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Published on: April 16, 2021
Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis
Lingyi Lynn Deng1, Donald E Humphries, Robert D Arbeit
1Department of Medicine, Boston University School of Medicine, Research Service, VA Boston Healthcare System, 150 S. Huntington Ave., Boston, MA 02130, USA. lynndeng@bu.edu
Abstract:
Mycobacterium tuberculosis is a major global pathogen whose threat has increased with the emergence of multidrug-resistant strains. The cell wall of M. tuberculosis is thick, rigid, and hydrophobic, which serves to protect the organism from the environment and makes it highly impermeable to conventional antimicrobial agents. There is little known about cell wall autolysins (also referred to as peptidoglycan hydrolases) of mycobacteria. We identified an open reading frame (Rv3915) in the M. tuberculosis genome designated cwlM that appeared consistent with a peptidoglycan hydrolase. The 1218-bp gene was amplified by PCR, cloned and expressed in E. coli strain HMS174(DE-3), and its gene product, a 47-kDa recombinant protein, was purified and partially characterized. Purified CwlM was able to lyse whole mycobacteria, release peptidoglycan from the cell wall of Micrococcus luteus and Mycobacterium smegmatis, and cleave N-acetylmuramoyl-L-alanyl-D-isoglutamine, releasing free N-acetylmuramic acid. These results indicate that CwlM is a novel autolysin and identify cwlM as the first, to our knowledge, autolysin gene identified and cloned from M. tuberculosis. CwlM offers a new target for a unique class of drugs that could alter the permeability of the mycobacterial cell wall and enhance the effectiveness of treatments for tuberculosis.
Insights
Researchers identified a novel cell wall autolysin, CwlM, in Mycobacterium tuberculosis. This discovery offers a new target for drugs to enhance tuberculosis treatment effectiveness.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mycobacterium tuberculosis poses a significant global health threat, exacerbated by multidrug-resistant strains.
- The unique, impermeable cell wall of M. tuberculosis presents challenges for conventional antimicrobial agents.
- Knowledge regarding mycobacterial cell wall autolysins (peptidoglycan hydrolases) is limited.
Purpose of the Study:
- To identify and characterize novel autolysins in Mycobacterium tuberculosis.
- To investigate the potential of identified autolysins as therapeutic targets for tuberculosis.
Main Methods:
- Bioinformatic analysis to identify potential peptidoglycan hydrolase genes (Rv3915/cwlM).
- Gene amplification, cloning, and expression of cwlM in E. coli.
- Purification and partial characterization of the recombinant CwlM protein.
- Enzymatic assays to assess CwlM's lytic activity on mycobacteria and peptidoglycan hydrolysis.
Main Results:
- The gene cwlM (Rv3915) was identified and successfully expressed as a 47-kDa recombinant protein.
- Purified CwlM demonstrated lytic activity against whole mycobacteria.
- CwlM released peptidoglycan from Micrococcus luteus and Mycobacterium smegmatis cell walls.
- The enzyme cleaved the N-acetylmuramoyl-L-alanyl-D-isoglutamine bond, releasing N-acetylmuramic acid.
Conclusions:
- CwlM is a novel mycobacterial autolysin, representing the first identified and cloned autolysin gene from M. tuberculosis.
- CwlM presents a new target for developing drugs that could modify mycobacterial cell wall permeability.
- Targeting CwlM may enhance the efficacy of existing tuberculosis treatments.
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