Screening And Identification Of Chlamydia Psittaci Inclusion Membrane Protein And Study On Its Inflammatory Mechanism

Objective: Chlamydia psittaci is a obligate intracellular parasitic pathogen,which can cause pneumonia,encephalitis and other diseases.Inclusion membrane proteins are a series of proteins secreted by Chlamydia and located in inclusion membrane,which play an important role in the structure and stability of inclusion membrane and the interaction between pathogen and host.Inclusion membrane proteins are instrumental to the growth,development and pathogenesis of Chlamydia,but little is known about the Cps inclusion membrane protein.This study intends to use the bioinformatics method to predict the Cps inclusion membrane protein,use indirect immunofluorescence to determine the subcellular localization of the predicted protein,and further explore the expression phase and basic function of the protein localized on the inclusion membrane.It will provide a new basis for elucidating the pathogenesis of Cps.Method: According to the characteristic bilobal hydrophobic domain of inclusion body membrane protein,the bioinformatics methodKyte-Doolittle algorithm was used to analyze the Cps 6BC amino acid sequence,and TMHMM and SMART were used to analyze the domain.Specific primers were designed according to the predicted protein gene sequence.Cps 6BC genomic DNA was used as template for PCR amplification.After enzyme digestion,the PCR products were ligated with pGEX-6p-1 or pET-28a（+）plasmid to construct a prokaryotic expression vector.After identification by cloning PCR and sequencing,the bacteria were cultured and expression of recombinant proteins were induced by IPTG.After the optimal induction conditions were determined,expression of recombinant proteins were induced and recombinant proteins were purified by GST binding resin or Ni-NTA resin.The protein concentration was determined by BCA method.The BALB/c mice were immunized with50 μg recombinant protein which was endotoxin removed by polymyxin B for 4 times,following by orbital blood was collected to prepare antiserum.IFA analysis was performed using polyclonal antibody as a primary antibody to determine the subcellular localization of the protein.The infected cells were collected at different time points for IFA,qRT-PCR and WB to analysis the protein expression phase.THP-1 cells were stimulated with endotoxin-free recombinant protein（CPSIT₀556 and CPSIT₀842）at 10 μg/mL for 24 h,and cells were collected for qRT-PCR to detect mRNA levels of proinflammatory cytokines IL-1β,IL-6,IL-8,IL-10 and TNF-α to determine whether these recombinant proteins can cause cellinflammatory response.THP-1 cells were stimulated with different concentrations of recombinant protein for 24 h or stimulated for different times with the same concentration of recombinant protein.Cells and culture supernatants were collected for qRT-PCR and ELISA,respectively,to determine the effect of recombinant protein stimulation concentration and stimulation time on the expression of pro-inflammatory cytokines.THP-1 cells were treated with recombinant protein for 0,15,30,60,120,180 min.Cells were collected and total cell proteins were extracted.Western blot analysis was performed to detect the phosphorylation levels of p38 kinase,JNK,ERK and NF-κB inhibitory molecule I-κB.THP-1cells which were stimulated with recombinant protein for 6 h were collected for IFA to analysis nuclear translocation of NF-κB p65 subunits.THP-1 cells were pretreated with specific inhibitor of p38,JNK,ERK and I-κBα for 30 min,following by were stimulated with recombinant protein.Cells and culture supernatants were analyzed by qRT-PCR and ELISA to determine whether these inhibitors could affect the expression of inflammatory cytokines,so as to further confirm the effect of MAPK pathway and NF-κB pathway on the expression of pro-inflammatory cytokines induced by recombinant protein.THP-1 cells were transfected with TLR2,TLR4,TLR6-siRNA and pDeNy-hMyD88,then were stimulated with recombinant protein.Cells and culture supernatants were collected for qRT-PCR and ELISA to determine the role of TLR receptorand its adaptor MyD88 in the expression of proinflammatory cytokines.Result:（1）Based on bioinformatics analysis,this study selected CPSIT₀201,CPSIT₀289,CPSIT₀321,CPSIT₀350,CPSIT₀357,CPSIT₀432,CPSIT₀532,CPSIT₀555,CPSIT₀556,CPSIT₀607,and CPSIT₀842 as prediction proteins;（2）According to the nucleotide sequences of these proteins,specific primers were designed,and the target fragments of Cps 6BC genome were successfully amplified using these primers.RCR showed that these target fragments were successfully inserted into the plasmid vector,and the sequencing results of the recombinant plasmid were identical with the nucleotide sequence published on NCBI,which confirmed that the prokaryotic expression vector of these proteins was constructed successfully.（3）Six GST recombinant protein and two His recombinant protein were successfully induced by IPTG,which were all detected in supernatant.The recombinant proteins were purified by GST-binding resin and Ni-NTA resin and were analyzed by Coomassie blue staining and WB which shown distinct bands at the corresponding molecular weights.（4）IFA results shown that CPSIT₀289,CPSIT₀357,CPSIT₀432,CPSIT₀607 and MOMP have similar distributions,all of which are located in inclusion,while distribution of CPSIT₀556 and CPSIT₀842 similar to Inc protein CPSIT₀846.（5）IFA results shown that the expression of CPSIT₀556and CPSIT₀842 can be observed at 18 h after infection,which arelocalized to the inclusion membrane;qRT-PCR results shown that transcription levels of CPSIT₀556 and CPSIT₀842 peaked at 12 h and 6h after infection,respectively;CPSIT₀556 protein expression were detected by WB at 12 h after infection,while expression of CPSIT₀842protein were detected at 6 h after infection.（6）CPSIT₀556 and CPSIT₀842 could induce the expression of IL-6 and IL-8 in THP-1 cells,and the optimal concentration of CPSIT₀556 was 15 μg/mL,the optimal time was 18 h,while the optimal concentration of CPSIT₀842 was 10μg/mL,the optimal time was 24 h.（7）Phosphorylation levels of p38,JNK,ERK and I-κBα and nuclear translocation of NF-κB p65 subunits were significantly increased when THP-1 cells were treated with CPSIT₀556and CPSIT₀842.After pretreatment by p38 kinase,JNK,ERK and I-κBαinhibitors,CPSIT₀556 and CPSIT₀842 induced IL-6 and IL-8expression were significantly decreased.（8）Expression of IL-6 and IL-8which were induced by CPSIT₀556 and CPSIT₀842 were significantly decreased when THP-1 cells were transfected by pDeNy-hMyD88;In addition,the expression of IL-6 and IL-8 induced by CPSIT₀556 were decreased after being interfered by the TLR4-siRNA,while both TLR2 and TLR4 could affect the expression of IL-6 and IL-8 induced by CPSIT₀842.Conclusion: 1.CPSIT₀289,CPSIT₀357,CPSIT₀432,CPSIT₀607 were located in the inclusion,while CPSIT₀556 andCPSIT₀842 were located in the inclusion membrane,and CPSIT₀556and CPSIT₀842 were Cps intermediate and early expressed proteins,respectively.2.CPSIT₀556 could induce the expression of IL-6 and IL-8in THP-1 cells via TLR4/MyD88-dependent MAPKs and NF-κB pathway;while CPSIT₀842 could be recognized by TLR2 and TLR4,and could induce the production of IL-6 and IL-8 in THP-1 cells by MyD88/MAPKs and MyD88/NF-κB pathway.