Large Clostridial Toxins: Mechanisms and Roles in Disease.

Large clostridial toxins (LCTs) are a family of bacterial exotoxins that infiltrate and destroy target cells. Members of the LCT family include Clostridioides difficile toxins TcdA and TcdB, Paeniclostridium sordellii toxins TcsL and TcsH, Clostridium novyi toxin TcnA, and Clostridium perfringens toxin TpeL. Since the 19th century, LCT-secreting bacteria have been isolated from the blood, organs, and wounds of diseased individuals, and LCTs have been implicated as the primary virulence factors in a variety of infections, including C. difficile infection and some cases of wound-associated gas gangrene. Clostridia express and secrete LCTs in response to various physiological signals. LCTs invade host cells by binding specific cell surface receptors, ultimately leading to internalization into acidified vesicles. Acidic pH promotes conformational changes within LCTs, which culminates in translocation of the N-terminal glycosyltransferase and cysteine protease domain across the endosomal membrane and into the cytosol, leading first to cytopathic effects and later to cytotoxic effects. The focus of this review is on the role of LCTs in infection and disease, the mechanism of LCT intoxication, with emphasis on recent structural work and toxin subtyping analysis, and the genomic discovery and characterization of LCT homologues. We provide a comprehensive review of these topics and offer our perspective on emerging questions and future research directions for this enigmatic family of toxins.

Genome-Wide CRISPR Screen Identifies Semaphorin 6A and 6B as Receptors for Paeniclostridium sordellii Toxin TcsL.

The exotoxin TcsL is a major virulence factor in Paeniclostridium (Clostridium) sordellii and responsible for the high lethality rate associated with P. sordellii infection. Here, we present a genome-wide CRISPR-Cas9-mediated screen using a human lung carcinoma cell line and identify semaphorin (SEMA) 6A and 6B as receptors for TcsL. Disrupting SEMA6A/6B expression in several distinct human cell lines and primary human endothelial cells results in reduced TcsL sensitivity, while SEMA6A/6B over-expression increases their sensitivity. TcsL recognizes the extracellular domain (ECD) of SEMA6A/6B via a region homologous to the receptor-binding site in Clostridioides difficile toxin B (TcdB), which binds the human receptor Frizzled. Exchanging the receptor-binding interfaces between TcsL and TcdB switches their receptor-binding specificity. Finally, administration of SEMA6A-ECD proteins protects human cells from TcsL toxicity and reduces TcsL-induced damage to lung tissues and the lethality rate in mice. These findings establish SEMA6A and 6B as pathophysiologically relevant receptors for TcsL.

Expression of the large clostridial toxins is controlled by conserved regulatory mechanisms.

The clostridia cause many human and animal diseases, resulting in significant morbidity and mortality. Host damage results from the action of potent exotoxins, an important group of which is the large clostridial toxins (LCTs) produced by Clostridium difficile, Clostridium sordellii, Clostridium perfringens and Clostridium novyi. Knowledge of the structure and function of these toxins has been attained, however, apart from C. difficile, the regulatory pathways that control LCT production remain largely unknown. Here we show that LCT production in C. sordellii and C. perfringens is temporally regulated and repressed by glucose in a similar manner to C. difficile. Furthermore, we show that the TpeL-encoding gene of C. perfringens is located in an uncharacterized Pathogenicity Locus (PaLoc), along with accessory genes predicted to encode a bacteriophage holin-type protein and a TcdR-family alternative sigma factor, TpeR. Inactivation of tpeR demonstrated that TpeR is critical for C. perfringens TpeL production, in a similar manner to C. difficile TcdR and C. sordellii TcsR, but cross-complementation showed that TpeR is not functionally interchangeable with TcdR or TcsR. Although conserved mechanisms are employed by the clostridia to control LCT production there are important functional differences that distinguish members of the TcdR-family of clostridial alternative sigma factors.