The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation.

Carolina Varela Chavez,Serge Pauillac,Bruno Baron,Patrick England,Alexandre Chenal,Georges Haustant,Arnaud Blondel,Michel-Robert Popoff

doi:10.3390/toxins8040090

Carolina Varela Chavez, Serge Pauillac + Show 6 more

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https://doi.org/10.3390/toxins8040090

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Journal: Toxins	Publication Date: Mar 25, 2016
Citations: 15	License type: CC BY 4.0

Affiliation: Institut Pasteur

Abstract

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.

Highlights

Clostridium sordellii is responsible for severe traumatic infections, as well non-traumatic myonecrosis and toxic shock in man and animals
Hydrophobicity analysis revealed residues on the the four-helix bundle by hydrophobic revealed residues on the tip of the four‐helix bundle surrounded by hydrophobic amino acids positively‐charged which extend along the helices
The effects of the mutation on cell viability may involve subtle impact on the TcsL-cat orientation that is still to be explored. These results show that the loops 1 and 3 on the tip of the N-terminal four-helix bundle of TcsL, which are rich in positively-charged residues, constitute a critical binding site for PS, and that binding to membranes via PS is required to direct the full enzymatic activity of

Summary

Introduction

Clostridium sordellii is responsible for severe traumatic infections, as well non-traumatic myonecrosis and toxic shock in man and animals. C. sordellii infections sporadically occur in humans, they are rapidly dramatic. C. sordellii infection of the genital tract in women results. Toxins 2016, 8, 90 in a fulminant toxic shock syndrome [1,2,3,4]. C. sordellii causes rapidly fatal enterotoxemia in livestock. C. sordellii lethal toxin (TcsL) is the major virulence factor of this pathogen. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which encompasses Clostridium difficile toxin A (TcdA) and toxin B (TcdB), Clostridium novyi alpha toxin (TcnA), and Clostridium perfringens large cytotoxin (TpeL) [5,6,7]. Some C. sordellii strains produce the hemorrhagic toxin (TcsH) in addition to

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