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Functional Analysis of VapC Toxins from Mycobacterium Tuberculosis

by
Melvilí Cintrón-Cotto
B.S., University of Puerto Rico, Mayagüez - 2010

Thesis Advisor: Nancy A. Woychik, Ph.D.
Graduate Program in Microbiology & Molecular Genetics

School of Public Health Building
Room 258
Piscataway

Friday, March 31, 2017
11:30 a.m.


Abstract

The Mycobacterium tuberculosis genome harbors an unusually high number of toxin-antitoxin (TA) systems. These small operons encode two proteins, a stable toxin and a labile antitoxin that form a stable complex which sequesters the activity of the toxin. When bacteria sense stress such as oxidative stress, nutrient starvation or antibiotics, the antitoxin is degraded by proteases and the toxin is released from its inhibition. This results in a phenomenon known as persistence, enabling bacteria to endure a variety of stressful insults by converting cells into a viable, but growth arrested state. Since TA systems have been linked to slow growth and persistence—which are also hallmarks of latent tuberculosis infection—we are interested in studying the enzymatic activity of toxins in the most abundant family of TA systems in M. tuberculosis, the VapBC (virulence associated protein) family. In this study, we characterized the mechanism of action of three M. tuberculosis VapC toxins, VapC-mt11 (Rv1561), VapC-mt19 (Rv2548), and VapC-mt20 (Rv2549c). Consistent with other previously characterized VapC toxins, VapC-mt11 and VapC-mt19 are tRNases. Unlike VapC-mt11, which inhibits translation and thus inhibits growth, the tRNase activity of VapC-mt19 does neither, i.e., VapC-mt19 has no discernable phenotype when overexpressed in M. tuberculosis. Nonetheless, VapC-mt20 has an unprecedented mechanism compared to other toxins within this family. VapC-mt20 cleaves the 23S rRNA at the sarcin-ricin loop (SRL). This is an evolutionarily conserved domain essential for protein synthesis and cell growth. Based on our results, we theorize that VapC toxins may initiate a cascade of events to facilitate transition of M. tuberculosis cells from active growth to latency during infection or vice versa.


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