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

Abstract

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.

VapC21 Toxin Contributes to Drug-Tolerance and Interacts With Non-cognate VapB32 Antitoxin in Mycobacterium tuberculosis

“…The transcript levels of esxK, esxL and hsdS.1 which were increased in the mutant strain were observed to be decreased in the overexpression strain. In agreement with earlier reports, transcript levels of non-cognate toxins and antitoxins such as vapC1, mazE3,vapB43,vapB22,vapC15,vapB17,Rv0366c,and vapB15 were also increased in the VapC21 overexpression strain ( Supplementary Table S3; Agarwal et al, 2018;Deep et al, 2018). DEGs annotated as regulatory proteins such as sigB, mce2R, whiB1, sigD, furA, whiB7, sigE, clgR were also upregulated in the overexpression strain ( Supplementary Table S3).…”

“…Several studies have shown that overexpression of toxins belonging to TA systems result in transcriptional reprogramming that might enable the bacteria to adapt to different stress conditions (Singh et al, 2010;Deep et al, 2018). We next performed RNA-seq analysis to compare the transcription profiles of parental and VapC21 overexpression strain.…”

“…Additionally, the structure of VapC toxin, Rv2549c that cleave 23S rRNA at the sarcin-ricin loop have also been solved (Winther et al, 2013;Deep et al, 2017). In our earlier reports, we have shown VapBC3, VapBC4, and VapBC11 are essential for M. tuberculosis to establish infection in guinea pigs (Agarwal et al, 2018;Deep et al, 2018). However, M. tuberculosis strain deficient in vapC28 did not exhibit a growth defect in the guinea pig model of infection (Agarwal et al, 2018).…”

“…The cellular targets for these ribonucleases have also been extensively characterized and include tRNA32 Gln−CTG , tRNA3 Leu−CAG , tRNA21 Cys−GCA , tRNA fmet , tRNA25 Ser−TGA , tRNA28 Ser−CGA , and tRNA7 Trp−CCA (Winther and Gerdes, 2011;Sharp et al, 2012;Cruz et al, 2015;Winther et al, 2016;Cintron et al, 2019). The crystal structures for some of these VapBC TA systems have been solved with the following PDB codes; 3H87 (Rv0300-Rv0301), 3DB0 (Rv0626-Rv0627), 4CHG (Rv2009-Rv2010), 4XGQ (Rv0623-Rv0624), 5X3T (Rv0581-Rv0582), and 6A7V (Rv1560-Rv1561) (Miallau et al, 2009;Min et al, 2012;Das et al, 2014;Lee et al, 2015;Kang et al, 2017;Deep et al, 2018). Additionally, the structure of VapC toxin, Rv2549c that cleave 23S rRNA at the sarcin-ricin loop have also been solved (Winther et al, 2013;Deep et al, 2017).…”

Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection

“…The results reported here support the notion that VapBC-1 (and TA modules in general) have evolved a number of mechanisms to maintain their activity and that their presence confers a significant advantage to the microorganism. Many studies have linked TA modules to the survival of bacterial pathogens in host tissues (7,(56)(57)(58)(59)(60). Achieving and maintaining persistence during an infection is central for both successfully evading the immune response and for nonspecific antibiotic tolerance, leading to a pathogen's survival of clinical therapy.…”