A virulence-associated small RNA MTS1338 activates an ABC transporter CydC for rifampicin efflux in Mycobacterium tuberculosis

Abstract

The efficacy of the tuberculosis treatment is restricted by innate drug resistance of Mycobacterial tuberculosis and its ability to acquire resistance to all anti-tuberculosis drugs in clinical use. A profound understanding of bacterial ploys that decrease the effectiveness of drugs would identify new mechanisms for drug resistance, which would subsequently lead to the development of more potent TB therapies. In the current study, we identified a virulence-associated small RNA (sRNA) MTS1338-driven drug efflux mechanism in M. tuberculosis. The treatment of a frontline antitubercular drug rifampicin upregulated MTS1338 by >4-fold. Higher intrabacterial abundance of MTS1338 increased the growth rate of cells in rifampicin-treated conditions. This fact was attributed by the upregulation of an efflux protein CydC by MTS1338. Gel-shift assay identified a stable interaction of MTS1338 with the coding region of cydC mRNA thereby potentially stabilizing it at the posttranscriptional level. The drug efflux measurement assays revealed that cells with higher MTS1338 abundance accumulate less drug in the cells. This study identified a new regulatory mechanism of drug efflux controlled by an infection-induced sRNA in M. tuberculosis.

Keywords: antimicrobial resistance; drug efflux; efflux protein; gene regulation; small RNAs.

Figure 1

Effect of rifampicin (A) treatment on the intracellular abundance of MTS1338 in Mycobacterium tuberculosis cells. Mid-exponentially grown cells (OD₆₀₀–0.9) were exposed to rifampicin (2 μg/mL) and then were harvested. Total RNA was isolated and MTS1338 abundance was estimated by RT-qPCR analysis. 5S gene was taken as an internal control. The relative amounts of the transcripts are presented as the average ± standard deviation (SD) from three separate experiments and normalized to the level of the untreated sample. One-way ANOVA was conducted and a difference of *p < 0.05 was considered statistically significant. (B) Total RNA (15 μg) isolated from the cells treated with/without rifampicin (2 μg/mL) were separated in a 2% agarose formaldehyde gel and analyzed by northern blot. The number under each lane represents the relative amount of MTS1338 when compared to untreated sample, which was set as 1. 5S rRNA gene was taken as a loading control. Northern blots were carried out three times and one representative blot is presented. (C) Northern blot experiments were carried out three times with 5′-Cy5 labeled DNA oligonucleotide probes for RNAs. Bands were quantitated by ImageJ. The amount of MTS1338 and 5S RNA in the untreated cell was set at 1.

Figure 2

A higher MTS1338 abundance increases the growth rate in the presence of rifampicin. (A) Tetracycline (1 μg/mL) was added to the exponentially grown M. tuberculosis cells (OD₆₀₀ = 0.9) harboring empty or recombinant pMTS1338 plasmids. Cells continued to grow for 72 h and the total RNA was extracted from each sample. MTS1338 abundance was estimated by RT-qPCR analysis. 5S gene was taken as an internal control. The relative amounts of the transcripts are presented as the average ± SD from three separate experiments and normalized to the level of the untreated sample. One-way ANOVA was conducted and a difference of *p < 0.05 was considered statistically significant. (B) Northern blot was carried out using 15 μg of total RNA isolated from the cells with/without overexpressing MTS1338. 5S RNA gene was taken as loading control. Northern blot experiments were carried out three times and identical results were obtained. A representative of Northern blots was presented. The number under overexpressed lane represents the relative amount of RNA when compared with control lane, which was set 1.0. (C) Bands of three independent northern blots were quantitated by ImageJ. Data were presented as the average ± SD. The amount of MTS1338 and 5S RNA in the untreated cell was set at 1. (D) H37Ra cells harboring recombinant pMTS1338 plasmid or empty vector were grown at 37°C. OD₆₀₀ was monitored in regular interval and was plotted against time. The data presented was the average ± S.D. of three independent experiments. Growth experiments were also carried out in presence of rifampicin (5 μg/mL) added at the day 1 (E).

