ResR/McdR-regulated protein translation machinery contributes to drug resilience in Mycobacterium tuberculosis.

Survival response of the human tuberculosis pathogen, Mycobacterium tuberculosis (Mtb) to a diverse environmental cues is governed through its versatile transcription regulatory mechanisms with the help of a large pool of transcription regulators (TRs). Rv1830 is one such conserved TR, which remains uncharacterized in Mtb. It was named as McdR based on an effect on cell division upon its overexpression in Mycobacterium smegmatis. Recently, it has been implicated in antibiotic resilience in Mtb and reannotated as ResR. While Rv1830 affects cell division by modulating the expression of M. smegmatis whiB2, the underlying cause of its essentiality and regulation of drug resilience in Mtb is yet to be deciphered. Here we show that ResR/McdR, encoded by ERDMAN_2020 in virulent Mtb Erdman, is pivotal for bacterial proliferation and crucial metabolic activities. Importantly, ResR/McdR directly regulates ribosomal gene expression and protein synthesis, requiring distinct disordered N-terminal sequence. Compared to control, bacteria depleted with resR/mcdR exhibit delayed recovery post-antibiotic treatment. A similar effect upon knockdown of rplN operon genes further implicates ResR/McdR-regulated protein translation machinery in attributing drug resilience in Mtb. Overall, findings from this study suggest that chemical inhibitors of ResR/McdR may be proven effective as adjunctive therapy for shortening the duration of TB treatment.

The physiological effect of rimI/rimJ silencing by CRISPR interference in Mycobacterium smegmatis mc2155.

N-terminal acetylation of proteins is an important post-translational modification (PTM) found in eukaryotes and prokaryotes. In bacteria, N-terminal acetylation is suggested to play various regulatory roles related to protein stability, gene expression, stress response, and virulence; however, the mechanism of such response remains unclear. The proteins, namely RimI/RimJ, are involved in N-terminal acetylation in mycobacteria. In this study, we used CRISPR interference (CRISPRi) to silence rimI/rimJ in Mycobacterium smegmatis mc2155 to investigate the physiological effects of N-terminal acetylation in cell survival and stress response. Repeat analysis of growth curves in rich media and biofilm analysis in minimal media of various mutant strains and wild-type bacteria did not show significant differences that could be attributed to the rimI/rimJ silencing. However, total proteome and acetylome profiles varied significantly across mutants and wild-type strains, highlighting the role of RimI/RimJ in modulating levels of proprotein acetylation in the cellular milieu. Further, we observed a significant increase in the minimum inhibitory concentration (MIC) (from 64 to 1024 µg ml-1) for the drug isoniazid in rimI mutant strains. The increase in MIC value for the drug isoniazid in the mutant strains suggests the link between N-terminal acetylation and antibiotic resistance. The study highlights the utility of CRISPRi as a convenient tool to study the role of PTMs, such as acetylation in mycobacteria. It also identifies rimI/rimJ genes as necessary for managing cellular response against antibiotic stress. Further research would be required to decipher the potential of targeting acetylation to enhance the efficacy of existing antibiotics.

Deciphering the Proteomic Landscape of Mycobacterium tuberculosis in Response to Acid and Oxidative Stresses.

The fundamental to the pathogenicity of Mycobacterium tuberculosis (Mtb) is the modulation in the control mechanisms that play a role in sensing and counteracting the microbicidal milieu encompassing various cellular stresses inside the human host. To understand such changes, we measured the cellular proteome of Mtb subjected to different stresses using a quantitative proteomics approach. We identified defined sets of Mtb proteins that are modulated in response to acid and a sublethal dose of diamide and H2O2 treatments. Notably, proteins involved in metabolic, catalytic, and binding functions are primarily affected under these stresses. Moreover, our analysis led to the observations that during acidic stress Mtb enters into energy-saving mode simultaneously modulating the acid tolerance system, whereas under diamide and H2O2 stresses, there were prominent changes in the biosynthesis and homeostasis pathways, primarily modifying the resistance mechanism in diamide-treated bacteria while causing metabolic arrest in H2O2-treated bacilli. Overall, we delineated the adaptive mechanisms that Mtb may utilize under physiological stresses and possible overlap between the responses to these stress conditions. In addition to offering important protein signatures that can be exploited for future mechanistic studies, our study highlights the importance of proteomics in understanding complex adjustments made by the human pathogen during infection.

Era, a GTPase-like protein of the Ras family, does not control ribosome assembly in Mycobacterium tuberculosis.

Era GTPase is universally present in microbes including Mycobacterium tuberculosis (Mtb) complex bacteria. While Era is known to regulate ribosomal assembly in Escherichia coli and predicted to be essential for in vitro growth, its function in mycobacteria remains obscured. Herein, we show that Era ortholog in the attenuated Mtb H37Ra strain, MRA_2388 (annotated as EraMT) is a cell envelope localized protein harbouring critical GTP-binding domains, which interacts with several envelope proteins of Mtb. The purified Era from M. smegmatis (annotated as EraMS) exhibiting ~90 % sequence similarity with EraMT, exists in monomeric conformation. While it is co-purified with RNA upon overexpression in E. coli, the presence of RNA does not modulate the GTPase activity of the EraMS as against its counterpart from other organisms. CRISPRi silencing of eraMT does not show any substantial effect on the in vitro growth of Mtb H37Ra, which suggests a redundant function of Era in mycobacteria. Notably, no effect on ribosome assembly, protein synthesis or bacterial susceptibility to protein synthesis inhibitors was observed upon depletion of EraMT in Mtb H37Ra, further indicating a divergent role of Era GTPase in mycobacteria.