System-Wide Analysis Unravels the Differential Regulation and In Vivo Essentiality of Virulence-Associated Proteins B and C Toxin-Antitoxin Systems of Mycobacterium tuberculosis

Talwar

et al. 2019

Preprint

32A worldwide increase in the frequency of multidrug-resistant and extensively drug-33 resistant cases of tuberculosis is mainly due to therapeutic noncompliance associated 34 with a lengthy treatment regimen. Depending on the drug susceptibility profile, the 35 treatment duration can extend from 6 months to 2 years. This protracted regimen is 36 attributed to a supposedly non-replicating and metabolically inert subset of the 37 Mycobacterium tuberculosis (Mtb) population, called 'persisters'. The mechanism 38 underlying stochastic generation and enrichment of persisters is not fully known. 39 We have previously reported that the utilization of host cholesterol is essential for 40 mycobacterial persistence. In this study, we have demonstrated that cholesterol-41 induced activation of a ribonuclease toxin (VapC12) inhibits translation by 42 targeting proT tRNA in Mtb. This results in cholesterol-specific growth modulation 43 that increases the frequency of the generation of persisters in a heterogeneous Mtb 44 population. Also, a null mutant strain of this toxin (ΔvapC12) failed to persist and 45 demonstrated an enhanced growth phenotype in a guinea pig model of Mtb 46 infection. Thus, we have identified a novel strategy through which cholesterol-47 specific activation of a toxin-antitoxin (TA) module in Mtb enhances persister 48 formation during infection. In addition to identifying the mechanism, the study 49 provides opportunity for targeting persisters, a new paradigm facilitating 50 tuberculosis drug development. 51 52 53 54 55 56 57 58 128the cholesterol-rich media. We have observed a decrease in the replication and metabolic 129 rates of wild-type (WT) H37Rv grown in the cholesterol-rich media by ten-and three-130 fold, respectively, compared with (WT) H37Rv grown in the glycerol-rich media ( Fig. 131 1A, 1B). An in-vitro time-kill curve assay revealed a cholesterol-specific increase in the 132 frequency of the generation of a rifamycin-tolerant sub-population (Fig. 1C). The TA loci 133 across bacterial species regulate growth (21,(29)(30)(31), therefore we speculated the 134 aforementioned phenotype to be regulated by one of the Mtb TA loci. For that we 135 analysed the data of a transposon mutagenesis screening performed in Mtb H37Rv to 136 identify genes essential for growth in a cholesterol-rich environment(32). Through 137

Section: Resultscontrasting

confidence: 99%

Host cholesterol dependent activation of VapC12 toxin enriches persister population duringMycobacterium tuberculosisinfection

Talwar

et al. 2019

Preprint

“…In contrast, most other toxins studied in Mtb exert a bacteriostatic effect (Singh et al, 2010;Tiwari et al, 2015;Agarwal et al, 2018;Sharrock et al, 2018;Tandon et al, 2019). This suggests that, among…”

Section: Discussionmentioning

confidence: 98%

Depletion of the DarG antitoxin in Mycobacterium tuberculosis triggers the DNA‐damage response and leads to cell death

Zaveri

Wang

Botella

et al. 2020

Molecular Microbiology

Toxin-antitoxin (TA) systems are ubiquitously present in prokaryotic genomes and consist of a toxic protein that inhibits an essential cellular process and a counteracting antitoxin that binds to and neutralizes the toxin (Yamaguchi et al., 2011). TA systems were originally discovered due to their ability to prevent plasmid loss by post-segregational killing (Ogura and Hiraga, 1983; Gerdes et al., 1986). They have subsequently been implicated in various cellular pathways including phage defense, genome stabilization, and bacterial per

“…VapC, the toxin, inhibits protein translation by cleaving either transfer RNA (tRNA) or the sarcin-ricin loop (SRL) of 23S ribosomal RNA (rRNA) or messenger RNA (mRNA), and this activity is neutralized by their cognate VapB antitoxins ( 24 ). The Mtb genome encodes for 90 TA systems, and the majority of these (50 TA modules) belong to the VapBC family ( 10 , 25 ). Interestingly, Mycobacterium smegmatis ( Msm ), a non-pathogenic mycobacterial species, harbours only a single VapBC module, suggesting their importance in Mtb pathogenesis ( 26 ).…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, Mycobacterium smegmatis ( Msm ), a non-pathogenic mycobacterial species, harbours only a single VapBC module, suggesting their importance in Mtb pathogenesis ( 26 ). Several structures, including VapBC3, VapBC5, VapBC15, VapC20, VapC21, VapBC26 and VapBC30 ( 27–33 ), have been resolved, but there is very limited information about their role in bacterial physiology ( 10 ). Though these toxins share poor sequence identity (<30%), they all adopt characteristic PIN domain architecture ( 34 ).…”

Section: Introductionmentioning

confidence: 99%

Structural, functional and biological insights into the role ofMycobacterium tuberculosisVapBC11 toxin–antitoxin system: targeting a tRNase to tackle mycobacterial adaptation

Deep

Tiwari

Agarwal

et al. 2018

Nucleic Acids Research

Self Cite

Toxin–antitoxin (TA) systems are involved in diverse physiological processes in prokaryotes, but their exact role in Mycobacterium tuberculosis (Mtb) virulence and in vivo stress adaptation has not been extensively studied. Here, we demonstrate that the VapBC11 TA module is essential for Mtb to establish infection in guinea pigs. RNA-sequencing revealed that overexpression of VapC11 toxin results in metabolic slowdown, suggesting that modulation of the growth rate is an essential strategy for in vivo survival. Interestingly, overexpression of VapC11 resulted in the upregulation of chromosomal TA genes, suggesting the existence of highly coordinated crosstalk among TA systems. In this study, we also present the crystal structure of the VapBC11 heterooctameric complex at 1.67 Å resolution. Binding kinetic studies suggest that the binding affinities of toxin–substrate and toxin–antitoxin interactions are comparable. We used a combination of structural studies, molecular docking, mutational analysis and in vitro ribonuclease assays to enhance our understanding of the mode of substrate recognition by the VapC11 toxin. Furthermore, we have also designed peptide-based inhibitors to target VapC11 ribonuclease activity. Taken together, we propose that the structure-guided design of inhibitors against in vivo essential ribonucleases might be a novel strategy to hasten clearance of intracellular Mtb.