Date Published: February 15, 2017
Publisher: Public Library of Science
Author(s): Waraphan Toniti, Toru Yoshida, Toshiharu Tsurumura, Daisuke Irikura, Chie Monma, Yoichi Kamata, Hideaki Tsuge, Michel R. Popoff.
Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia–actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and β/γ-actin.
Human food-borne gastroenteritis caused by the Clostridium perfringens enterotoxin (CPE) has been widely studied. C. perfringens is a Gram-positive, rod-shaped, anaerobic, spore-forming, pathogenic bacterium of the genus Clostridium that can be classified into five types (A–E) based on the type of toxin secreted. The CPE is known to cause diarrhea in humans and other animals. Most CPE-producing C. perfringens are classified as type A. On the other hand, type E produces alpha and iota toxins and leads to antibiotic–associated enterotoxemia in bovine and ovine species .
Interestingly, CPILE-a showed different sensitivities towards α- and β/γ-actin. Against α-actin, the maximum activity of CPILE-a was the same as that of Ia. However, the maximum activity of CPILE-a towards β/γ-actin was 36% compared with α-actin. Thus, discussion of the role of mutated residues was only based on α-actin results in the next section. In contrast, Ia showed somewhat different sensitivity compared with CPILE-a and the sensitivity towards β/γ-actin is 58% that for α-actin.