Defining the catalytic domain of toxin A through structural and functional studies of Clostridium difficile toxins A and B

Date

1998-05

Journal Title

Journal ISSN

Volume Title

Publisher

Texas Tech University

Abstract

Clostridium difficile is one of the most frequently encountered pathogens in human enteric diseases. The major virulence factors produced by C. difficile are toxins A and B. These two toxins, as well as the lethal toxin and the hemorrhage toxin of C. sordellii, and the atoxin of C. novyi constitute a family of large clostridial cytotoxins. All these toxins are glucosyltransferases, which use a co-substrate UDPsugar to glucosylate and inactive small GTPases of the Rho or Ras subfamily proteins. The enzymatic activity of these toxins is apparently responsible for their cytotoxicity. However, mechanistic details including the catalytic region(s) of these toxin molecules and the enzymatically essential amino acid residues are unknown. Toxin A was the principal focus of this study, because of its prime clinical relevance in mediating the major tissue damage in C. difficileassociated diseases by enterotoxic and cytotoxic activities. Some additional information from the preliminary study of toxin B is also presented, as it relates to toxin A.

A catalytic and receptor binding, two-domain structure model was proposed for both C. difficile toxins. This study tests the hypothesis that the N-terminal portion of toxin A is responsible for the enzymatic activity by examining a series of toxin A deletion mutant proteins. By correlation of the glucosylation activity with the deleted regions of the toxin mutant proteins, the catalytic domain of toxin A was localized within its first N-terminal 659 amino acids — less than 25% of the length of native toxin A.

Previous studies showed that the cytotoxicity of toxins A and B was inactivated by chemical modification using the arginine-specific 1,2-cyclohexanedione. Therefore, this study hypothesized that the loss of cytotoxicity resulted from the loss of enzymatic activity. Indeed, both toxins and active toxin A mutant derivatives lost enzymatic activity upon 1,2-cyclohexanedione treatment. Importantly, toxin A inactivation by 1,2-cyclohexanedione was prevented in the presence of excess amount of the co-substrate, UDP-glucose, suggesting functional arginine(s) are in or very near the UDP-glucose binding site. Four of 21 candidate arginines within the toxin A enzymatic domain are conserved, based on comparisons with the large clostridial cytotoxins. Therefore, site-directed mutagenesis studies were used to further identify essential arginines. Since, substitution of alanine for arginine 272 or 405 abolished enzymatic activity, the need for the positive charges was then confirmed by lysine replacement, which resulted in lower enzymatic activity for both mutants. However, the double mutant, R272K+R405K, had no detectable enzymatic activity, suggesting disposition of the positive charges is important for enzymatic activity. These results are consistent with an atomic crystal structure of T4 DNA-glucosyltransferase with its co-substrate, UDPglucose, in which positive charges of three arginines interact with negative phosphates of the co-substrate, thus stabilizing it in the catalytic site of the enzyme.

Description

Keywords

Proteins -- Toxicology, Clostridium difficile, Toxins -- Physiological effect, Glucosyltransferases, Arginine -- Physiological effect, Cytotoxins

Citation