Prediction, Identification And Characters Of Effector Proteins Secreted Via Type Ⅲ Secretion System Of Chlamydophila Psittaci

Objective: To predict and screen effector proteins of type III secretion system inChlamydophila psittaci, and investigate the intracellular localization andcharacterization of these putative Chlamydophila psittaci effector proteins.Methods: In combination of computeral methods such as Effective T3and BPBAactool, and the relevent T3SS effector proteins literatures, effector protein code genesproteins were predicted in Chlamydophila psittaci. DNA was extracted as the temple,and specific pairs of Primers were synthesized, then PCR was used to amplify thesegenes. After digestion, they were ligated into pGEX-6P-1vector, and cloned into tothe vector. Sequencing was used to verify the identities. The protein expression wasinduced with IPTG in E.coli BL21, then identifed in Western blot. After theoptimization of time and temperature, we harvested the soluble proteins. Thepurification was proceeded according to GST*tag assay kit. After the concentrationsdetermination by BCA assay, the recombinated proteins were immunized BALB/cmice to produce polyclonalantibodies. Indirect ELISA were developed to test the titreof antibodies The subcellular localization was observed with Immunofluorescenceassay（IFA）. The expression and secretion was monitored at various time points. Thespecific reaction was detected with other chlamydia. Moreover, THP-1cells weretreated with effector proteins at different concentrations and various time points to testthe production of IL-1β, IL-6and TNF-α by ELISA.Results: In the cross-validation of Effective T3and BPBAac tool,9Cps T3SS geneswere selected: CPSIT₀357, CPSIT₀429, CPSIT₀461, CPSIT₀46, CPSIT₀490,CPSIT₀594, CPSIT₀785, CPSIT₀844, CPSIT₀846and CPSIT₀019, CPSIT₀020,CPSIT₀968, CPSIT₀969, CPSIT₀942were also included in our study according tothe other references. The sequencing verified the100%similarity. Except ofCPSIT₀357, CPSIT₀429, CPSIT₀968, CPSIT₀969, the other proteins were expressed in the soluble state. Specific humoral response were induced byrecombinant proteins in BALB/c mice, and the specific antibody titer of CPSIT₀785,CPSIT₀844,CPSIT₀846, CPSIT₀019, CPSIT₀020reach to1∶64000. IFA wassuccessfully to detect the asubcellular localization, and the results were showed asfollowing: CPSIT₀844and CPSIT₀846were observed in the inclusion membrance,while CPSIT₀461, CPSIT₀463, CPSIT₀490, CPSIT₀594, CPSIT₀785,CPSIT₀019, CPSIT₀020and CPSIT₀942were through the inclusion. In thesecretion monitoring with IFA, CPSIT₀844and CPSIT₀846were detected in theinclusion menbrance at18h pi. In the study of transcription and expression,CPSIT₀846was detected as early as2h, while CPSIT₀844was observed at12h, andthe lever were increased gradually, and Western blot indicated the same results.Western blot proved that the recombinant proteins could specifically react withChlamydophila psittaci antiserum,but failed with Chlamydia pneumoniae antiserumand Chlamydia trachomatis antiserum. The level of IL-1β, IL-6and TNF-α wereproduced with the stimulation of CPSIT₀844and CPSIT₀846.Conclusions：1. After the screening of T3SS effector proteins with Effective T3and BPBAac tool,the localization analysis by microscope are helpful to the prediction of T3SS effectorproteins.2. CPSIT₀844and CPSIT₀846were observed in the inclusion membrance, whileCPSIT₀461, CPSIT₀463, CPSIT₀490, CPSIT₀594, CPSIT₀785, CPSIT₀019,CPSIT₀020and CPSIT₀942were mainly throughout the inclusion.3. As effector proteins, CPSIT₀844and CPSIT₀846may play some roles in themid-cycle and in the early-cycle respectively. They can induce the production ofinflammatory cytokines. Besides, The recombinant CPSIT₀844and CPSIT₀846protein could specifically react with Chlamydophila psittaci antiserum, but failed withChlamydia pneumoniae antiserum and Chlamydia trachomatis antiserum.