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Jared D. Sharp
University of Delaware
Thesis Advisor: Nancy A. Woychik, Ph.D.
Graduate Program of Molecular Genetics, Microbiology and Immunology
7th floor conference room
Wednesday, September 16, 2009
Mycobacterium tuberculosis has the unique ability to persist for long periods of time in its host as a latent infection. While the molecular switches that trigger M. tuberculosis, latency are poorly understood, increasing evidence suggests a role for toxin-antitoxin (TA) systems. Detailed characterization of TA systems in Escherichia coli has revealed that TA toxins impart a state of reversible dormancy with striking similarities to the slowly or non-replicating state exhibited by M. tuberculosis in latent tuberculosis. The unusual abundance of TA systems in M. tuberculosis further implicates a role for them in the slow growth rate associated with this pathogen since an inverse relationship between growth rate and the number of TA modules has been noted.
TA systems appear to have evolved in order to allow free-living bacteria to cope with unfavorable environmental conditions. This is accomplished through the concerted action of two genes encoding a labile antitoxin and a stabile toxin. Under normal conditions, the antitoxin sequesters the toxin in a stable protein complex in order to neutralize its activity. However, under stressful conditions the level of the antitoxin in the cell is markedly reduced, freeing the toxin to act on its intracellular target. As a result, the toxin induces a state of reversible dormancy enabling the cell to survive when stressed until environmental conditions improve.
Although the mechanism of action is known for many of the TA systems found in M. tuberculosis, the precise function of the VapC toxins has remained elusive. Here we determined the mode of action of the VapC toxin from the VapBC-mt4 (Rv0596c-Rv0595C) TA system. Expression of VapC-mt4 drastically inhibited translation while replication was affected to a lesser extent. The inhibition of translation was accompanied by a gradual decrease in the levels of several mRNAs and primer extension analysis revealed that VapC-mt4 cleaved mRNA at ACGC and AC(A/U)GC sequences. These results were confirmed in vitro as purified VapC-mt4 exhibited ribonuclease activity that was specifically blocked by the addition of VapB-mt4. Surprisingly, cleavage of mRNA was not responsible for the inhibition of protein synthesis as VapC-mt4 inhibited protein synthesis in a cell free system without cleaving mRNA. We propose that VapC-mt4 inhibits protein synthesis by binding and/or cleaving a subset of tRNAs containing ACGC sequences, thereby inhibiting their essential role in protein synthesis.