Mutations in the Interdomain Loop Region of thetetA(A) Tetracycline Resistance Gene Increase Efflux of Minocycline and Glycylcyclines

Abstract

A novel class of tetracyclines, the glycylcyclines, have been shown to be active against bacterial strains harboring genes encoding tetracycline efflux pumps. However, two veterinary Salmonella isolates that carried tetracycline resistance determinants of the tetA(A) class were found to have reduced susceptibility to glycylcyclines, especially two early investigational glycylcyclines, DMG-MINO and DMG-DMDOT. These isolates were also quite resistant to tetracycline and minocycline. The isolates, one a strain of S. cholerasuis and the other, S. typhimurium, both carried the same novel tetA(A) variant, based on DNA sequencing, with one determinant plasmid encoded and the other located on the chromosome. This tetA(A) variant was cloned and shown to provide reduced susceptibility to the glycylcycline class although GAR-936, a glycylcycline currently in clinical development, was the least affected. The novel tetA(A) gene carries two mutations in the largest cytoplasmic loop of the efflux pump, which causes a double frameshift in codons 201, 202, and 203. This "interdomain region" of the efflux pump has generally been regarded as having no functional role in the efflux of tetracycline but the double frameshift is most likely responsible for the enhanced resistance observed and points to an interaction that was previously unrecognized. Mutants of the tetA(B) class with decreased susceptibility to the glycylcyclines were also generated in vitro. These all carried mutations in the portion of the tetA(B) gene encoding a transmembrane spanning region of the efflux pump. The laboratory-generated mutants point to the tight constraints in substrate recognition of the transmembrane-spanning region and may suggest that it will be the interdomain region of the pump that is likely to be the locus of future glycylcycline resistance mutations as these compounds enter clinical use.

Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)

“…These results indicate that tigecycline is not practically recognized by Tet(B) at low concentrations; therefore, tigecycline exhibits potent antibacterial activity, although at high concentrations it would be recognized with a low affinity. Tuckman et al (26) reported that a tet(A) mutation, i.e., a double frameshift, in the interdomain loop region resulted in resistance to glycylcyclines, although tigecycline was the least affected. It might be on April 7, 2019 by guest http://aac.asm.org/ possible that such a mutant with a higher affinity would emerge from a clinical situation in the future.…”

Combined ramR Mutation and Presence of a Tn 1721 -Associated tet (A) Variant in a Clinical Isolate of Salmonella enterica Serovar Hadar Resistant to Tigecycline

“…Molecular analysis for the presence of known tet genes (2) revealed the presence of tet(A). Sequencing of the full open reading frame of the tet(A) gene revealed a previously described (29) double frameshift mutation compared to the RP1-linked tet(A) gene (gene id number gi:42508), leading to the substitution of amino acids 201, 202, and 203 (serine, phenylalanine, and valine to alanine, serine, and phenylalanine, respectively) in the interdomain loop. This variant was found to elevate the MIC for the glycylcycline GAR-936, now termed tigecycline (29).…”

“…Although the exact mechanisms of resistance could not be definitely determined, a common finding in these sporadic isolates as well as in the naturally resistant species is overexpression of different efflux pumps: AcrAB RND-type efflux pumps in Enterobacteriaceae, certain other RND-type efflux pumps in Pseudomonas aeruginosa (MexXY) and Acinetobacter species (AdeABC and AdeIJK), and MATE family efflux pumps in Staphylococcus aureus (7,8,12,13,15,(24)(25)(26)30). Although genes conferring resistance to tetracyclines do not seem to have an effect on susceptibility to tigecycline (9,11), mutants of Tet(A) and Tet(B) with altered substrate specificities have been isolated that demonstrated low-level resistance against an early glycylcycline (10) and tigecycline (29). So far, tigecycline resistance due to Tet(A) or Tet(B) variants has not been described in clinical isolates.…”

Second-site Suppressor Mutations for the Serine 202 to Phenylalanine Substitution within the Interdomain Loop of the Tetracycline Efflux Protein Tet(C)

“…Moreover, some resistance can be obtained by cloning the two domains of the Tet protein separately in the cell without the interdomain loop (18,19). However, previous studies pointed out that a double frameshift mutation in the interdomain loop of TetA modified the substrate specificity of the protein (23) and that local mutations within the loop greatly reduced the Tc resistance phenotype (1,17,22).…”

“…Four mutations, D190C, E192C, S201C, and M210C, located within the interdomain loop, caused greatly reduced tetracycline resistance levels (8 -32-fold) (22). A double frameshift within the interdomain of Tet(A) changed its substrate specificity and increased the efflux of minocycline and glycylcyclines (23). In Tet(C), the interdomain mutation S202F leads to a 12-fold increase in tetracycline susceptibility of the Escherichia coli cells bearing the tet gene on a low copy number plasmid (1).…”