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

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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.