鼠衣原体质粒缺失株生物学特性及免疫保护性研究

Partial characterization of plasmid-free Chlamydiamuridarum organism and evaluation of its protective efficacy in a mouse model

目的以鼠衣原体(Chlamydia muridarum,CM)质粒缺失株CMUT3和CM972为对象研究质粒缺失对CM生物学特性的影响,评价质粒缺失株的免疫保护作用。方法碘染色观察衣原体包涵体中糖原聚集情况,间接免疫荧光法(IFA)和Western blot方法检测糖原合酶(GlgA)的表达,离心辅助感染试验检测衣原体对HeLa细胞的感染力。C3H/HeJ小鼠阴道感染CM,感染后60d内每隔3或7d取生殖道分泌物,检测包涵体形成单位(IFU);感染60d后处死小鼠,分离生殖道组织,观察输卵管水肿程度。CBA/J小鼠阴道感染CMUT3,感染后60d用CM菌株进行再次感染,观察质粒缺失菌株抗CM感染的免疫保护效果。结果 CMUT3和CM972菌株碘染色呈阴性;质粒影响GlgA的表达,质粒缺失后GlgA表达水平下降;离心因素能显著提高新分离质粒缺失株CMUT3对HeLa的感染力;C3H/HeJ小鼠下生殖道感染质粒缺失株CMUT3和CM972后不发生输卵管积水,而质粒缺失株在小鼠下生殖道的增殖与野生株比较差异不明显;CBA/J小鼠下生殖道免疫CMUT3后再感染CM,其单侧输卵管积水发生率为15%(2/13),而先行感染CM后再次感染CM的小鼠单侧或双侧输卵管积水发生率89%(8/9)。结论 CM质粒缺失株在小鼠生殖道中的致病力减弱,小鼠下生殖道免疫CMUT3后可抑制CM再感染所致的病理变化,CM质粒缺失株具有潜在的疫苗研究价值。

Objectives To ascertain the effect of plasmid depletion on the biological function of Chlamydia muridarum and to assess the immunoprotective efficacy of plasmid-free C.muridarumin an intravaginal C.muridarum model.Methods HeLa cells infected with C.muridarum were stained with iodine to examine glycogen accumulation within intracytoplasmic inclusions.The level of GlgA expression in plasmid-free HeLa cells infected with C.muridarum was determined with an indirect immunofluorescence assay（IFA）and Western blot analysis.C.muridarum was titrated on HeLa cell monolayers with or without centrifugation for in vitro infection.The number of live organisms was expressed as IFUs per ml.The ratio of IFUs was calculated based on the titer of centrifuged C.muridarumto that of non-centrifuged C.muridarum.Female C3H/HeJ mice were intravaginally infected with 2.0 × 105 IFUs of plasmid-free and wild-type C.muridarumto evaluate pathology in the genital tract.To verify and track genital infection,cervical-vaginal swabs were collected from mice on day 3 postinfection,and the course of infection was followed on days 3,7,10,and 14 postinfection and every 7 days thereafter for 60 days postinfection.Bacteria recovered from vaginal/cervical swabs were titrated on fresh monolayer cells,and the results were expressed as log10 IFUs per swab.Sixty days after infection,all mice were sacrificed to observe gross pathology and hydrosalpinx.Female CBA/J mice were intravaginally vaccinated with plasmidfree C.muridarumand subsequently challenged vaginally with virulent C.muridarumto evaluate the protective efficacy of plasmid-free C.muridarum. Results The plasmid-free C.muridarumstrains CMUT3 and CM972 were unable to accumulate glycogen detectable with iodine within intracellular inclusions.The chlamydial plasmid affected glgA gene expression.Plasmid-free C.muridarumstrains CMUT3 and CM972 failed to secrete a detectable level of GlgA in IFA,and reduced GlgA expression was noted in Western blot analysis.Centrifugation enhanced the infectivity of all C.muridarum strains,regardless of whether the plasmid was present,but the fresh,plasmid-free C.muridarumstrain CMUT3 depended significantly on centrifugation for infection.The titer of CMUT3 increased〉70-fold with centrifugation while the titer of wild-type C.muridarumincreased up to 40-fold under the same conditions（P〈0.05）.C3 H/HeJ mice intravaginally infected with plasmid-free C.muridarumdisplayed minimal oviduct pathology while 4 mice infected with wild-type C.muridarumdeveloped hydrosalpinx on one or both sides（P〈0.05）.Live C.muridarum with or without the plasmid was shed in a similar amount from the lower genital tract of mice.Female CBA/J mice primarily infected with plasmidfree CMUT3 and challenged with virulent C.muridarumexhibited minimal pathology;2 of 13 mice exhibited hydrosalpinx.In contrast,8 of 9 mice infected with virulent C.muridarumand challenged with C.muridarumexhibited hydrosalpinx. Conclusion The oviducts were unaffected by plasmid-free C.muridarum.The oviducts of female CBA/J mice primarily infected with the plasmid-free strain CMUT3 were protected from disease despite a challenge with virulent C.muridarum.The pathogenicity of C.muridarum was highly attenuated in the genital tract of mice,and plasmid-free C.muridarum may serve as a live attenuated vaccine for administration in the genital tract.