Virulence-associated protein B and C toxin-antitoxin (TA) systems are widespread in prokaryotes, but their precise role in physiology is poorly understood. We have functionally characterized the VapBC22 TA system from Mycobacterium tuberculosis. Transcriptome analysis revealed that overexpression of VapC22 toxin in M. tuberculosis results in reduced levels of metabolic enzymes and increased levels of ribosomal proteins. Proteomics studies showed reduced expression of virulence-associated proteins and increased levels of cognate antitoxin, VapB22 in the ΔvapC22 mutant strain. Furthermore, both the ΔvapC22 mutant and VapB22 overexpression strains of M. tuberculosis were susceptible to killing upon exposure to oxidative stress and showed attenuated growth in guinea pigs and mice. Host transcriptome analysis suggests upregulation of the transcripts involved in innate immune responses and tissue remodeling in mice infected with the ΔvapC22 mutant strain. Together, we demonstrate that the VapBC22 TA system belongs to a key regulatory network and is essential for M. tuberculosis pathogenesis.
Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific diagnostic tools, novel therapeutics, and vaccines for the eradication of TB. In the present study, a chemical screen of a pharmacologically active compound library was performed to identify antimycobacterial compounds. The phenotypic screen identified a few novel small-molecule inhibitors, including NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium tuberculosis protein kinase D (PknD) and protein kinase G (PknG). Comparative structural and sequence analysis along with docking simulation suggest that the unique binding site stereochemistry of PknG and PknD accommodates NU-6027 more favorably than other M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment induces the expression of proapoptotic genes in macrophages. Finally, we demonstrate that NU-6027 inhibits M. tuberculosis growth in both macrophage and mouse tissues. Taken together, these results indicate that NU-6027 can be optimized further for the development of antimycobacterial agents.
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