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Publication Date

2013-12-03

Availability

UM campus only

Embargo Period

2013-12-03

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Microbiology and Immunology (Medicine)

Date of Defense

2013-11-21

First Committee Member

Kenneth A. Fields

Second Committee Member

Kurt R. Schesser

Third Committee Member

Gregory V. Plano

Fourth Committee Member

Gennaro D'Uros

Fifth Committee Member

Pedro J. Salas

Sixth Committee Member

Travis Jewett

Abstract

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted disease as well as the leading cause of preventable blindness worldwide. Virulence of C. trachomatis depends on the secretion and translocation of effector proteins into host cells through a type III secretion system. One such effector is CT694, a protein that is specific to C. trachomatis and C. muridarum. Previous studies demonstrated that CT694 localizes to the plasma membrane of HeLa cells, where the C-terminus of CT694 interacts with the giant human protein Ahnak. Expression of CT694 also disrupts actin stress fibers; however, this is at least partially independent of interaction with the cytoskeletal protein-associated Ahnak, suggesting that a separate domain is responsible for this phenotype. Evidence is presented herein that endogenous CT694 localizes to host cell membranes and that this localization is dependent on a discrete domain, termed the membrane localization domain (MLD). Further, localization and localization-dependent activity of ectopically-expressed CT694 is also manifested in two unrelated species of yeast, suggesting that the host cell target of CT694 is highly conserved. Truncation analyses in fission yeast Schizosaccharomyces pombe further revealed the presence of a toxicity domain located between amino acids 151 and 222, which was confirmed by epifluorescent imaging of HeLa cells expressing CT694 deletion mutants. A synthetic genetic analysis in S. pombe was undertaken to identify pathways targeted by CT694 expression. As a result, the Elongator complex and Cdc42 were identified as novel host cell targets of CT694.

Keywords

chlamydia trachomatis; pathogen; sexually transmitted disease; genetic screen; cdc42

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