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Updated: Oct 3, 2025
Preparation of Mycobacterium Tuberculosis Culture Filtrate to Understand TB Pathogenesis
Published on: March 28, 2025
A Phagosomally Expressed Gene, rv0428c, of Mycobacterium tuberculosis Demonstrates Acetyl Transferase Activity and
Aashish Sharma1,2, Arbind Kumar1,2, Mudasir Rashid3
1Department of Biotechnology, Panjab University, BMS Block-1, South Campus, Chandigarh, 160014, India.
Abstract:
Mycobacterium tuberculosis genome is composed of several hypothetical gene products that need to be characterized for understanding the physiology of bacteria. Rv0428c was one of the 11 proteins exclusively identified within the phagosomal compartment of macrophages infected with mycobacteria and marked as hypothetical. The expression of rv0428c gene was upregulated under acidic and nutritive stress conditions in M. tuberculosis H37Ra, which was supported by potential sigma factor binding sites in the region upstream to the rv0428c gene. The bioinformatics analysis predicted it to be a GCN5- acetyl transferase, belonging to the Histone acetyl transferase (HAT) family. The docking analysis predicted formation of hydrogen bonds and hydrophobic interactions between donor acetyl-co-A and histone H3 tail region. rv0428c gene was cloned and expressed in E. coli. The protein was purified to homogeneity and was fairly stable over a wide range of pH 5.0-9.0 and temperature up to 40 °C. The HAT activity of purified Rv0428c was confirmed by in vitro acetylation assay using recombinant H3 histone expressed in bacteria as substrate, which increased in time dependent manner. The results suggested that it is the second confirmed acetyl transferase in M. tuberculosis H37Rv. Furthermore, rv0428c was over expressed in surrogate host M. smegmatis, which led to enhanced growth rate and altered colony morphology. The expression of rv0428c in M. smegmatis promoted the survival of bacteria under acidic and nutritive stress conditions. In conclusion, Rv0428c, a phagosomal acetyl transferase of M. tuberculosis, might be involved in survival under stress conditions.
Insights
Rv0428c, a hypothetical protein in Mycobacterium tuberculosis, functions as a histone acetyl transferase (HAT). This enzyme enhances bacterial survival under stress conditions, offering new insights into tuberculosis pathogenesis.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mycobacterium tuberculosis possesses numerous hypothetical proteins requiring functional characterization.
- Rv0428c is a hypothetical protein found in the phagosomal compartment of infected macrophages.
- rv0428c gene expression increases under acidic and nutrient-poor conditions.
Purpose of the Study:
- To characterize the function of the hypothetical protein Rv0428c from Mycobacterium tuberculosis.
- To investigate the potential histone acetyl transferase (HAT) activity of Rv0428c.
- To assess the role of Rv0428c in bacterial survival under stress.
Main Methods:
- Bioinformatic analysis predicted Rv0428c as a GCN5-like acetyltransferase.
- The rv0428c gene was cloned, expressed in E. coli, and the protein purified.
- In vitro acetylation assays using recombinant H3 histone and overexpression in M. smegmatis were performed.
Main Results:
- Rv0428c exhibited HAT activity, confirmed by in vitro acetylation of histone H3 in a time-dependent manner.
- The purified Rv0428c protein was stable across a pH range of 5.0-9.0 and temperatures up to 40°C.
- Overexpression of Rv0428c in M. smegmatis enhanced growth rate, altered colony morphology, and improved survival under stress.
Conclusions:
- Rv0428c is identified as the second confirmed acetyltransferase in Mycobacterium tuberculosis.
- Rv0428c plays a role in enhancing bacterial survival under acidic and nutrient stress conditions.
- Rv0428c, a phagosomal acetyltransferase, is implicated in the stress response of Mycobacterium tuberculosis.
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