Transcriptional Expression Of Chlamydia Psittaci In-vitro And In-vivo Persistent Infection Models

Objective:Transcriptome analysis was performed on the whole genes of Chlamydia psittaci?C.psittaci?acute and persistent infection models to explore differentially expressed genes?DEGs?and related pathways involved in regulating persistent infections.By constructing a model of C.psittaci acute and persistent infection in BALB/c mice,it was explored whether DEGs obtained by cDNA microarray could be verified in C.psittaci infected mice.The systematic study of the mechanism of persistent infection of C.psittaci has important guiding significance for further exploring the prevention and treatment measures of persistent infection of C.psittaci.Methods:1.The cDNA microarray results of the acute and persistent infection models of the research group were compiled,and DEGs,Venn,GO classification,KEGG pathway analysis,and protein interaction network analysis were performed using bioinformatics software or website.2.C.psittaci 6BC was infected with a single layer of HeLa cells,and 2h later,it was replaced with fresh Chlamydia medium containing 35ng/mL rhIFN-?to construct a persistent infection model.Total RNA from cells with acute and persistent infection was extracted at 12h,24h,36h,48h,and 60h after infection,and some DEGs were verified using qRT-PCR technology.3.All BALB/c mice were anesthetized with ether,and each group of mice was infected with 5×10⁵ IFUs C.psittaci 6BC standard strain by nasal infection.Starting from the fifth day after infection,the four groups of mice were given sterile water,2mg/kg,20mg/kg,and 40mg/kg amoxicillin by gavage twice daily for 7d.The appetite and activity of the mice were observed daily,and their growth status,weight and survival rate were dynamically monitored.4.The infected mice were sacrificed 12d after the infection,and the lung tissue was taken aseptically.The C.psittaci load in the equal amount of lung tissue homogenate was simultaneously counted by indirect immunofluorescence and qRT-PCR to predict the growth status of C.psittaci in the lung tissue.H&E staining and S-P immunohistochemistry were performed on part of the lung tissue of 6 mice in each group to evaluate the pathological changes and the distribution of chlamydia.The morphology of C.psittaci particles of chlamydia inclusion in lung tissue of mice was observed by TEM.5.Lung tissue RNA was extracted and the transcription of C.psittaci gene in lung tissue was detected by qRT-PCR technology.Result:1.By cDNA microarray analysis,screen for DEGs with|log2FC|?1,At12h,24h,36h,48h,and 60h,the number of differentially expressed genes was 247,267,561,263,and 641,respectively.Venn analysis revealed that 177 consecutive differentially expressed genes were 12⁶0h,of which 68 genes were up-regulated and 109 genes are down-regulated.2.GO function enrichment analysis and KEGG pathway analysis were performed on DEGs.3.Analysis of the functions of DEGs found that up-regulated genes are mainly responsible for amino acid synthesis and metabolism,gene translation,lipid metabolism,nucleotide metabolism,carbohydrate and energy metabolism,and down regulation genes are mainly related to TCA cycle,gene expression and regulation,protein secretion,membrane protein,virulence,proteolysis and peptide transport,cell division and other related genes.QRT-PCR detected the expression of 20 target genes euo,prmC,ahpC,pth,pyk,atpE,pmpH,ydho,glgA,oppA₄,rpoN,gmk,yyaL,clpC,fliN,glmU,pbp3,ligA,hsp60,sucB₁,among which the results of 18 genes and the cDNA microarray were consistent?P<0.05?,and the up-regulation and down-regulation of 2 genes were not statistically significant?P>0.05?.4.After C.psittaci infection,the mice in the sterile Water group and the Amox 2 group showed severe symptoms of acute infection.H&E staining and S-P immunohistochemistry showed severe pathological damage to the lung tissue.In the Amox 20 group,the symptoms of the mice were reduced and not obvious,and the pathological damage to the lung tissue was mild.After the mice were given 40 mg/kg Amox,the symptoms were alleviated,and normal mice were restored without pathological damage to the lung tissue.Observed by transmission electron microscope,the morphology of inclusion in the lung tissue of the Water group was larger and filled with a large number of EB particles and a small amount of RB particles.The morphology of inclusion in the lung tissue of the Amox 20group was significantly smaller than that in the Water group,and it was found that they were filled with a large,loose electron density RB.5.QRT-PCR detection of 20 target genes euo,prmC,ahpC,pth,pyk,atpE,pmpH,ydho,glgA,oppA₄,rpoN,clpC,fliN,glmU,pbp3,ligA,hsp60,sucB₁ in lung tissue.In the case,8 genes were consistent with the cDNA microarray results?P<0.05?,and 12 genes were not statistically significant?P>0.05?.Conclusion:1.1008 C.psittaci genes were transcribed through cDNA microarray technology,and up-regulated genes include translation,amino acid metabolism,lipid metabolism,nucleotide metabolism,and glucose metabolism genes.Down-regulated genes include TCA cycle,expression regulation and transcription,protein secretion,proteolysis and peptide transport,membrane proteins,virulence,and cell division genes.2.Construct a mouse model of C.psittaci persistent infection by inducing amoxicillin.C.psittaci has a weak pathological response under persistent infection and is a hidden infection.The expressions of ahpC,euo,prmC of C.psittaci genes were up-regulated,and the expressions of pbp3,sucB₁,oppA₄,pmpH,and ligA were down-regulated.