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"Mechanisms of LtxA-mediated Cell Death"

by
Kristina DiFranco
Oral Biology Program
B.S. 2008, Bucknell University


Thesis Advisor: Scott Kachlany, Ph.D.
Associate Professor
Department of Oral Biology

Thursday, October 3, 2013
10:00 AM, MSB C600


Abstract

Leukotoxin (LtxA) is a protein secreted from the oral bacterium Aggregatibacter actinomycetemcomitans. LtxA helps the bacterium avoid immune detection by binding to the 2 integrin lymphocyte-associated function antigen-1 (LFA-1) on human white blood cells (WBCs), resulting in cell death. However, the mechanism by which LtxA kills has not been well understood. In this study, the diverse ways LtxA initiates cell death in various WBCs was evaluated. In human lymphocytes, LtxA kills by a novel mechanism involving both LFA-1 and death receptor Fas, where LtxA interaction with these receptors initiates a FADD- independent/caspase-8 dependent signal transduction pathway ultimately leading to cell death. When monocytes are treated with LtxA, they undergo an entirely different mechanism, where LtxA activates two cell death pathways simultaneously. First, there is a rapid activation of caspases, but LtxA could overcome the inhibition of their activation and still intoxicate. However, inhibiting either vesicular trafficking or cathepsin D release from the lysosome resulted in significant inhibition of cytotoxicity, indicating that monocytic cells primarily die by a lysosomal-mediated pathway and that caspase activation is secondary. In addition, binding of LtxA to LFA-1 resulted in internalization of both LtxA and LFA-1, with LtxA localizing specifically to the lysosomal compartment.
To further elucidate the role that LFA-1 plays in LtxA-mediated cell death, we upregulated the receptor in low expressing cell lines and downregulated levels in high expressing ones. We found that while LFA-1 was important, it was not the determining factor in the kinetics or the mechanism of LtxA-mediated cell death; cell type appeared to be more significant. In addition, to describing the cell death pathway of human WBCs, we showed that rodent cells are killed by LtxA in an LFA-1-dependent manner and initiate death by an analogous lysosomal-mediated mechanism involving cathepsin D release.


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