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Biochemical Characterization of VapC46 Toxin from Mycobacterium tuberculosis
Madhurima Roy1, Madhuparna Bose1, Kamakshi Bankoti2
1Department of Biotechnology, Indian Institute of Technology, Kharagpur, India.
Abstract:
Emergence of multidrug resistant strains and extremely drug resistant strains of Mycobacterium tuberculosis is due to its ability to form persister cells. The formation of persister cells is assumed to be triggered due to the presence of large number of toxin-antitoxin (TA) systems in its genome. Mtb genome encodes 47 VapBC TA systems. In this work, we aim to biochemically characterize VapC46 toxin of the VapBC46 TA operon from Mycobacterium tuberculosis. Heterologous expression of VapC46 in E. coli is shown to exhibit bacteriostasis and toxicity alters the surface morphology of the E. coli cells. VapC46 is shown to possess ribonuclease activity in a magnesium-dependent manner. Using FRET and pull down assay, VapC46 is shown to interact with VapB46 antitoxin. A model of VapC46 is shown to resemble PIN domain family of proteins and reveals the putative active site required for its ribonuclease activity.
Insights
Mycobacterium tuberculosis forms persister cells, leading to drug resistance. Researchers characterized the VapC46 toxin, revealing its ribonuclease activity and interaction with VapB46 antitoxin, crucial for understanding tuberculosis drug resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Multidrug resistance in Mycobacterium tuberculosis is a significant global health threat.
- The formation of persister cells is a key mechanism contributing to tuberculosis drug resistance.
- Toxin-antitoxin (TA) systems are implicated in the regulation of persister cell formation in M. tuberculosis, with 47 VapBC TA systems encoded in its genome.
Purpose of the Study:
- To biochemically characterize the VapC46 toxin from the VapBC46 TA operon of Mycobacterium tuberculosis.
- To investigate the functional properties and interactions of VapC46.
Main Methods:
- Heterologous expression of VapC46 in E. coli.
- Assays to determine ribonuclease activity.
- Förster resonance energy transfer (FRET) and pull-down assays to study protein interactions.
- Bioinformatic analysis and molecular modeling of VapC46.
Main Results:
- Heterologous expression of VapC46 induced bacteriostasis and altered E. coli cell morphology.
- VapC46 exhibits magnesium-dependent ribonuclease activity.
- VapC46 directly interacts with its cognate antitoxin, VapB46.
- Structural modeling suggests VapC46 belongs to the PIN domain family and identifies a putative active site.
Conclusions:
- VapC46 is a functional toxin with ribonuclease activity, contributing to the VapBC46 toxin-antitoxin system in Mycobacterium tuberculosis.
- The characterization of VapC46 provides insights into the molecular mechanisms underlying persister cell formation and drug resistance in tuberculosis.
- Understanding VapC46 function can inform the development of novel strategies to combat multidrug-resistant tuberculosis.
