Graduate Thesis Or Dissertation


Analysis of chlamydial and host proteins associated with infection Public Deposited

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  • Chlamydiae are obligate intracellular bacteria that infect a variety of eukaryotic hosts and affect normal host processes. Within host cells, their developmental cycle takes place inside non-acidified vacuoles termed inclusions. An inclusion membrane composed primarily of secreted chlamydial synthesized proteins called Incs encloses the inclusion. At this location, Incs have a direct access to the host cytosol and are hypothesized to mediate important host- chlamydial interactions. One such Inc is IncA, a protein that participates in fusion of chlamydial inclusions. Host proteins that are localized to the inclusion are also hypothesized to participate in these interactions. Such interactions are thought to facilitate chlamydial intracellular development and survival. As a genetic system to manipulate chlamydiae does not currently exist, characterizations of such proteins rely on the use of contemporary molecular techniques. In this work, these techniques were utilized to (1) identify and characterize host phosphoproteins whose abundance and subcellular localization were altered as a result of chlamydial infection and (2) analyze the transcription of the incA mRNA in clinical isolates that have incA sequence polymorphisms, lack detectable IncA, and have chlamydial inclusions that are non-fusogenic. Adducin, Raf-1 and PKB (protein kinase B) are previously unreported host proteins whose abundance and subcellular localization are altered in Chlamydia- infected cells. Adducin is a cytoskeleton- associated, actin- capping protein whose phospho- protein abundance is depleted in the Triton X-100 soluble (TS) fractions of infected cells, specifically at mid-to-late time points post- infection. However, adducin abundance is unchanged in the total protein lysates, suggesting that the subcellular localization of the phosphorylated protein is affected by chlamydial infection. A fraction of adducin is also localized at the margin of the chlamydial inclusion and localization is independent of intact microtubules or actin. Raf-1 is a signaling protein whose abundance is also depleted in the TS fractions of infected cells. While phospho- PKB abundance is comparable in the total protein lysates between C. trachomatis- infected and mock- infected cells, the phospho- PKB is depleted in the total protein lysates of C. caviae- infected cells. This result suggests that C. caviae infection affects PKB phosphorylation events. The transcription analysis of incA demonstrated that incA is transcribed in cells infected by the wild type or non- fuser strains, initiating at a common transcriptional start site. However, the abundance of incA transcripts in cells infected by a non- fuser strain is reduced relative to those infected by the wild type strains.
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