Abstract
Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.
Highlights
A group of Gram-positive microorganisms, including Clostridium botulinum, C. difficile, C. perfringens, C. spiroforme, and Bacillus cereus, produces actin-ADP-ribosylating “binary toxins”
Budding yeast S. cerevisiae is a single-cell eukaryotic organism, which is used for studies on molecular mechanisms of bacterial virulence factors [20]
The only actin-coding gene ACT1 of S. cerevisiae is located on chromosome VI and is translated into a protein, which is ~87–90% identical to human actin isoforms
Summary
A group of Gram-positive microorganisms, including Clostridium botulinum, C. difficile, C. perfringens, C. spiroforme, and Bacillus cereus, produces actin-ADP-ribosylating “binary toxins”. These toxins consist of a binding component, which is involved in toxin up-take, and an enzymatically active component, which harbors ADP-ribosyltransferase activity. Both components are secreted as separated proteins [1,2]. The binding component of binary toxins is proteolytically activated, forms heptamers, and binds to membrane receptors of eukaryotic target cells. In an acidic endosomal compartment, the toxin heptamer inserts into membranes and forms a pore, which allows the translocation of the enzyme component into the cytosol
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