Mycobacterium tuberculosis Rv3160c is a TetR-like transcriptional repressor that regulates expression of the putative oxygenase Rv3161c

Abstract

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a major health threat listed among the top 10 causes of death worldwide. Treatment of multidrug-resistant Mtb requires use of additional second-line drugs that prolong the treatment process and result in higher death rates. Our team previously identified a 2-pyridone molecule (C10) that blocks tolerance to the first-line drug isoniazid at C10 concentrations that do not inhibit bacterial growth. Here, we discovered that the genes rv3160c and rv3161c are highly induced by C10, which led us to investigate them as potential targets. We show that Rv3160c acts as a TetR-like transcriptional repressor binding to a palindromic sequence located in the rv3161c promoter. We also demonstrate that C10 interacts with Rv3160c, inhibiting its binding to DNA. We deleted the rv3161c gene, coding for a putative oxygenase, to investigate its role in drug and stress sensitivity as well as C10 activity. This Δrv3161c strain was more tolerant to isoniazid and lysozyme than wild type Mtb. However, this tolerance could still be blocked by C10, suggesting that C10 functions independently of Rv3161c to influence isoniazid and lysozyme sensitivity.

Figure 1

Expression of the rv3160c-rv3161c operon is significantly induced by C10-IMD. Mtb Erdman WT was cultivated in Sauton’s medium at 37 °C until its OD₆₀₀ reached 0.3. The cells were then exposed to 0.1% DMSO, 0.1 µg/ml INH and 25 µM C10-IMD separately or in combination for 48 h at 37 °C. Differences in expression of the rv3160c-rv3161c operon were determined by qRT-PCR analysis performed by targeting rv3161c coding region. Expression of the sigA gene was determined by qRT-PCR analysis and used as housekeeping control. Relative fold-change in rv3160c-rv3161c operon expression was calculated for each biological replicate by the Livak (2^−ΔΔCt) method. Bar graphs were plotted based on average and standard deviation obtained from three independent biological replicates and statistical analysis comparing to the DMSO control was determined by one-tail t-test that was performed using GraphPad Prism version 8.4.3 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com (**p < 0.01).

Figure 2

Rv3160c acts as transcriptional repressor of the rv3160c-rv3161c operon. (A) Schematic representation of the rv3160c-rv3161c operon and the relative positions of the targets for CRISPRi and qRT-PCR. (B,C) qRT-PCR analysis of rv3160c and rv3161c mRNA expression in Mtb Erdman WT strain harboring either vector control (pJR965) or the rv3160c-CRISPRi (pSA253) construct in absence (−) or presence (+) of 100 ng/ml aTc. sigA mRNA expression was used as control and relative fold change in rv3160c and rv3161c mRNA expression were calculated for each biological replicate by the Livak (2^−ΔΔCt) method. Average value of 3 technical replicates was used for each biological replicate. Bar graphs were plotted based on average and standard deviation obtained from three independent biological replicates and statistical analysis was determined by one-tail t-test that was performed using GraphPad Prism version 8.4.3 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com (*p < 0.05, **p < 0.01).

Figure 3

Binding of Rv3160c to the rv3160c-rv3161c operon is blocked by C10-IMD. (A) Schematic depicting model of the Rv3160c dimer binding to the palindromic sequence present in the rv3161c upstream fragment. (B) EMSA assay using an 86 bp rv3161c upstream fragment (1 pmol; lanes 1–3) and a 204 bp cfp10 upstream fragment as negative control fragment (1 pmol; lanes 4–5). Lane 1 and 5: no protein added. Lanes 2, 3 and 5: binding reaction with 10 pmol Rv3160c. Lane 3: 50 nmol C10-IMD. DNA Ladder: Thermo Scientific GeneRuler 1 kb Plus (SM1331).

Figure 4

C10-IMD binds to a single site in Rv3160c and the interaction does not cause changes in the secondary structure of the protein. (A) Fluorescence quenching spectra of Rv3160c. The 10 µM Rv3160c in PBS solution is excited and quenching of fluorescence is recorded in the presence of varying concentrations of C10-IMD (1–100 μM). (B) Stern–Volmer plot of decrease in mean fluorescence intensity of Rv3160c in the presence of varying concentrations of C10-IMD derived from three independent experiments. The dynamic quenching rate constant K_sv is evaluated from the slope of the line. (C) Logarithmic plot of mean relative fluorescence quenching of Rv3160c derived from three independent experiments against logarithmic concentrations of C10-IMD. K_D is calculated from the intersection of the line with the y axis and the number of binding sites (n) from the slope of the line. (D) CD spectra of Rv3160c in the presence of 1 µM or 10 µM 86 bp rv3161c upstream fragment (DNA) (Fig. 2C). (E) CD spectra of Rv3160c in the presence of 10 µM or 100 µM C10-IMD. Error bars in (B) and (C) indicate standard deviation.

Figure 5

Deletion of the rv3161c gene results in higher resistance to isoniazid and lysozyme. (A) Representative images showing effect of INH and lysozyme on bacterial growth scored by pellet formation. Mtb Erdman WT and ∆rv3161c strains were incubated 2 weeks in Sauton’s medium with 0.02% DMSO or 5 µM C10-IMD in addition to various concentration of INH (left; 0.002–0.064 µg/ml) or lysozyme (right; 3.9–125 µg/ml). (B) Representative images showing effect of INH and lysozyme treatment on bacterial survival scored by spotting bacteria exposed to INH (left; 0.002–0.064 µg/ml) or lysozyme (right; 3.9–125 µg/ml) in the presence of 0.02% DMSO or 5 µM C10-IMD on antibiotic-free 7H10 plates.