The global rise in antibiotic-resistant tuberculosis has prompted an urgent search for new drugs. Toxin-antitoxin (TA) systems allow bacteria to adapt rapidly to environmental changes, and Mycobacterium tuberculosis encodes more TA systems than any known pathogen.

The recalcitrance of mycobacteria to antibiotic therapy is in part due to its ability to build proteins into a multi-layer cell wall. Proper synthesis of both cell wall constituents and associated proteins is crucial to maintaining cell integrity, and intimately tied to antibiotic susceptibility. How mycobacteria properly synthesize the membrane-associated proteome, however, remains poorly understood. Recently, we found that loss of lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. LepA is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cellular processes is not clear. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to strains with lepA. We find that LepA is important for the steady-state abundance of a number of membrane-associated proteins, including an outer membrane porin, MspA, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane which leads to the drug tolerance phenotype of the lepA mutant. In mycobacteria, the translation factor LepA modulates mycobacterial membrane homeostasis, which in turn affects antibiotic tolerance. Importance The mycobacterial cell wall is a promising target for new antibiotics due to the abundance of important membrane-associated proteins. Defining mechanisms of synthesis of the membrane proteome will be critical to uncovering and validating drug targets. We found that LepA, a universally conserved translation factor, controls the synthesis of a number of major membrane proteins in M. smegmatis. LepA primarily controls synthesis of the major porin MspA. Loss of LepA results in decreased permeability through the loss of this porin, including permeability to antibiotics like rifampin and vancomycin. In mycobacteria, regulation from the ribosome is critical for the maintenance of membrane homeostasis and, importantly, antibiotic susceptibility.

Peptidyl tRNA hydrolase (Pth) is an enzyme that cuts unfinished peptides off tRNA that has been prematurely released from a stalled ribosome. Pth is essential in nearly all bacteria, including the pathogen Mycobacterium tuberculosis (Mtb), but it has not been clear why.

Therapeutic advances in the 20th century significantly reduced tuberculosis (TB) mortality. Nonetheless, TB still poses a massive global health challenge with significant annual morbidity and mortality that has been amplified during the COVID-19 pandemic. Unlike most common bacterial infectious diseases, successful TB treatment requires months-long regimens, which complicates the ability to treat all cases quickly and effectively. Improving TB chemotherapy by reducing treatment duration and optimizing combinations of drugs is an important step to reducing relapse. In this review, we outline the limitations of current multidrug regimens against TB and have reviewed the genetic tools available to improve the identification of drug targets. The rational design of regimens that sterilize diverse phenotypic subpopulations will maximize bacterial killing while minimizing both treatment duration and infection relapse. Importantly, the TB field currently has all the necessary genetic and analytical tools to screen for and prioritize drug targets in vitro based on the vulnerability of essential and nonessential genes in the Mtb genome and to translate these findings in in vivo models. Combining genetic methods with chemical screens offers a formidable strategy to redefine the preclinical design of TB therapy by identifying powerful new targets altogether, as well as targets that lend new efficacy to existing drugs.

Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen,Mycobacterium tuberculosis(Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA ofMtb, using tRNA sequencing (tRNA-seq) and genome-mining. Homology searches identified 18 candidate tRNA modifying enzymes that are predicted to create 13 tRNA modifications across all tRNA species. Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of 9 modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion ofMtbgenes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species. Furthermore, the absence ofmnmAattenuatedMtbgrowth in macrophages, suggesting that MnmA-dependent tRNA uridine sulfation contributes toMtbintracellular growth. Our results lay the foundation for unveiling the roles of tRNA modifications inMtbpathogenesis and developing new therapeutics against tuberculosis.