The Pathogenic Machanism Of Cryptic Plasmid In Chlamydia Trachomatis

PARTâ… PROTEIN-PROTEIN INTERACTION ANALYSIS:A PLASMID-ENCODED PROTEIN IN CHLAMYDIA TRACHOMATISObjective To analyze the interactions among proteins encoded by the plasmid in Chlamydia trachomatis.Methods The DNA fragments of C. trachomatis plasmid genes, Pgpl-4 and Pgp8, were cloned into the vectors (pRAC and pRAC) and the target genes were in-frame fused to a-NTD and A. CI, respectively. The expression of cloned genes in Escherichia coli(E.coli) were determined by Western blotting. The protein-protein interactions were analyzed by a transcription-based bacterial two-hybrid system.Resultes The fusion proteins of a-Pgpl, a-Pgp3,CI-Pgp1,CI-Pgp3 and CI-Pgp4 were expressed in E.coli by the addition of Isopropyl P-D-1-thiogalactopyranoside (IPTG). In the presence of Pgp3 (encoded by plasmid pBR a-Pgp3), the expression of CI-Pgp3 activated transcription from a reporter gene in the bacterial two-hybrid assay strain. We did not detect interactions of Pgp3 with other test proteins.Conclusion Pgp3 interacts with Pgp3 in C. trachomatis.PARTâ…¡DECIPHERING PATHOGENIC ROLE OF PLASMID-ENCODED PROTEIN PGP3 AND PGP4 IN CHLAMYDIA TRACHOMATISObjective To evaluate the exact pathogenic roles of the Pgp3 and Pgp4 in HeLa 229 epithelial cells.Methods Used C. trachomatis wild type LGV L2, transformed strain L2GFP, and its isogenic strains carrying plasmid with Pgp3 or Pgp4 null mutation as models. The strains were identified by PCR; The EB progeny yields were determined by IFUs assay; The inclusion morphological were analyzed by IFA; The expression of MOMP were determined by Western blotting. The level of Glycogen were analyzed by anthrone assay; The capability of activitied TLR2 and NF-k B signals were determined by SEAP Quanti-Blue assay.Results We find that these C. trachomatis strains display a similar growth fashion in HeLa cells. Pgp3 and Pgp4 mutant release the contents of inclusion in early stage, although only Pgp4 exhibit altered inclusion morphology similar to the plasmidless strain. We demonstrate that deletion of Pgp4 leads to MOMP overproduction, in addition to reduction of the glycogen levels in C. trachomatis. The enhanced MOMP production in Pgp4 mutant does not correlate with its less capacity to activate TLR2 and NF-k B signals.Conclusion Pgp4 is implicated in cell surface characteristics, with Pgp3 correlating with properties related with survival, but not necessary to the immune stimulation in vitro.