Screening Of The Type Ⅲ Secretion System Putative Effector Proteins Of Chlamydophila Psittaci And Primary Study On The Effects Of Proinflammatory

Chlamydophila psittaci（ C. psittaci） belongs to the family Chlamydiaceae causing respiratory disease in pet birds and poultry. Pet bird owners, veterinary surgeons, and poultry workers are at risk of becoming infected by this zoonotic agent. Its symptom is similar with the common cold, so it is not easy to be found and leads to misdiagnosis in clinic. The flu-like symptoms and chronic infection may result in severe complications such as pneumonia, pulmonary edema, myocarditis, endocarditis, etc. Studies on chlamydial pathogenesis and illuminating an nosogenesis of effector protein become an area of great value in prevention and control the C. psittaci infection.The T3 SS effector proteins are highly associated with many chlamydial diseases and play an important role in Chlamydia pathogenesis. Inclusion membrane protein, Tarp, and Ct 694 interact with host cells in the process of C. psittaci infection that interferes with the normal metabolic activity of cells. Screening T3 SS effector proteins and studies on their biological characteristics provide important experimental basis for the Cps interaction with host cells. Therefore, we carried out the following experiments on clamydial pathogenesis and the nosogenesis of Cps T3 SS effector proteinsPartⅠ: Cps T3 SS is involved in the host inflammatory response by activating the JNK/ERK signaling pathwayChlamydia psittaci is a human zoonotic pathogen and the transmission of C. psittaci from birds to human results in severe respiratory disease. Objective: The aim is to investigate the role and mechanism of the type III secretion system（T3SS） of C. psittaci in regulating the inflammatory response in host cells using an in vitro infection model in the present study. Methods: C. psittaci-infected human mononuclear THP-1 cells were incubated with different reagents, including the specific T3 SS inhibitor INP0007, Fe SO4, inhibitors of c-Jun N-terminal kinase（JNK）, the extracellular signal-regulated kinase（ERK） or p38, and the levels of inflammatory cytokines including IL-1β, IL-6, IL-8, and TNF-α were analyzed using quantitative polymerase chain reaction（Q-PCR） and enzyme-linked immunosorbent assay（ELISA）.The levels of ERK, p38 and JNK phosphorylation were analyzed with Western blotting analysis. Our results verified that INP0007 inhibited Chlamydial growth in vitro, but the co-addition of exogenous iron completely reversed the growth deficit. INP0007 was demonstrated to inhibit the growth of C. psittaci and decrease the levels of interleukin（IL）-8, IL-6, tumor necrosis factor（TNF）-α and IL-1β. The addition of iron to INP0007-treated THP-1 cells infected with C. psittaci restored the Chlamydial growth; however, iron did not restore the production of inflammatory cytokines. These results demonstrated that the expression of inflammatory cytokines during infection was associated with the T3 SS. The cytokines production related with C. psittaci infection was reduced by incubation with the ERK and JNK inhibitors, whereas not by incubation with the p38 inhibitor. We concluded that Chlamydial T3 SS elicited the C. psittaci-infected host inflammatory responses by activating the JNK or ERK signaling pathways, but not the p38 signaling pathway.Part Ⅱ: Screening and identification of the T3 SS effector proteins of Chlamydophila psittaciThe aim is to screen and identify the T3 SS effector proteins of Chlamydophila psittaci. Methods: Using bioinformatics methods and combined with some relevent literatures, we screened three kinds of Cps T3 SS effector protein code genes. The genes were cloned by PCR and inserted into the expression vector p ET30 a to construct p ET30a-CPSIT₀531, p ET30a-CPSIT₀018 and p ET30a-CPSIT_p7 recombinant plasmids which were transformed into E. coli BL21, and then expressed and purified His-CPSIT₀531, His-CPSIT₀018 and His-CPSIT_p7 fusion proteins. The purified proteins were used to immunize BALB/c mice to produce polyclone antibodies, which were subsequently used to localize the endogenous proteins CPSIT₀531, CPSIT₀018 and CPSIT_p7 in C. psittaci-infected cells via an indirect immunofluorescence assay（IFA）. ELISA were carried out to identify the immunogenicity. Results: The recombinant expression plasmids p ET30a-CPSIT₀531, ET30a-CPSIT₀018 and p ET30a-CPSIT_p7 were successfully constructed, and then expressed and purified the fusion proteins. Specific immune response were induced by recombinant proteins in BALB/c mice, and ELISA results showed the specific antibody titer against CPSIT_p7 reached 1:1 000 000, CPSIT₀531 and CPSIT₀018 reached 1:640 000. IFA showed that the distribution pattern of the CPSIT₀018 and CPSIT₀531 is similar to that of the major outer membrane protein（MOMP）, but not to that of inclusion membrane protein A（Inc A）. CPSIT_p7 can be seen not only in inclusion body, but also in the cytoplasm of Cps-infected He La cells, which was limited in the inclusion body of Cps after it was treated with the T3 SS inhibitor. Conclusion: 1) CPSIT_p7, CPSIT₀531 and CPSIT₀018 recombinant proteins were successfully expressed and purified in prokaryotic expression system. 2) The three proteins had a good immunongenicity. 3) CPSIT₀018 and CPSIT₀531 are located on the bacterial organism in Cps-infected He La cells. CPSIT_p7 is a T3 SS secretory protein that can distribute in cytoplasm from inclusion body of Cps, but CPSIT₀018 and CPSIT₀531 are maybe structural proteins of Cps.Part Ⅲ: CPSIT_p7 is involved in the host inflammatory response by activating the JNK/ERK signaling pathwayIn the present study, we investigated the role and mechanism of the CPSIT_p7 from C. psittaci in regulating the inflammatory response in host cells using an in vitro infection model. Methods: CPSIT_p7-treated THP-1 cells were incubated with different reagents, including 30μM specific inhibitors of ERK, p38 or JNK, and the levels of inflammatory cytokines were analyzed by ELISA. The levels of ERK, p38 and JNK phosphorylation were analyzed with Western blotting analysis. We first analyzed the effects of CPSIT_p7 on CKs in THP-1 cells. The results showed that CPSIT_p7 induced THP-1 cells to produce CKs with a dose-dependent manner. When THP-1 cells were incubated for 0, 6, 12, 24 or 36 h with 10 μg/m L of CPSIT_p7, IL-6, IL-8 or IL-1β expression was highest at 24 h and TNF-α expression was highest at 12 h. CPSIT_p7 promoted phosphorylation of ERK and JNK, not p38 in THP-1 cells. The ERK and JNK inhibitors decreased the levels of IL-8, IL-6, TNF-α or IL-1β induce by CPSIT_p7. We conclude that CPSIT_p7 induces THP-1 cells to produce inflammatory cytokines such as IL-1β, IL-6, IL-8 or TNF-α by activating the JNK or ERK signaling pathways.