16S rRNA Mutations That Confer Tetracycline Resistance inHelicobacter pyloriDecrease Drug Binding inEscherichia coliRibosomes

Abstract

Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors

“…To relax the selective pressure on ribosome function in the presence of the U1052G mutation, a randomized plasmid library of positions 965-967 was subsequently constructed. Equivalent libraries have been used to isolate mutants with tetracycline resistance (24). Using this approach, multiple tetracycline-resistant mutants with sequence variations at positions 965-967 were identified.…”

Mechanisms ofHelicobacter pyloriantibiotic resistance: An updated appraisal

“…the crucial part of primary acceptor site (site P) is recognized as the major mechanism of tetracy cline resistance. The main point mutation is a substitution of an AGA with a TTC triplet [59,60] and it reduces the affinity of 24%-52% [61] . Levels of resistance are proportional to the number of changes in the AGA 965-967.…”

“…Purine-rich sequences in the loop of helix 31 are more frequently observed in susceptible strains, whilst pyrimidine-rich loops are in the resistant strains. It is possible that pyrimidine-rich sequences in helix 31 are not compatible with tetracycline conformation, leading to a decreasing affinity [60] . Another study found a deletion of G942 in all resistant strains.…”

rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines

“…These mutations have been shown to occur in the tetracycline highaffinity binding sites on the ribosomal 30S subunit (which are often base-paired regions) and are believed to lead to conformational changes that hamper base pairing and affect drug binding (10,23,58). They have also been shown to decrease drug binding in direct proportion to the number of base mutations (63,64). Hence, all the resistance mechanisms against the tetracyclines appear to involve decreased drug binding in some way and indicate the involvement of these binding sites in the mechanism of action of the tetracyclines.…”