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Molecular Analysis of Escherichia coli Toxin-Antitoxin Systems

Chun-Yi Lin
M.S., National Taiwan University, 2002

Thesis Advisors: Masayori Inouye, Ph.D.
Graduate Program in Cellular & Molecular Pharmacology

Center for Advanced Biotechnology & Medicine (CABM)
Seminar Room 010

Thursday, May 1, 2014
12:00 p.m.


Bacterial subpopulations can survive under stresses, such as antibiotics treatment, without genetic change. This is identified as persistence. These persister cells are clinically multidrug tolerant. However, the mechanism of molecular switching to the persister phenotype is still unclear. The function of HipA, the toxin of HipBA Toxin-Antitoxin (TA) system, is relevant to persistence formation based on its inhibition of protein translation. However, this hypothesis only provided a weak explanation for the persistence-guided molecules, RelA and SpoT.
All free-living bacteria have TA systems in their genomes or extrachromosomal plasmids. These TA protein complexes are negatively regulated by their respective promoters. In the HipBA TA system, HipB is a DNA binding protein and negatively regulates four HipB-binding palindromic sequences located on the hipBA promoter. Based on bioinformatics data, we identified HipB as a global transcriptional regulator, which also regulates the relA promoter.
We investigated the toxicity of HipA which was identified independent of persistence. We carried out a series of biochemical experiments to identify the targets relevant to HipA toxicity. We truncated HipA to create the amino terminal domain HipA and the carboxyl terminal domain HipA to identify its targets. Moreover, we co-expressed the intact HipA with the proposed targets to confirm the interactions in vivo. Finally, we confirmed the protein, which demonstrated the ability to change the toxicity of the truncated HipA mutant when co-expressed.
Additionally, we are interested in applying TA systems in the fields of healthcare, such as cancer therapy. YeeV, the toxin of YeeUV TA system, is relevant to inhibit the polymerization of bacterial cytoskeletons, MreB and FtsZ. Based on the similar structures between the filaments of MreB and eukaryotic actin, we examined the physiological changing in human cells by expressing YeeV protein in cells.
Our results of HipBA not only revealed the possible pathway of HipA-mediated toxicity but also provide the new model for HipBA associated persistence. The YeeV-mediated human cell death provides a potential tool in advanced application of cancer therapy.

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