Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis

Abstract

The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the -10 and -35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20-180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 microg/mL < minimum inhibitory concentration (MIC) <or=40 microg/mL] but not to amikacin (MIC <4 microg/mL). Additionally, 80% of clinical isolates examined in this study that exhibited low-level kanamycin resistance harbored eis promoter mutations. These results have important clinical implications in that clinical isolates determined to be resistant to kanamycin may not be cross-resistant to amikacin, as is often assumed. Molecular detection of eis mutations should distinguish strains resistant to kanamycin and those resistant to kanamycin and amikacin. This may help avoid excluding a potentially effective drug from a treatment regimen for drug-resistant TB.

Fig. 1.

Characterization of eis promoter and expression. (A) eis promoter sequence and predicted promoter elements in M. tuberculosis. Mutations identified in clinical isolates are denoted by arrows. The mutation identified in the K204 eis promoter region is noted by the asterisk. The eis transcription start site is denoted by a bent arrow. The −10 and −35 regions are underlined, and the start codon is boxed. The extended −10 region (TGn motif) is located directly upstream of the −10 region. (B) Immunoblot analysis of cell lysates generated from strains harboring either WT (H37Rv, K214, K215) or the C-14T eis allele (K204, K315, K317). The lysates were probed with either anti-Eis or anti-Groel2 serum. (C) RNA from H37Rv (lanes 1–4) and K204 (lanes 5–8) was used in 1-step RT-PCR reactions. Odd numbered lanes contain primer pair AZ198 (anneals at −20 to −1 of the eis start codon) and AZ87; even lanes contain primers AZ199 (anneals at +1 to +20) and AZ87. H37Rv genomic DNA (lanes 9 and 10) was used as a positive control for each primer set. The + and − signs indicate whether reverse transcriptase was added to the reaction and serve as negative controls for each primer set and RNA sample.

Fig. 2.

Analysis of eis expression and acetyltransferase activity. The ratio of eis/sigA transcripts (A–D) was determined by qRT-PCR and normalized to H37Rv. (A) H37Rv, K204, and allelic exchange derivatives. (B) H37RvΔeis complemented with eis promoter truncation constructs; bp (base pairs) indicates the length of sequence upstream from the eis start codon present on the complementing plasmid. (C) H37RvΔeis complemented with eis promoter alleles. (D) Clinical isolates. Acetyltransferase activity (E–H) was measured from crude cell lysates and is expressed in nmol per mg per min. (E) H37Rv, K204, and allelic exchange derivatives. (F) H37RvΔeis complemented with eis promoter truncation constructs. (G) H37RvΔeis complemented with different eis promoter alleles (H) Clinical isolates. In all panels, error bars represent 3 independent experiments.

Fig. 3.

Eis acetylates KAN and AMK. (A) Acetylation rates for KAN, AMK, and SM. (B) Michaelis-Menton plots for KAN and AMK. Acetyltransferase reactions were performed using either (C) KAN or (D) AMK as substrate, and the products were developed on TLC plates. Lane 1: unmodified antibiotic; lane 2: no acetyl-CoA control; lane 3: complete acetyltransferase reaction; lane 4: no Eis control. R_f values under these solvent conditions: unmodified KAN, 0.31; acetyl-KAN, 0.44; unmodified AMK, 0.09; acetyl-AMK, 0.46.