Inhibition of Bacterial Toxin Activity by the Nuclear Stain, Drug 5
Advisor: Angela Brown
View: UGRS Research Poster (PDF)
The repeats-in-toxin (RTX) family of toxins includes proteins produced by Gram-negative bacteria such as a-HlyA, ClyA, and LtxA, secreted by Escherichia coli, Bordetella pertussis, and Aggregatibacter actinomycetemcomitans respectively. These toxins contribute to the pathogenesis of organisms by killing host cells. In the case of LtxA produced by A. actinomycetemcomitans, white blood cells are targeted, allowing the bacteria to avoid clearance by the host immune system. In these cells, LtxA binds to the cell membrane and is internalized via a lysosomal-mediated pathway.
The motivation for this project comes from a previous finding in our lab that Draq5, a membrane-permeant nuclear stain, prevents the internalization of LtxA. Unfortunately, Draq5 is toxic to host cells, as it binds to DNA and interferes with replication. In this project, the mechanism by which Draq5 inhibits LtxA internalization was investigated in pursuit of an alternative inhibitor of LtxA activity and A. actinomycetemcomitans pathogenicity.
Differential scanning calorimetry results demonstrated that Draq5 does not alter the onset of the gel-to-liquid transition temperature (TM) of the membrane, however various laurdan fluorescence experiments showed a decrease in membrane fluidity with increasing Draq5 concentrations. A subsequent experiment demonstrated that this Draq5-induced decrease in fluidity inhibits the ability of LtxA to destabilize the bilayer structure of the membrane, preventing a key step in LtxA activity. Furthermore, based on cytotoxicity results, the Draq5-induced fluidity change inhibits LtxA toxicity. Together, our results demonstrate that Draq5-mediated changes in membrane structure inhibit the ability of LtxA to bind to host cells, thus inhibiting the toxin’s activity.
About Joshua Webb:
Joshua N. Webb, a senior at Lehigh University, has been working in Dr. Angela Brown’s lab for almost three years. His work, focusing on the inhibition of bacterial toxin activity, is being published in The Journal of Membrane Biology and has been presented at numerous conferences and symposiums, including the Biophysical Society Annual Meeting, the American Institute of Chemical Engineers Annual Meeting, and the Biomedical Engineering Society Annual Conference. In May, Josh plans to graduate with a Bachelors of Science degree in Bioengineering and a minor in Health, Medicine, and Society. Josh’s love of research has fueled his decision to continue school in pursuit of a higher degree.