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.
Highlights
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a leading cause of mortality worldwide [1]
We show that the VapBC11 TA system is dispensable for in vitro growth but is essential for Mtb to establish disease in vivo
These results demonstrate the essentiality of VapBC11 TA systems for Mtb to establish infection in guinea pigs
Summary
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a leading cause of mortality worldwide [1]. Mtb is a highly successful intracellular pathogen because of its ability to exist in an altered metabolic state that is phenotypically drug-tolerant [5]. Numerous studies have identified various metabolic pathways, such as protein kinases, stringent response, two-component systems, sigma factors and toxin–antitoxin (TA) modules, as the key determinants of bacterial virulence [6,7,8,9,10]. The overexpression of Type II toxins inhibit bacterial growth in either a bactericidal or bacteriostatic manner [15,16]. In Type II TA systems, both toxin and antitoxin form a tight TA complex that binds to the operator region and results in auto-repression [17]. The N-terminal domain of antitoxins possess DNA-binding properties, and the C-terminal domain neutralizes the toxin [18,19]. Several DNA-binding motifs, such as ribbon-helix-helix (RHH), helix-turn-helix, AbrB and PhD/YefM, have been mapped
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