RNase HI depletion strongly potentiates cell killing by rifampicin in mycobacteria

Abstract

Multidrug resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are non-viable. This essentiality supports it as a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two RNase HI enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin and rifampicin was increased, with sensitivity to the transcriptional inhibitor rifampicin strikingly increased by nearly 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and transcriptional inhibition. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action, and providing the first step in developing a new class of anti-mycobacterial drug.

The C-terminal acid phosphatase module of the RNase HI enzyme RnhC controls rifampicin sensitivity and light-dependent colony pigmentation ofMycobacterium smegmatis

“…ΔrnhC sensitivity was detected in two independent rnhC in-frame mutants (Figure S3C) and ectopic expression of rnhC in the ΔrnhC mutant restored the WT sensitivity (Figures 4C and S3D) indicating that RnhC confers rifampicin tolerance. This finding confirms recently published results using an M. smegmatis rnhC mutant that was hypersensitive to Rifampin using an MIC based assay (35).…”

“…The RNase HI enzyme encoded by rnhC, while coessential with RnhA, also contains an autonomous C terminal acid phosphatase domain that does not contribute to its essential function (21,22). Al-Zubaidi et al recently demonstrated that the rnhC mutant of M. smegmatis led to the accumulation of R-loops and a sensitivity to rifampicin (35), a finding (rif sensitivity) we replicate in this study. However, our data indicates that the RNase HI activity of the RnhC protein is not required for the rifampicin resistance phenotype and that the acid phosphatase activity is the important biochemical activity.…”

“…However, our data indicates that the RNase HI activity of the RnhC protein is not required for the rifampicin resistance phenotype and that the acid phosphatase activity is the important biochemical activity. These data suggest that the accumulation of R-loops, while clearly shown to be due to loss of rnhC (35) and likely due to a defect of the RNase HI activity of RnhC, may not be linked to rifampicin sensitivity.…”

“…Rifampicin is a cornerstone antibiotic used to treat Mtb. As proposed by Al-Zubaidi et al (35), The higher sensitivity of the M. smegmatis ΔrnhC mutant to rifampicin opens the possibility of targeting RnhC to potentiate rifampicin and reduce the effective dose or duration of rifampicin treatment as well as attenuate the frequency of resistance acquisition. However, based on our data, compounds that sensitize to rifampicin should not be optimized based on the RNase HI activity but instead the acid phosphatase activity of RnhC.…”