The Natural Ketolides Methymycin and Pikromycin: Binding Sites, Modes of Action, Mechanisms of Resistance
thesisposted on 2015-10-21, 00:00 authored by Mashal M. Almutairi
Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, due in part to the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide, pikromycin and a related compound methymycin, are produced by Streptomyces venezuelae strain ATCC 15439. The producer avoids the inhibitory effects of its own antibiotics by expressing two paralogous genes pikR1 and pikR2 with seemingly redundant functions. We show here that the PikR1 and PikR2 enzymes mono- and dimethylate the N6-amino group in 23S rRNA nucleotide A2058, respectively. PikR1 monomethylase is constitutively expressed; it confers low resistance at low fitness cost and is required for ketolide-induced activation of pikR2 to attain high level of resistance. The regulatory mechanism controlling pikR2 expression has been evolutionary optimized for preferential activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism. We studied the binding site and mode of action of methymycin and pikromycin. We found that they both target the same ribosomal site located within the nascent chain exit tunnel which also the target site of all macrolide and ketolide antibiotics. Interestingly, our biochemical experiments revealed that methymycin establishes idiosyncratic interactions with rRNA nucleotides of the PTC not characteristic of other macrolides, including pikromycin. The differential binding modes of methymycin and pikromycin to the nascent chain exit tunnel may be one reason for the ability of these compounds to inhibit the translation of somewhat distinct subsets of cellular proteins. Furthermore, we observed that methymycin inhibits the growth of a narrower spectrum of bacterial species in comparison with pikromycin, a feature that probably benefits the producer by allowing it to adjust the type of antibiotic it produces to specifically target competitor bacteria according to its needs.