Depleting Mycobacterium tuberculosis of the transcription termination factor Rho causes pervasive transcription and rapid death

Abstract

Rifampicin, which inhibits bacterial RNA polymerase, provides one of the most effective treatments for tuberculosis. Inhibition of the transcription termination factor Rho is used to treat some bacterial infections, but its importance varies across bacteria. Here we show that Rho of Mycobacterium tuberculosis functions to both define the 3′ ends of mRNAs and silence substantial fragments of the genome. Brief inactivation of Rho affects over 500 transcripts enriched for genes of foreign DNA elements and bacterial virulence factors. Prolonged inactivation of Rho causes extensive pervasive transcription, a genome-wide increase in antisense transcripts, and a rapid loss of viability of replicating and non-replicating M. tuberculosis in vitro and during acute and chronic infection in mice. Collectively, these data suggest that inhibition of Rho may provide an alternative strategy to treat tuberculosis with an efficacy similar to inhibition of RNA polymerase.

Small RNA Mcr11 regulates genes involved in the central metabolism ofMycobacterium tuberculosisand requires 3’ sequence along with the transcription factor AbmR for stable expression

“…A recent study of Rho function in Mtb (41) found that depletion of Rho did not impact transcriptional boundaries at predicted RITs (43, 44), demonstrating a clear separation in the populations of transcripts terminated by RITs and Rho-dependent mechanisms. While we found that TSEs promote expression of Mcr11, their underlying mechanism remains unclear and our data do not precisely fit either the current RIT or Rho-dependent termination models (Table 1).…”

“…Several mycobacterial sRNAs have multiple reported 3’ ends, suggestive of Rho-dependent termination and/or post-transcriptional processing (23, 30, 31). However, relatively little is known about transcriptional termination in Mtb, particularly at sRNAs (41-45).…”

Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis

“…A recent study of Rho function in Mtb (Botella, Vaubourgeix, Livny, & Schnappinger, ) found that depletion of Rho did not impact transcriptional boundaries at predicted RITs (Gardner et al, ; Mitra et al, ), demonstrating a clear separation in the populations of transcripts terminated by RITs and Rho‐dependent mechanisms. While we found that TSEs promote expression of Mcr11, their underlying mechanism remains unclear and our data do not precisely fit either the current RIT or Rho‐dependent termination models (Table ).…”

“…In contrast, Mcr11 TSEs 2–4, which contributed strongly to the termination of Mcr11, all possess C‐rich loops in their predicted secondary structures that could be rut sites (Figure ). Rho‐significant regions (RSRs) reported in the Botella et al study (Botella et al, ) include an RSR that begins 6 nt downstream of the mapped 3′ end of Mcr11, that is characterized as a region of general antisense transcription. The presence of this RSR is consistent with the possibility that mcr11 expression is Rho‐terminated.…”

Identifying the essential genes ofMycobacterium aviumsubsp.hominissuiswith Tn-Seq using a rank-based filter procedure

“…This includes the targets of the mycobacterial drugs cycloserine (alanine racemase, D-alanine – D-alanine ligase), rifamycins (RNA polymerase beta subunit), macrolides (50S ribosome), aminoglycosides (30S ribosome), fluoroquinolones (type IV topoisomerases and gyrases), bedaquiline (ATP synthase), and ethambutol (arabinosyltransferase). Additional compounds that have been reported to have some activity against mycobacteria include tryptophan synthase inhibitors 11 , ClpP inhibitors 12 , and Rho inhibitors (albeit only shown to be effective through genetic manipulation) 13 . A brief literature search also reveals many compounds that inhibit non-mycobacterial orthologs of these genes but appear to lack published results for killing activity in mycobacteria including inhibitors of GroEL 14 , RibBA 15 , SecA 16 , and LigA 17,18 .…”