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

Abstract

The prokaryotic ubiquitous Toxin-antitoxin (TA) modules encodes for a stable toxin and an unstable antitoxin. VapBC subfamily is the most abundant Type II TA system in M. tuberculosis genome. However, the exact physiological role for most of these Type II TA systems are still unknown. Here, we have comprehensively characterized the VapBC21 TA locus from M. tuberculosis. The overexpression of VapC21 inhibited mycobacterial growth in a bacteriostatic manner and as expected, growth inhibition was abrogated upon co-expression of the cognate antitoxin, VapB21. We observed that the deletion of vapC21 had no noticeable influence on the in vitro and in vivo growth of M. tuberculosis. Using co-expression and biophysical studies, we observed that in addition to VapB21, VapC21 is also able to interact with non-cognate antitoxin, VapB32. The strength of interaction varied between the cognate and non-cognate TA pairs. The overexpression of VapC21 resulted in differential expression of approximately 435 transcripts in M. tuberculosis. The transcriptional profiles obtained upon ectopic expression of VapC21 was similar to those reported in M. tuberculosis upon exposure to stress conditions such as nutrient starvation and enduring hypoxic response. Further, VapC21 overexpression also led to increased expression of WhiB7 regulon and bacterial tolerance to aminoglycosides and ethambutol. Taken together, these results indicate that a complex network of interactions exists between non-cognate TA pairs and VapC21 contributes to drug tolerance in vitro.

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“…Notably, the VapBC TA family is the most abundant type of TA system encoded by Mtb (60). Several studies have demonstrated that host-mediated stress, such as hypoxia and activated macrophages, induces transcriptional activation of multiple VapBC TA loci (61)(62)(63). Hudock et al identified the transcriptional profile from granuloma samples of active and latent TB patients; the expression of eight dosR regulon members (Rv0080, Rv0081, Rv1736c, Rv1737c, Rv2032, Rv2625c, and Rv2630) along with the induction of four pairs of toxin/antitoxin (vapBC19, vapBC21, vapBC33, and vapBC34) were observed within the granulomas of active and latent TB patients (61).…”

“…Hudock et al identified the transcriptional profile from granuloma samples of active and latent TB patients; the expression of eight dosR regulon members (Rv0080, Rv0081, Rv1736c, Rv1737c, Rv2032, Rv2625c, and Rv2630) along with the induction of four pairs of toxin/antitoxin (vapBC19, vapBC21, vapBC33, and vapBC34) were observed within the granulomas of active and latent TB patients (61). Sharma et al demonstrated that VapC21 overexpression hinders mycobacterial growth, and co-expression of antitoxin VapB21 reverses this effect (62). Moreover, VapC21 overexpression mutant and Mtb cultured in stress conditions, such as nutrient deprivation and hypoxia, exhibited similar transcriptional profiles (62).…”

“…Sharma et al. demonstrated that VapC21 overexpression hinders mycobacterial growth, and co-expression of antitoxin VapB21 reverses this effect ( 62 ). Moreover, VapC21 overexpression mutant and Mtb cultured in stress conditions, such as nutrient deprivation and hypoxia, exhibited similar transcriptional profiles ( 62 ).…”

“…demonstrated that VapC21 overexpression hinders mycobacterial growth, and co-expression of antitoxin VapB21 reverses this effect ( 62 ). Moreover, VapC21 overexpression mutant and Mtb cultured in stress conditions, such as nutrient deprivation and hypoxia, exhibited similar transcriptional profiles ( 62 ). Furthermore, VapC21 overexpression resulted in upregulated WhiB7 regulon, inducing antibiotic tolerance to aminoglycosides and EMB ( 62 ).…”

“…Moreover, VapC21 overexpression mutant and Mtb cultured in stress conditions, such as nutrient deprivation and hypoxia, exhibited similar transcriptional profiles ( 62 ). Furthermore, VapC21 overexpression resulted in upregulated WhiB7 regulon, inducing antibiotic tolerance to aminoglycosides and EMB ( 62 ). Talwar et al.…”