| dc.description.abstract |
<p>The obligate intracellular pathogen, Chlamydia trachomatis, is becoming an ever
greater public health threat worldwide. Despite aggressive public health awareness
campaigns and treatment with antibiotics, chlamydial infections continue to be the
most frequently reported sexually transmitted infection in the United States and the
cause of 3% of worldwide blindness. While research into understanding various mechanisms
of chlamydial pathogenesis is ongoing, efforts to identify critical protein targets
are hampered by the lack of facile genetic manipulation systems available for Chlamydia.
Without the ability to perform genetic studies, researchers have employed chemical
biology tools to close the gap in understanding how Chlamydia survives and thrives
in the host cell.</p><p>Chlamydial protease-like activity factor (CPAF) has been identified
as a central virulence factor in chlamydial pathogenesis. Several studies have indicated
a role for CPAF-mediated degradation of host proteins in the late stages of infection.
CPAF is hypothesized to interfere with myriad host cell processes, including inflammation,
cell proliferation, cytoskeletal development, and immunity presentation. However,
recent studies have called into question the methods used to previously identify bona
fide in vivo CPAF targets, as CPAF has been shown to retain proteolytic activity even
in the presence of broad spectrum protease inhibitors. As a result of these new finding,
there is a renewed call to carefully identify CPAF substrates using methods that ensure
total inhibition of post-lysis proteolysis.</p><p>This dissertation aims to clarify
the role of CPAF in chlamydial pathogenesis and to identify mechanisms by which CPAF
exhibits substrate specificity. Because enzymes can manifest specificity through kinetic
mechanisms, sequence recognition, secondary site substrate binding, or protein structure
level specificity, multiple methods of biochemical characterization were employed
to distinguish between these modes of specificity. </p><p>Optimized HPLC-based and
fluorescence quenching assays were developed and used to investigate the chemical
and kinetic mechanism of CPAF proteolysis, as well as to characterize CPAF resistance
to broad spectrum protease inhibitors. Peptide library proteomics were designed to
probe active site sequence recognition of specific amino acids. Bioinformatic approaches
were used to recognize and annotate a cryptic PDZ-like domain in CPAF, which bears
strong structural similarity to human epithelial tight junction proteins. Using a
new endocervical cellular model of infection, a recently developed C. trachomatis
mutant lacking CPAF activity was investigated. Mass spectrometry proteomics analysis
was employed to detect differential cleavage of host proteins in endocervical cells
infected with CPAF+ and CPAF- strains of C. trachomatis. Lastly, methods for N-terminal
labeling and enrichment were adapted for further identifying CPAF substrates in a
cellular infection model. The subtiligase system for biotinylation of N-terminal amines
was adapted for integration with C. trachomatis infection assays and downstream mass
spectrometry proteomics. Ultimately, the dissertation offers clarification of the
role of CPAF in chlamydial infection and provides chemical biology tools for further
study of protease function in bacterial pathogenesis.</p>
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