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.
Tiamulin 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.
Long
Katherine S
KS
Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark. long@bio.ku.dk
Poehlsgaard
Jacob
J
Hansen
Lykke H
LH
Hobbie
Sven N
SN
Böttger
Erik C
EC
Vester
Birte
B
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D004224:Diterpenes / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D008826:Microbial Sensitivity Tests; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism*; D011083:Polycyclic Compounds; D012329:RNA, Bacterial / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D012407:rRNA Operon; D000097567:Pleuromutilins
D000900:Anti-Bacterial Agents; D004224:Diterpenes; D010458:Peptidyl Transferases; D011083:Polycyclic Compounds; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D000097567:Pleuromutilins; C117845:valnemulin; C014224:tiamulin