Nonmutational compensation of the fitness cost of antibiotic resistance in mycobacteria by overexpression of tlyA rRNA methylase

Abstract

Several studies over the last few decades have shown that antibiotic resistance mechanisms frequently confer a fitness cost and that these costs can be genetically ameliorated by intra- or extragenic second-site mutations, often without loss of resistance. Another, much less studied potential mechanism by which the fitness cost of antibiotic resistance could be reduced is via a regulatory response where the deleterious effect of the resistance mechanism is lowered by a physiological alteration that buffers the mutational effect. In mycobacteria, resistance to the clinically used tuberactinomycin antibiotic capreomycin involves loss-of-function mutations in rRNA methylase TlyA or point mutations in 16S rRNA (in particular the A1408G mutation). Both of these alterations result in resistance by reducing drug binding to the ribosome. Here we show that alterations of tlyA gene expression affect both antibiotic drug susceptibility and fitness cost of drug resistance. In particular, we demonstrate that the common resistance mutation A1408G is accompanied by a physiological change that involves increased expression of the tlyA gene. This gene encodes an enzyme that methylates neighboring 16S rRNA position C1409, and as a result of increased TlyA expression the fitness cost of the A1408G mutation is significantly reduced. Our findings suggest that in mycobacteria, a nonmutational mechanism (i.e., gene regulatory) can restore fitness to genetically resistant bacteria. Our results also point to a new and clinically relevant treatment strategy to combat evolution of resistance in multidrug-resistant tuberculosis. Thus, by utilizing antagonistic antibiotic interactions, resistance evolution could be reduced.

Keywords: Mycobacterium tuberculosis; antibiotic resistance; compensation; fitness; rRNA methylation; tlyA.

FIGURE 1.

Capreomycin affects post-transcriptional methylation in 16S rRNA and 23S rRNA. Primer extension analysis of Cm1409*, helix 44 (A) and Cm1920*, helix 69 (B) in the rRNA of M. smegmatis wild type and M. smegmatis ΔtlyA mutant. The strains were grown in liquid culture for 20 h. Wild type strains were grown without drug and in the presence of 0.5 and 1 µg/mL capreomycin. The size marker indicates positions from A1408 to C1412 and A1919 to C1925, respectively. Densitometric analysis of position Cm1409 (C) and Cm1920 (D).

FIGURE 2.

tlyA mRNA levels measured by quantitative PCR. (A) TlyA mRNA levels in M. smegmatis; wild type (white bar) and A1408G (gray bar). 16S rRNA (left) and EF-Tu (right) were used as internal controls. The average expression levels of three wild-type strains and three A1408G strains are given, SDs are indicated, P < 0.01. (B) TlyA mRNA expression in laboratory strain M. tuberculosis H37Rv ATCC27294. Averaged expression levels of triplicate measurements (three wild type clones, five A1408G mutants), SDs are indicated, P < 0.01. (C) TlyA mRNA expression in M. tuberculosis wild-type versus A1408G clinical isolates. Averaged expression levels of triplicate measurements (six clinical isolates of each M. tuberculosis wild type and M. tuberculosis A1408G). Mean and SD are given, P < 0.001. (Inset) TlyA mRNA expression at the level of individual clinical isolates. Mean and SD are given, P < 0.01.

FIGURE 3.

Generation times. M. smegmatis wild type; ΔtlyA mutant; A1408G mutant; and ΔtlyA, A1408G double mutant. Average generation times and SDs are indicated. The average generation times were as follows: wild type 2.99 ± 0.01, ΔtlyA 3.01 ± 0.07, A1408G 3.14 ± 0.05, ΔtlyA, A1408G 3.32 ± 0.06. Each strain was measured in triplicate; P-values are indicated.