Abstract
Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.
Highlights
Microbial type II toxin-antitoxin (TA) systems, which are stress regulators, are generally composed of two genes organized in an operon, encoding a toxin (T) and an antitoxin (A) protein [1,2,3]
It is likely that: (I) toxin expression enhances Fos sensitivity, and such effect does not seem to correlate with increased rate of cell lysis; (II) neither toxin expression nor Fos addition deplete the uridine diphosphate-N-acetylglucosamine (UNAG) pool, because ε2 antitoxin expression facilitates the exit of the dormant state of a large fraction of cells; and (III) unregulated levels of murein synthesis, by transient toxin expression and Fos addition, do not induce massive cell autolysis
We propose that δ toxin and Fos decrease the UNAG pool, but when ε2 antitoxin expression halts δ toxin activity, by forming an inactive complex, the remaining active fraction of UNAG is processed by MurA enzymes and cells exit the δ-induced dormant state with resumption of growth as expected for a toxin that is bacteriostatic in nature
Summary
Microbial type II toxin-antitoxin (TA) systems, which are stress regulators, are generally composed of two genes organized in an operon, encoding a toxin (T) and an antitoxin (A) protein [1,2,3]. The toxins of the δ family are highly conserved, δ (286 amino acids long polypeptide) shares 42% of sequence identity with PezT (253 amino acids long). Despite this low sequence identity, the C-terminal region of PezA folds into a three helix bundle similar to that of ε [7,8] Both Firmicutes toxins, δ and PezT might interact with ATP-Mg2+ or GTP-Mg2+, with uridine diphosphate-N-acetylglucosamine (UNAG) and with ε2 (PezA) antitoxin [7,9]. These toxins, as part of the biological non-toxic heterotetrameric (δε2δ or PezTPezA2PezT) complex, interact with UNAG [9]. It is likely that toxin concentrations or time of exposure shows hormesis, complicating the understanding of δ toxin mode of action
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