IDENTIFICATION OF NATIVE MYCOBACTERIUM SMEGMATIS CELL WALL DEGRADATIVE ENZYMES USING ELECTROPHORETIC FLUORESCENT ASSAYS

Matthew B. Au, Lisa Siswando, Jeffery Hall, James S. Weinberg, Lingyi Lynn Deng

Abstract


Mycobacterium is an important bacterial genus containing many serious pathogens, including Mycobacterium tuberculosis. The mycobacterial cell wall is thick, rigid, waxy and extremely important for its survival in a hostile environment. Mycobacterium species contain multiple cell wall hydrolases, which are critical for cell growth, cell division and latent cell reactivation. Additionally, cell wall hydrolases can be considered as a group of potential drug targets and therapeutic agents for mycobacterial pathogens. However, the conventional hydrolytic enzyme assays are slow, laborious and inaccurate. In this study, we used a fast-growing, non-pathogenic bacterial strain - Mycobacterium smegmatis - to develop a more sensitive and efficient assay. With this assay, we were able to simultaneously quantify the enzymatic activity and localize the enzymatic protein bands. Furthermore, this new assay speeds up the purification and identification of native cell wall hydrolases. In this study we isolated three proteins showing hydrolytic activity with estimated molecular masses of 30 kDa, 70 kDa and 110 kDa. Overall, the knowledge obtained from this study should be translated to clinical research and ultimately used for the diagnosis and treatment of mycobacterial pathogens, including M. tuberculosis.


Keywords


Mycobacterium smegmatis, M. tuberculosis, hydrolase, cell wall degradation enzymes, cell wall hydrolase assays, zymography

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