Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket

Abstract

SummaryTiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

Expression of distinct maternal and somatic 5.8S, 18S, and 28S rRNA types during zebrafish development

“…In the PTC there is just 1 nt difference: U3780C. But even a single difference in such a conserved region is worth noticing, knowing that in the prokaryote PTC-equivalent, individual nucleotides have specific roles in the translation process and some mutations result in a lethal phenotype (Beringer 2008;Polacek and Mankin 2008;Long et al 2009Long et al , 2010Yang et al 2009;Chirkova et al 2010).…”

Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

“…Additional mutations have been investigated in M. smegmatis strains modified to contain a single rRNA operon (31,32,47,57), where one model system allows for the introduction of mutations regardless of whether the mutations confer antibiotic resistance or not (32). This genetic system has been used to test whether rRNA mutations that confer linezolid resistance in other organisms (G2576U) also do so in M. smegmatis and to validate rRNA mutations associated with linezolid resistance in the literature without genetic proof (A2503G, G2505A, C2571G, and C2612A) (56).…”

Mycobacterium tuberculosis: Dormancy, Persistence and Survival in the Light of Protein Synthesis

“…It should also be mentioned that mycobacterial species often harbour only one or two rRNA operons in contrast to seven rRNA operons in E. coli. Thus mycobacteria provide a very good system to test the effect of mutations in the significant positions of rRNA towards the viability of the bacteria (Hobbie et al, 2007;Long et al, 2009). Also, homogeneous populations of the ribosomes carrying mutations in the rRNA could be isolated and characterized relatively easily from the mutated mycobacterial strains.…”