Abstract
Clostridium sordellii infections cause gangrene and edema in humans and gastrointestinal infections in livestock. One of the principle virulence factors is TcsL, a large protein toxin which glucosylates host GTPases to cause cytopathic and cytotoxic effects. TcsL has two enzymatic domains, an N-terminal glucosyltransferase domain (GTD) and an autoprocessing domain responsible for release of the GTD within the cell. The GTD can then use its N-terminal membrane localization domain (MLD) for orientation on membranes and modification of GTPases. This study describes the use of conditionally immortalized murine pulmonary microvascular endothelial cells as a model for the study of TcsL functional activities. Point mutations that disrupt the glucosyltransferase, autoprocessing, or membrane localization activities were introduced into a recombinant version of TcsL, and the activities of these mutants were compared to those of wild-type toxin. We observed that all mutants are defective or impaired in cytotoxicity but differ in their modification of Rac1 and Ras. The data suggest a model where differences in GTPase localization dictate cellular responses to intoxication and highlight the importance of autoprocessing in the function of TcsL. IMPORTANCE Clostridium sordellii is a bacterium that can infect humans and cause serious disease and death. The principle virulence factor associated with clinical symptoms is a large protein toxin known as lethal toxin. The mechanism of lethal-toxin intoxication is assumed to be similar to that of the homologous toxins from C.difficile, but very few studies have been done in the context of endothelial cells, a relevant target in C.sordellii infections. This study was designed to test the role of the lethal-toxin enzymatic activities and membrane localization in endothelial cell toxicity and host substrate modification.
Highlights
Clostridium sordellii infections cause gangrene and edema in humans and gastrointestinal infections in livestock
The autoprocessing domain is activated by host inositol hexakisphosphate and cleaves the glucosyltransferase domain (GTD) [15,16,17,18], presumably to permit access to substrates residing at the plasma membrane
Previous studies investigated the different roles of host GTPases in the cellular response to intoxication and showed that glucosylation of H, K, and NRas leads to the apoptosis seen in cells by causing cell cycle arrest [26,27,28]
Summary
Clostridium sordellii infections cause gangrene and edema in humans and gastrointestinal infections in livestock. One of the principle virulence factors is TcsL, a large protein toxin which glucosylates host GTPases to cause cytopathic and cytotoxic effects. The mechanism of lethal-toxin intoxication is assumed to be similar to that of the homologous toxins from C. difficile, but very few studies have been done in the context of endothelial cells, a relevant target in C. sordellii infections. This study was designed to test the role of the lethal-toxin enzymatic activities and membrane localization in endothelial cell toxicity and host substrate modification. Previous studies investigated the different roles of host GTPases in the cellular response to intoxication and showed that glucosylation of H, K, and NRas leads to the apoptosis seen in cells by causing cell cycle arrest [26,27,28]. We introduced mutations into TcsL to inhibit the glucosyltransferase and autoprocessing enzymatic functions and studied the changes these mutations exhibit in lung endothelial cells
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