Study On The Role And Mechanism Of Glycogen Synthase A In Chlamydia Trachomatis-induced Hydrosalpinx

Objective:Chlamydia is one of the leading causes of sexually transmitted diseases in humans,and female genitourinary Chlamydia infection can cause inflammatory lesions that can lead to a variety of diseases,including hydrosalpinx,miscarriage,and ectopic pregnancy.GlgA is a novel secreted protein of Chlamydia,which is found in the cytoplasm of host cells and in the body of chlamydia,it may be an important pathogenic factor of hydrosalpinx induced by chlamydia.The aim of this study was to investigate the effect of GlgA on the pathogenicity of Chlamydia in mice,and to analyze the role of GlgA in the pathogenesis of Genital Tract Lesions induced by Chlamydia,and to provide a new theoretical basis for the treatment and prevention of Chlamydia infection.Methods:Female Balb/C Mice of 6-8 weeks were inoculated with purified wild-type chlamydia muridarum(WT),plasmid-free chlamydia muridarum(PF)and GlgA point mutation(P1D1 and P2B10)respectively,establishment of chlamydia infection model of mouse reproductive tract.At the 3rd,7th,10 th,14th,21 st,28th and 35 th day after infection,chlamydia was collected from mouse vaginal exfoliated cells and inoculated into monolayer Hela cells.After 24 h growth,the inclusion bodies were measured by immunofluorescence,to analyze the growth of different chlamydia in mouse reproductive tract.On the 60 th day after infection,intact genital tract tissue was isolated and the severity of hydrosalpinx lesions was evaluated.The extent of tubal dilatation was observed under microscope after H&E staining.In vitro,different chlamydia were inoculated into single-layer Hela cells,and the glycogen storage capacity of these different chlamydia were compared with each other by PAS staining,the infection ability of each strain of chlamydia in vitro was analyzed.In a parallel experiment,Mice were killed on the 10 th day after vaginal infection with chlamydia muridarum.The intact genital tract tissue was separated and divided into three parts,including ovary-oviduct segment(OV),uterus-uterus Horn segment(UH)and vagina-cervix segment(VC),to investigate the effect of GlgA deficiency on the ascending dispersal ability of chlamydia muridarum in mouse reproductive tract,the contents of chlamydia muridarum in different tissues were measured,and the homogenate of Mouse ovary-oviduct tissue was collected for multifactor analysis,to compare the levels of inflammatory cytokines in the upper genital tract of mice after different Chlamydia infection.Results:The incidence and severity of hydrosalpinx induced by PF,P1D1 and P2B10 were significantly lower than those induced by WT.The pathological changes of the fallopian tubes of mice infected with chlamydia muridarum for 60 days were observed under the microscope.It was found that the fallopian tubes of mice infected with WT strain were obviously dilated,the lumen of the fallopian tubes were obviously thinned,and many tissues were fibrotic,the fallopian tubes of mice infected with PF and GlgA point mutant strains were only slightly dilated or not.At the same time,there was obvious inflammatory cell infiltration in oviduct tissue of mice infected with wild-type chlamydia muridarum strain,but only slight inflammatory reaction was induced by plasmid deletion or GlgA mutation,there was no obvious inflammatory infiltration in mouse oviduct tissue.In addition,compared with WT,the shedding of chlamydia vaginalis in mice was significantly reduced at 3,7,10 and 14 days after infection with PF,and the shedding of GlgA mutants was significantly reduced at 7,10 and 14 days after infection,the duration of lower genital tract infection was shorter than that of wild plant.The inclusion bodies of chlamydia muridarum were stained with PAS.The inclusion bodies of wild chlamydia muridarum showed deep purple-red color and rich in glycogen.However,the glycogen content in the inclusion bodies of GlgA deficient chlamydia decreased significantly.In addition,the number of inclusion bodies of PF,P1D1 and P2B10 progenies was significantly lower than that of WT in the reinfection assay after 30 hours of chlamydia infection.In the parallel experiment,compared with WT,PF,/P1D1/P2B10 in Mouse ovary-oviduct tissue were significantly reduced,and the contents of PF and P2B10 in vagina-cervix tissue were lower than WT.Multifactor analysis of tissue homogenate showed that,compared with WT strain,almost all cytokines were significantly reduced after PF infection,and the contents of IL-12P40,IL-12P70,MIP-1β 和 IL-1α in group P1D1,IL-12P40,IL-12P70 and MIP-1β in group P2B10 were significantly reduced.Conclusions:(1)After vaginal infection,the degree of hydrosalpinx and the degree of tissue lesion in mice induced by GlgA deficiency chlamydia were significantly reduced.GlgA deficient chlamydia infection were quickly cleared by the Mice’ immune systems and were unable to grow and reproduce in the reproductive tract.(2)GlgA point mutation at different sites could decrease the ability of glycogen storage of chlamydia in different degree,and the ability of reproduction and infection of chlamydia in vitro were decreased.(3)GlgA deficiency can inhibit the upward spread of chlamydia in the mouse reproductive tract,decrease the content of cytokines in the tissue,and weaken the inflammatory response in the mouse oviduct tissue,these results suggest that GlgA is an important pathogenic factor in the pathogenesis of chlamydia induced hydrosalpinx in mice.