Hemolytic and sphingomyelinase activities of Clostridium perfringens alpha-toxin are dependent on a domain homologous to that of an enzyme from the human arachidonic acid pathway

Abstract

The N-terminal domain of Clostridium perfringens alpha-toxin, homologous with the nontoxic phospholipase C of BaciUus cereus, was expressed in Escherichia coli and shown to retain all of the phosphatidylcholine hydrolyzing activity of the alpha-toxin, but not the sphingomyelinase, hemolytic, or lethal activities. The C-terminal domain of alpha-toxin showed sequence and predicted structural homologies with the N-terminal region of arachidonate 5-lipoxygenase, an enzyme from the human arachidonic acid pathway which plays a role in inflammatory and cardiovascular diseases in humans.

The carboxy‐terminal C2‐like domain of the α‐toxin from Clostridium perfringens mediates calcium‐dependent membrane recognition

“…This was especially intriguing as phospholipases C are involved in the generation of arachidonic acid, which is converted into leukotrienes by arachidonate 5-lipoxygenase. Most prominent among the conserved amino acid residues were four tyrosines, and Titball has suggested that these could play an important role in recognizing substrate hydrocarbon chains (Titball et al, 1991;Titball, 1993). This is consistent with the finding that chemical modification of the tyrosine residues in ␣-toxin abolished its haemolytic and lethal activities as well as platelet aggregation (Sakurai et al, 1989).…”

“…This region of ␣-toxin contains seven tyrosine residues, three of which are conserved in the C. bifermentans enzyme, four in C. novyi and four in lipoxygenase. Originally, five tyrosine residues were thought to be conserved in both lipoxygenase and ␣-toxin (Titball et al, 1991) but, from the refined alignment of clostridial phospholipases, it is now clear that there are only four ( Fig. 1).…”

“…It clearly potentiates all enzymatic activities as recombinant derivatives of ␣-toxin lacking the C-terminal domain, and thus corresponding to PC-PLC, or ␣-toxin fusion proteins in which this domain has been replaced by the reporter enzymes, alkaline phosphatase or ␤-lactamase, showed negligible activities and, where tested, were non-toxic (I. Guillouard et al unpublished results;Titball et al, 1991). Immunization of mice with a recombinant version of the C-terminal segment resulted in full protection against challenge with a lethal dose of native ␣-toxin indicating that it probably contains protective epitopes .…”

“…A possible clue to the function of the C-terminal domain was provided by sequence comparisons, since significant similarity was detected with residues 1-113 of the large lipid-modifying enzyme, arachidonate 5-lipoxygenase from humans (Titball et al, 1991), as 29% of the residues in this segment were identical to those found in ␣-toxin ( Fig. 1).…”

Highly efficient extracellular expression of naturally cytoplasmic Leuconostoc mesenteroides sucrose phosphorylase

“…Three bacterial PLCs capable of hydrolyzing the major phospholipid of the E. coli cell membrane, phosphatidylethanolamine, 24 were selected for co-expression studies: the PLCs from B. cereus, L. monocytogenes and C. perfringens. [21][22][23] They were expressed in E. coli BL21(DE3), and their cell localization was investigated, except for that of B. cereus PLC, which was investigated in a previous study. 12 After L. monocytogenes PLC expression was induced in E. coli BL21(DE3) for 24 h, the extracellular and intracellular L. monocytogenes PLC activities were 7.5 U mL −1 and 0.3 U mL −1 , respectively.…”