Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis.

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.

Beyond antibiotic resistance: The whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria.

Pathogenic mycobacteria are a significant cause of morbidity and mortality worldwide. The conserved whiB7 stress response reduces the effectiveness of antibiotic therapy by activating several intrinsic antibiotic resistance mechanisms. Despite our comprehensive biochemical understanding of WhiB7, the complex set of signals that induce whiB7 expression remain less clear. We employed a reporter-based, genome-wide CRISPRi epistasis screen to identify a diverse set of 150 mycobacterial genes whose inhibition results in constitutive whiB7 expression. We show that whiB7 expression is determined by the amino acid composition of the 5' regulatory uORF, thereby allowing whiB7 to sense amino acid starvation. Although deprivation of many amino acids can induce whiB7, whiB7 specifically coordinates an adaptive response to alanine starvation by engaging in a feedback loop with the alanine biosynthetic enzyme, aspC. These findings describe a metabolic function for whiB7 and help explain its evolutionary conservation across mycobacterial species occupying diverse ecological niches.

Beyond antibiotic resistance: the whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria.

Pathogenic mycobacteria are a significant cause of morbidity and mortality worldwide. These bacteria are highly intrinsically drug resistant, making infections challenging to treat. The conserved whiB7 stress response is a key contributor to mycobacterial intrinsic drug resistance. Although we have a comprehensive structural and biochemical understanding of WhiB7, the complex set of signals that activate whiB7 expression remain less clear. It is believed that whiB7 expression is triggered by translational stalling in an upstream open reading frame (uORF) within the whiB7 5' leader, leading to antitermination and transcription into the downstream whiB7 ORF. To define the signals that activate whiB7, we employed a genome-wide CRISPRi epistasis screen and identified a diverse set of 150 mycobacterial genes whose inhibition results in constitutive whiB7 activation. Many of these genes encode amino acid biosynthetic enzymes, tRNAs, and tRNA synthetases, consistent with the proposed mechanism for whiB7 activation by translational stalling in the uORF. We show that the ability of the whiB7 5' regulatory region to sense amino acid starvation is determined by the coding sequence of the uORF. The uORF shows considerable sequence variation among different mycobacterial species, but it is universally and specifically enriched for alanine. Providing a potential rationalization for this enrichment, we find that while deprivation of many amino acids can activate whiB7 expression, whiB7 specifically coordinates an adaptive response to alanine starvation by engaging in a feedback loop with the alanine biosynthetic enzyme, aspC. Our results provide a holistic understanding of the biological pathways that influence whiB7 activation and reveal an extended role for the whiB7 pathway in mycobacterial physiology, beyond its canonical function in antibiotic resistance. These results have important implications for the design of combination drug treatments to avoid whiB7 activation, as well as help explain the conservation of this stress response across a wide range of pathogenic and environmental mycobacteria.

Mutations in rv0678 Confer Low-Level Resistance to Benzothiazinone DprE1 Inhibitors in Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading cause of death from any bacterial infection, causing 1.5 million deaths worldwide each year. Due to the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) there have been significant efforts aimed at developing novel drugs to treat TB. One promising drug target in Mtb is the arabinogalactan biosynthetic enzyme DprE1, and there have been over a dozen unique chemical scaffolds identified which inhibit the activity of this protein. Among the most promising lead compounds are the benzothiazinones BTZ043 and PBTZ169, both of which are currently in or have completed phase IIa clinical trials. Due to the potential clinical utility of these drugs, we sought to identify potential synergistic interactions and new mechanisms of resistance using a genome-scale CRISPRi chemical-genetic screen with PBTZ169. We found that knockdown of rv0678, the negative regulator of the mmpS5/L5 drug efflux pump, confers resistance to PBTZ169. Mutations in rv0678 are the most common form of resistance to bedaquiline and there is already abundant evidence of these mutations emerging in bedaquiline-treated patients. We confirmed that rv0678 mutations from clinical isolates confer low level cross-resistance to BTZ043 and PBTZ169. While it is yet unclear whether rv0678 mutations would render benzothiazinones ineffective in treating TB, these results highlight the importance of monitoring for clinically prevalent rv0678 mutations during ongoing BTZ043 and PBTZ169 clinical trials.

CRISPRi chemical genetics and comparative genomics identify genes mediating drug potency in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) infection is notoriously difficult to treat. Treatment efficacy is limited by Mtb's intrinsic drug resistance, as well as its ability to evolve acquired resistance to all antituberculars in clinical use. A deeper understanding of the bacterial pathways that influence drug efficacy could facilitate the development of more effective therapies, identify new mechanisms of acquired resistance, and reveal overlooked therapeutic opportunities. Here we developed a CRISPR interference chemical-genetics platform to titrate the expression of Mtb genes and quantify bacterial fitness in the presence of different drugs. We discovered diverse mechanisms of intrinsic drug resistance, unveiling hundreds of potential targets for synergistic drug combinations. Combining chemical genetics with comparative genomics of Mtb clinical isolates, we further identified several previously unknown mechanisms of acquired drug resistance, one of which is associated with a multidrug-resistant tuberculosis outbreak in South America. Lastly, we found that the intrinsic resistance factor whiB7 was inactivated in an entire Mtb sublineage endemic to Southeast Asia, presenting an opportunity to potentially repurpose the macrolide antibiotic clarithromycin to treat tuberculosis. This chemical-genetic map provides a rich resource to understand drug efficacy in Mtb and guide future tuberculosis drug development and treatment.