Date of Award
12-1-2014
Degree Name
Master of Science
Department
Molecular Biology Microbiology and Biochemistry
First Advisor
Fisher, Derek
Abstract
Chlamydia spp. are obligate intracellular, gram negative bacterial pathogens responsible for a variety of diseases in humans and animals. While vaccines are available for C. felis and C. abortus, they are not available for other chlamydial species. Antibiotics are commonly used to treat chlamydial infections, but resistance has been seen in some species, and low dosage can lead to persistence. These problems support the need for further research into factors mediating chlamydial infections, which may identify novel therapeutic targets and/or vaccine strategies. Protein phosphorylation on serine, threonine, and/or tyrosine residues has been identified in prokaryotes (performed by Hanks-type kinases), and these modifications serve important roles in development and virulence. Three genes with homology to Hanks-type kinases were found when analyzing the C. trachomatis serovar D genome (PknD, Pkn1, and Pkn5). Amino acid alignments were performed to compare the similarity of these three kinases between nine different chlamydial species. To identify kinase-substrate pairings, the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system was used to test putative kinase substrates along with development of a C. trachomatis BACTH expression library. To further validate kinase substrate pairings and to perform kinase inhibitor screens, a kinase assay was developed. Mutant kinases were generated to validate kinase activity. Collectively, this work has built a framework for directed and library-based kinase-substrate interaction testing and for high throughput kinase activity analysis to aid in inhibitor discovery.
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