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EXPRESSION OF AN EPITOPE TAGGED TARP EFFECTOR IN CHLAMYDIA TRACHOMATIS
Title
EXPRESSION OF AN EPITOPE TAGGED TARP EFFECTOR IN CHLAMYDIA TRACHOMATIS.
Creator
Nguyen, Brenda, Jewett, Travis, University of Central Florida
Abstract / Description
Previous studies performed on Chlamydia trachomatis have demonstrated how these obligate intracellular microbes invade host cells through the utilization of secreted effector proteins. One secreted effector called Tarp (translocated actin recruiting protein) is implicated in cytoskeleton rearrangements that promote bacterial entry into the host cell. The focus of our study is to create a plasmid that carries the tarP gene that when transcribed and translated from within Chlamydia trachomatis...
Show morePrevious studies performed on Chlamydia trachomatis have demonstrated how these obligate intracellular microbes invade host cells through the utilization of secreted effector proteins. One secreted effector called Tarp (translocated actin recruiting protein) is implicated in cytoskeleton rearrangements that promote bacterial entry into the host cell. The focus of our study is to create a plasmid that carries the tarP gene that when transcribed and translated from within Chlamydia trachomatis will generate a c-Myc epitope tagged Tarp. The tag will be used in future studies to track the progression of the protein through the infectious process and will allow us to distinguish this protein from the Tarp effector expressed from the endogenous wild type gene. The epitope-tagged Tarp expression plasmid will be used as a template to construct Tarp deletion mutants. The mutant forms will be created in regions that have been biochemically characterized and predicted to be important to the invasion process of the pathogen. Observations on the potential phenotypes of these mutants and the possibility of allelic exchange will also be pursued in the future.
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Date Issued
2013
Identifier
CFH0004385, ucf:45022
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004385
Chlamydia trachomatis Transformants Show a Significant Reduction in Rates of Invasion upon Removal of Key Tarp Domains
Title
Chlamydia trachomatis Transformants Show a Significant Reduction in Rates of Invasion upon Removal of Key Tarp Domains.
Creator
Parrett, Christopher, Jewett, Travis, Roy, Herve, Moore, Sean, University of Central Florida
Abstract / Description
Chlamydia trachomatis is an obligate, intracellular bacterium which is known to cause multiple human infections including nongonococcal urethritis (serovars D-K), lymphogranuloma venereum (serovars L1, L2, L3) and trachoma (serovars A-C). The infectious form of the bacterium, called the elementary body (EB), harbors a type III secreted effector known as Tarp (translocated actin recruiting phosphoprotein) which is a candidate virulence factor and is hypothesized to play a role in C....
Show moreChlamydia trachomatis is an obligate, intracellular bacterium which is known to cause multiple human infections including nongonococcal urethritis (serovars D-K), lymphogranuloma venereum (serovars L1, L2, L3) and trachoma (serovars A-C). The infectious form of the bacterium, called the elementary body (EB), harbors a type III secreted effector known as Tarp (translocated actin recruiting phosphoprotein) which is a candidate virulence factor and is hypothesized to play a role in C. trachomatis' ability to invade and grow within epithelial cells in a human host. C. trachomatis L2 Tarp harbors five unique protein domains which include the Phosphorylation Domain, the Proline Rich Domain, the Actin Binding Domain, and two F-Actin Binding Domains. Tarp has been biochemically characterized in vitro, but it has yet to be characterized in vivo due to a lack of genetic tools in C. trachomatis. Through the recent generation of a chlamydial transformation system, we have created transformants which express epitope tagged wild type or mutant Tarp effectors. In this thesis, C. trachomatis transformants expressing Tarp lacking one of the five biochemically defined protein domains were used to examine both bacterial invasion and bacterial development within mammalian host cells. Our results demonstrate that those EBs which harbor mutant Tarp missing either its Phosphorylation Domain or its Actin Binding Domain were less capable of host cell invasion. However, these transformants, once internalized, were capable of normal development when compared to wild type C. trachomatis or C. trachomatis harboring an epitope tagged wild type Tarp effector. These results suggest that transformant expressed Tarp lacking the Phosphorylation Domain or Actin Binding Domain may be acting as a dominant-negative effector protein. Ultimately, these results support the hypothesis that Tarp is a virulence factor for Chlamydia trachomatis. Furthermore, this data indicates that through the manipulation of the Tarp effector, C. trachomatis pathogenesis may be attenuated.
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Date Issued
2016
Identifier
CFE0006159, ucf:51142
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006159