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"IDENTIFICATION OF PROTEINS AND PATHWAYS REGULATING LEUKOTOXIN MEDIATED KILLING OF LEUKEMIC CELLS"

by
Manpreet Kaur
Oral Biology Program
B.S. 2007, York College, CUNY


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

Wednesday, July 16, 2014
12:00 P.M,, MSB C-600


Abstract

Leukotoxin (LtxA), a protein toxin secreted by an oral bacterium Aggregatibacter actinomycetemcomitans specifically targets and kills WBCs expressing lymphocyte function antigen-1 (LFA-1), a ß2 integrin that serves as a receptor for LtxA. To characterize proteins that regulate LtxA killing in monocytes, two molecular approaches were used. The first approach of genome wide silencing, followed by selection of genes conferring resistance to apoptosis identified proteins associated with vesicular trafficking in THP-1 cells. Rab3Gap2 was one protein that upon siRNA-mediated silencing partially inhibited LtxA killing by inhibiting internalization of LFA-1/LtxA complexes into the lysosomes. The knockdown of Rab3Gap2 also made the lysosomes more acidic. The second approach involving 2D difference gel electrophoresis and mass spectrometry, identified cofilin-an actin binding protein that regulates actin dynamics- to undergo dephosphorylation in response to LtxA in THP-1 and HL-60 cells. Cofilin phosphorylation is regulated by an upstream LIM kinase (LIMK), because of which LIMK inhibitor (LIMKi) was used to study the role of dephosphorylated cofilin in cell death. LIMKi treatment led to cofilin dephosphorylation, and enhanced LtxA killing and LtxA mediated lysosomal disruption by increasing surface levels and clustering of LFA-1. Both LIMKi and LtxA treatment also induced actin depolymerization, which could play a role in trafficking and surface distribution of LFA-1. Also, cofilin dephosphorylation was found to be LFA-1 dependent and specific to LtxA/LFA-1 interaction. Another approach of pharmacological inhibition was utilized to study the role of known survival proteins during LtxA mediated killing of Jurkat T lymphocytes. Inhibition of NF-kB pathway (by Bay11-7082), MEK/ERK pathway (by UO126) and PI3K/Akt pathway (by Wortmannin) significantly increased LtxA killing of Jurkat T cells. U0126 and Wortmannin enhanced LtxA killing by upregulating LtxA –mediated dephosphorylation of Erk and Akt respectively. Bay11-7082 enhanced LtxA cytotoxicity by inhibiting LtxA induced translocation of NF-kB into the nucleus and thus inhibiting expression of pro-survival proteins.
In conclusion, we have identified novel proteins and their potential role in vesicular trafficking of integrins and its impact on LtxA mediated cytotoxicity of monocytes. In T lymphocytes, we have shown the involvement of survival pathways in resisting LtxA killing.


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