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Characterization and Application of the DinJ-YafQ Toxin-Antitoxin System

by
Christopher J. Mozdzierz
B.A. Rutgers University



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

School of Public Health (SPH)
2nd floor Conference Room 25

Wednesday, August 25, 2010
12:30 p.m.


Abstract

Toxin-antitoxin (TA) systems on the chromosomes of free-living bacteria appear to facilitate cell survival during intervals of stress by inducing a state of reversible growth arrest. However, upon prolonged stress, TA toxin action leads to cell death. They have been implicated in several clinically important phenomena--bacterial persistence during antibiotic treatment, biofilm formation and bacterial pathogenesis--and serve as attractive new antibiotic targets for pathogens. We determined the mode of action of the YafQ toxin of the DinJ-YafQ TA system. YafQ expression resulted in inhibition of translation, but not transcription or replication. Purified YafQ exhibited robust ribonuclease activity in vitro that was specifically blocked by the addition of DinJ. However, YafQ associated with ribosomes in vivo and facilitated rapid mRNA degradation near the 5` end via cleavage at AAA lysine codons followed by a G or A. YafQ(H87Q) mutants lost toxicity and cleavage activity but retained ribosome association. Finally, LexA bound to the dinJ-yafQ palindrome and triggered module transcription after DNA damage. YafQ function is distinct from other TA toxins: it associates with the ribosome through the 50S subunit and mediates sequence-specific and frame-dependent mRNA cleavage at 5`AAA - G/A3` sequences leading to rapid decay through the mRNA degradosome.

Since their target in eukaryotes is conserved, TA toxins are excellent new candidates for breast and prostate cancer treatment because they not only enlist a potent toxin, but their cognate antitoxin can be recruited to block toxin activity in non-cancerous cells. Towards this cause, we identified the applicability of YafQ as a novel cancer therapeutic. Ectopic overexpression of YafQ in human embryonic kidney cells led to a progressive cytopathic effect, cellular detachment, and a 90% decrease in viability; these effects were rescued by co-expression of the DinJ antitoxin. Levels of GAPDH and -actin mRNA were significantly reduced and new protein synthesis was inhibited upon YafQ expression. Lastly, the cleaved form of poly-ADP-ribose polymerase was detected upon YafQ expression, indicating apoptotic cell death. The levels of apoptotic death do not corroborate the levels of total cell death suggesting another form of cell death occurs, possibly autophagy. Taken together, the bacterial DinJ-YafQ TA system shows promise for use in the development of new cancer treatments.


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