Figure 3

(A) Relative expression of cydC mRNA was estimated by RT-qPCR analysis in the exponentially grown M. tuberculosis cells (OD₆₀₀ = 0.9) harboring control or recombinant pMTS1338 vectors. 5S gene was taken as an internal control. The relative amounts of cydC mRNA are presented as the average ± standard deviation from three separate experiments and normalized to the level of the untreated sample. One-way ANOVA was conducted and a difference of *p < 0.05 was considered statistically significant. (B) Northern blot was carried out using 5’-Cy5-labeled DNA probe to detect cydC mRNA present in the total RNA isolated from the cells with/without overexpressing MTS1338. 5S was taken as a loading control. A representative of three independent northern blots with identical results was presented. The number under each lane represents the relative amount of cydC when compared with control. (C) Bands of three independent northern blots were quantitated by ImageJ. Data were presented as the average ± SD. The amount of cydC and 5S RNA in the control cell was set at 1.

Figure 4

Accumulation of cydC in M. tuberculosis while treated with rifampicin (2 μg/mL). (A) Mid-log phase H37Ra cells (OD₆₀₀ = 0.9) were exposed to antibiotics for 30 min at 37°C. Relative amount of cydC in the total RNA was measured by RT-qPCR analysis. 5S gene was taken as an internal control. The relative amounts of cydC mRNA are presented as the average ± standard deviation from three separate experiments and normalized to the level of the untreated sample. One-way ANOVA was conducted and a difference of *p < 0.05 was considered statistically significant. (B) Total RNA (15 μg) from rifampicin treated and untreated samples were separated in in a 2% agarose formaldehyde gel and analyzed by northern blot. The number under each lane represents the relative amount of cydC when compared to untreated sample, which was set as 1. 5S RNA gene was taken as a loading control. Northern blots were carried out three times and one representative blot is presented. (C) Northern blot experiments were carried out three times with 5′-Cy5 labeled DNA oligonucleotide probes for RNAs. Bands were quantitated by ImageJ. The amount of MTS1338 and 5S RNA in the untreated cell was set at 1.

Figure 5

(A) Base pairing region between MTS1338 and cydC mRNA fragment as predicted by IntaRNA program. The numbering of nucleotides on MTS1338 and cydC mRNA is relative to the transcription start site of each RNA. (B,C) Interaction between MTS1338 and cydC mRNA (B) and katG (C). Fragment of cydC and katG mRNA (10 pmol) was incubated with 10, 20 and 30 pmol of MTS1338 (lanes 4, 5, and 6 respectively) in a 20 μL reaction. Numbers at the bottom of the gels represent the lanes (C).

Figure 6

Stability of cydC mRNA with/without MTS1338 overexpression. Cells harboring either empty or pMTS1338 plasmids were grown to exponential phase (OD₆₀₀–0.9) and then tetracycline (1 μg/mL) was added to cells. Cells were further grown for 72 h and were treated with 500 mg/liter rifampicin. Samples were withdrawn at the indicated time points and the amount of cydC mRNA was determined by RT-qPCR analysis.

Figure 7

(A) Accumulation of EtBr in M. tuberculosis cells with/without MTS1338 overexpression. Cells (OD₆₀₀ = 0.9) containing empty or recombinant pMTS1338 plasmids were allowed to grow in presence of tetracycline (1 μg/mL) for 72 h followed by the addition of EtBr (1 μg/mL). After 10 min CCCP (6.25 μM) was added to the culture. Cells were then pelleted down and resuspended in PBS containing CCCP. Intracellular EtBr fluorescence was measured by fluorimetry. Data was presented as the average ± SD from three separate experiments. One-way ANOVA was conducted and a difference of *p < 0.05 was considered statistically significant. (B) After the treatment with EtBr and CCCP, cells were resuspended in PBS and intracellular accumulation of EtBr was measured by fluorimetry at indicated time points. Data was presented as the average ± SD from three separate experiments.