Date on Master's Thesis/Doctoral Dissertation


Document Type

Doctoral Dissertation

Degree Name

Ph. D.


Microbiology and Immunology

Committee Chair

Worley, Micah J.

Author's Keywords

Chaperones; Secretion; Microbiology; Chlamydia; Type III; TARP




Chlamydia trachomatis is an obligate intracellular pathogen that utilizes a type III secretion system to enter mammalian cells and establish an intracellular niche. TARP, the translocated actin recruitment protein, is a chlamydial invasion protein known to be type III secreted by the metabolically inert elementary body upon docking to the mammalian cell surface. Because immediate secretion of TARP into host cells is necessary for entry, I hypothesized that a chlamydial chaperone binds to TARP and facilitates its translocation through the type III secretion apparatus. Most effector-binding type III secretion chaperones are small (14-18 kDa), have an acidic pI, and share a specific secondary structure of alternating alpha-helices (a) and beta-sheets (~). Typically, type III secretion chaperones dimerize and interact with their effectors as a complex of two molecules of chaperone to one effector molecule. Only 3 Chlamydia trachomatis proteins have been identified in EB' s that are predicted to be putative chlamydial type III effector chaperones. These are CT043, CT663, and CT088, which I have designated as SIc 1, SIc2, and Scc 1, respectively. These chaperones were tested for their interaction with the N-terminal 200 amino acids of TARP (HIS6 TARpl-200) by co immunoprecipitation. HIS6 TARpl-200 interacted specifically with SlcI, but not Scc1 or Slc2. This interaction was enhanced by coexpression of the recombinant proteins. To confirm this interaction and rule out the possibility of Slc 1 heterodimerization enhancing the interaction with TARP, I employed a 2-hybrid system to test for TARP: chaperone and chaperone:chaperone interactions. I confirmed the specific interaction between Cya18- TARpl-200 and Cya25-Slc 1. I was also able to detect SIc1 interaction with itself as well as confirm a few other previously described chaperone-chaperone interactions. Analysis by crosslinking and gel filtration chromatography indicated that Slc 1 forms a stable dimer in solution. Complexes of the Slci chaperone dimer with TARP in a 2:1 stoichiometry were detected following purification from co-expressing bacteria, but not following addition of singly purified species. Expression ofbeta-Iactamase fused to TARl-200 by the heterologous system Yersinia enterocolitica allowed for secretion of TARP into type-III inducing media (low calcium). Furthermore I was able to detect SIc I-dependent translocation of T ARP into HeLa cells via the heterologous type III secretion system of Y enterocolitica, and also by the SPI-2 system of Salmonella enterica serovar typhimurium.