The production and secretion of antibiotics creates a protective environment for the producing microorganism, and an inhospitable environment for invading microorganisms — a microbial form of chemical warfare. But how do the producers avoid committing suicide? One answer to this question has recently been provided by a team at the University of Wisconsin. Reporting in Science, Jon Thorson and colleagues have elucidated the mechanism by which actinomycetes are self-resistant to the antibiotic calicheamicin γ1 — an enediyne that acts as a DNA-cleaving agent — in which the CalC protein is sacrificed.
It had previously been shown that the calC gene is required to confer resistance to calicheamicin γ1, and that CalC is involved in inhibition of calicheamicin γ1-induced DNA scission. However, the mechanism by which this resistance is conferred was unknown.
Using a molecular break-light assay to monitor enediyne-induced DNA scission, the authors established that CalC is specific for calicheamicin γ1; it does not inhibit the action of all enediynes and it does not simply prevent the cleavage of DNA. Furthermore, tryptophan fluorescence in the presence of dithiothreitol was used to detect the calicheamicin γ1–CalC interaction, and susbsequent SDS–PAGE analysis showed that, on binding, the CalC protein is cleaved at Gly113, generating two peptides.
The authors postulated that this self-resistance mechanism proceeds by hydrogen abstraction at Gly113 of CalC, and the results of site-directed mutants of Gly113 and liquid chromatography–mass spectroscopy analysis confirm this view. In the absence of CalC, the active form of calicheamicin γ1 removes hydrogen from both the sense and antisense strands of double-stranded DNA. However, the presence of CalC provides an alternative and competing substrate, and therefore mechanism, for this active form of calicheamicin γ1, which removes a hydrogen from Gly113 of CalC to generate a hydrogen backbone C-α radical species.
Like other self-resistance mechanisms, the CalC self-sacrifice mechanism could be common among bacteria, and its elucidation could aid in the battle against multi-drug resistance pathogenic microorganisms.
ORIGINAL RESEARCH PAPERS
Biggins, J. B., Onwueme, K. C. & Thorson J. S. Resistance to enediyne antitumor antibiotics by CalC self-sacrifice. Science 301, 1537–1541 (2003).
Whitwam, R. E. et al. The gene calC encodes for a non-heme iron metalloprotein responsible for calicheamicin self-resistance in Micromonospora. J. Am. Chem. Soc. 122, 1556–1557 (2000).
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Saunders, J. Chemical warfare. Nat Rev Microbiol 1, 88 (2003). https://doi.org/10.1038/nrmicro768