also involved GT-defective C. difficile mutants in the 630 strain, which is genetically tractable, but causes only mild disease symptoms in animal models [17]. In contrast, the BI/ NAP1/PCR-ribotype 027 epidemic strains have been associated with multiple human epidemics and cause significant pathology in animal models of infection [16][17][18][19]. Among several differences, the 630 strain has been characterized as one with low expression levels of TcdA and TcdB [17,19].…”