Discipline: Biological Sciences
Marie Theresa Mazzeo - Hunter College, The City University of New York
Co-Author(s): Sangeeta Tiwari, Albert Einstein College of Medicine, Bronx, NY; William R. Jacobs Jr., Albert Einstein College of Medicine, Howard Hughes Medical Institute, Bronx, NY
Tuberculosis (Tb), caused by Mycobacterium tuberculosis, remains one of the top ten causes of mortality in the world and is the leading cause of death due to an infectious disease. The devastation of this pandemic necessitates improved methods of prevention and treatment. Furthermore, effective treatment for Tb must sterilize emergent drug-resistant ‘persister’ Mtb subpopulations. Amino acid auxotrophic mutants, including methionine, tryptophan, pantothenate, leucine, and biotin, have been explored; however, the bactericidal activity of all of these mutants have yet to be fully characterized. Since drug screening against amino acid biosynthesis targets in a Biosafety Level 3 facility is cumbersome and Mtb grows slowly, we decided to use Mycobacterium smegmatis as a model organism because it is fast growing and shares a large degree of genetic homology with Mtb.
To further investigate the role of amino acid starvation, we decided to explore the effects of arginine starvation, as it has been shown to be essential for other bacteria, including Salmonella typhimurium. Therefore, we constructed allelic exchange substrates (AES) and used specialized transduction to establish the mutant strain mc27156 with a gene deletion in MSMEG_3772, which encodes an ornithine transcarbamylase in the arginine biosynthesis pathway. This mutant strain was then subject to arginine starvation and cell death was assessed through fluorescence microscopy, spectrophotometry, and plating. Our preliminary data demonstrate that arginine starvation via deletion of MSMEG_3772 has a bactericidal effect on Mycobacterium smegmatis, which demonstrates the potential of these strains as a drug-screening model. Understanding the implications of various gene deletions in Mycobacterium smegmatis will aid in the identification of putative drug targets for Mycobacterium tuberculosis and will also provide a promising direction for future research.
Funder Acknowledgement(s): I would like to acknowledge the John P. McNulty Scholars Program at Hunter College for funding and support. I would also like to acknowledge the Summer Undergraduate Research Program at the Graduate School of Biomedical Sciences at Albert Einstein College of Medicine for funding and support. Funding for this experiment was provided by an HHMI grant to William R. Jacobs Jr.
Faculty Advisor: Sangeeta Tiwari, firstname.lastname@example.org
Role: The project described was my individual summer project at Albert Einstein College of Medicine. I collected and interpreted all of the results described, for each experimental trial ran I cultured the bacteria, purified and washed the bacteria, starved one of the samples for arginine and supplemented the other with arginine, I measured the optical density using spectrophotometry, plated the bacteria in the morning and evening for the length of the experiment and measured the number of colony forming units.