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Genetics of leukotoxin biosynthesis in Aggregatibacter actinomycetemcomitans

by
Maria P. Isaza
Interdisciplinary Ph.D Program

B. S., 2002, College of Saint Elizabeth

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

MSB B554

Monday, September 29, 2008
12:00 p.m.


Abstract

Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis (LAP) and extra-oral infections including infective endocarditis. A. actinomycetemcomitans expresses several virulence factors including leukotoxin (LtxA), a potent toxin that kills white blood cells. Although A. actinomycetemcomitans leukotoxin has been well-studied, little is known about its genetics and production. We developed two screens to isolate leukotoxin mutants. We took advantage of our recent observation that LtxA causes hemolysis on certain growth media. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system (PTS). HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cAMP production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild type ptsH in trans and real-time PCR (RT-PCR) analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA compared to the wild type strain. The levels of cAMP in the ptsH mutant were significantly lower than the wild type strain and LtxA production could be restored by adding exogenous cAMP to the culture.
In addition to identifying genes that are required for the biosynthesis of leukotoxin we wanted to study biological effects of LtxA on host cells. To elucidate this we have engineered HL-60 cells that stably express firefly luciferase and produce light that can be detected using an in vivo imaging system (IVIS). Treatment of HL-60luc cells with leukotoxin revealed that the bioluminescent viability assay is able to detect cell death earlier than the trypan blue dye exclusion assay. HL-60luc cells administered intraperitoneally (i.p) or intravenously (i.v.) were visualized in living mice. The rapidity and ease of detecting HL-60luc cells in biological fluid indicates that this cell line could be used in high throughput screens for the identification of drugs with anti-leukemia activity under physiological conditions.


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