Aminoglycoside cross-resistance in Mycobacterium tuberculosis due to mutations in the 5' untranslated region of whiB7

Abstract

Since the discovery of streptomycin's bactericidal activity against Mycobacterium tuberculosis, aminoglycosides have been utilized to treat tuberculosis (TB). Today, the aminoglycosides kanamycin and amikacin are used to treat multidrug-resistant (MDR) TB, and resistance to any of the second-line injectable antibiotics, including kanamycin, amikacin, or capreomycin, is a defining characteristic of extensively drug-resistant (XDR) TB. Resistance to kanamycin and streptomycin is thought to be due to the acquisition of unlinked chromosomal mutations. However, we identified eight independent mutations in the 5' untranslated region of the transcriptional activator whiB7 that confer low-level resistance to both aminoglycosides. The mutations lead to 23- to 145-fold increases in whiB7 transcripts and subsequent increased expression of both eis (Rv2416c) and tap (Rv1258c). Increased expression of eis confers kanamycin resistance in these mutants, while increased expression of tap, which encodes an efflux pump, is a previously uncharacterized mechanism of low-level streptomycin resistance. Additionally, high-level resistance to streptomycin arose at a much higher frequency in whiB7 mutants than in a wild-type (WT) strain. Although whiB7 is typically associated with intrinsic antibiotic resistance in M. tuberculosis, these data suggest that mutations in an uncharacterized regulatory region of whiB7 contribute to cross-resistance against clinically used second-line antibiotics. As drug resistance continues to develop and spread, understanding the mechanisms and molecular basis of antibiotic resistance is critical for the development of rapid molecular tests to diagnose drug-resistant TB strains and ultimately for designing regimens to treat drug-resistant cases of TB.

Fig 1

5′ untranslated region of whiB7 and mutations. The locations of mutations identified in spontaneous mutants are denoted by arrowheads; deletions by solid arrowheads, insertions by an open arrowhead, and transversions by gray arrowheads. The whiB7 start codon is boxed. A predicted IdeR binding region is underlined, and an inverted repeat element is denoted by arrows.

Fig 2

Mutations confer increased expression of whiB7, eis, and tap. The ratio of gene transcripts to sigA (A and B) was determined by qRT-PCR and normalized to H37Rv. Error bars represent the standard errors of the mean (SEM) from at least 3 experiments. (A) whiB7 mutants and parental strains. The mutation harbored in each strain is listed in Table 1. (B) Gene expression from one representative strain of each type of whiB7 mutation and the corresponding allelic exchange strain reverted to the wild-type sequence: deletion strain K301 (ΔC+133), insertion strain K205 (+C+133), and transversion strain K209 (A+237G). Parental strains CDC1551 and H37Rv are included for comparison. (C and D) Acetyltransferase activity due to Eis was measured from crude cell lysates and is expressed in nmol/mg/min. (C) Mutants and parental strains; (D) whiB7 mutants and representative allelic exchange strain. (E and F) Immunoblot analysis of cell lysates from mutants and parental strains (E) and representative allelic exchange isolates (F). Lysates were probed with either anti-Eis (αEis) or anti-GroES (αGroES) serum.

Fig 3

Antibiotic resistance patterns of CDC1551 deletion strains. M. tuberculosis strains were streaked on 7H10 agar containing either 5 μg/ml KAN (A) or 2 μg/ml STR (B) and incubated for 28 days at 37°C.

Fig 4

eis and tap expression in a whiB7 knockout and acetyltransferase activity in Kan^r clinical isolates. (A) The relative expression of gene transcripts was determined by qRT-PCR and normalized to sigA in H37Rv. Error bars represent the SEM from at least 3 experiments. (B) Immunoblot analysis of cell lysates. Twenty micrograms of cell lysate was probed with anti-Eis antibodies to ensure that basal-level Eis expression could be detected. (C) Sixteen clinical isolates with unexplained KAN resistance were analyzed by sequencing the eis, rrs, and whiB7 loci. With the exception of isolate CL-27 (which harbors a +C+133 whiB7 mutation), all of the loci were wild type. Acetyltransferase activity due to Eis was measured from crude cell lysates from 12 of the isolates and is expressed in nmol/mg/min. However, the remaining four clinical isolates grew very poorly in 7H9 medium and did not reach an OD₆₀₀ sufficient to perform the acetyltransferase assays. Isolates CL-27, MLB100, and MLB126 displayed low-level cross-resistance to KAN and STR. H37Rv is a wild-type laboratory strain, and K204 harbors an eis C−14T mutation.