Antigenic variation of Mycoplasma genitalium during persistent genital tract infection of pig-tailed macaques

项目摘要 生殖支原体（MG），一种性传播的细菌病原体，越来越多地被认为是 重大公共卫生问题。MG的患病率范围从基于人群的研究中的1-4%到更多 超过20%的患者在高风险获得性传播感染。MG的疾病谱是 类似于淋病奈瑟菌和沙眼衣原体，包括男性尿道炎和女性宫颈炎。 妇女特别值得关注的是，MG感染与严重的上生殖道后遗症有关， 妇女包括盆腔炎、不孕、早产和自然流产。令人担忧的是， MG的耐药性正在增加：40-100%的菌株对阿奇霉素完全耐药， 有些感染是完全无法用美国批准的疗法治疗的。最近FDA批准了两种MG 诊断测试肯定会提高公众对MG的认识和对改善治疗的需求。一个 迫切需要动物模型来了解MG感染的自然史，包括 持久性和免疫逃避，由于必须治疗症状性 感染我们已经优化了持续性生殖道感染的猪尾猕猴模型， 建议使用该模型来研究抗原变异在免疫回避中的作用。扩展我们以前的 研究了免疫显性的MgpB和MgpC粘附素的抗原性和相变化机制 蛋白质，我们将确定是否需要变异在生殖道的持久性。首先，使用全基因组 通过测序，我们将把感染18周期间变异的出现与 在三种MG感染的灵长类动物和以前的存档标本中， 灵长类动物实验第二，相同菌株与变异菌株重新感染清除生殖器感染的动物的能力。 将评估肠道感染以了解菌株特异性免疫。第三，不可变，“锁定” 将在体外构建和表征不能经历抗原变异的MG菌株。三 将用野生型和“锁定”MG的混合接种物对灵长类动物进行颈部接种， 持久性和上道Ascension的两个菌株同时超过18周的我们的模型。所有 受感染的灵长类动物将进行尸检，以检查子宫颈、子宫和输卵管的大体病理学 和组织学，并评估MG在上生殖道中的存在。这些实验不会 仅确定基因变异是否是持久性所必需的，但也将提供额外的观察结果， 个别灵长类动物的自然历史的下道持久性，上道Ascension，和免疫 反应（包括细胞因子、细胞浸润和保守型和可变型MG特异性抗体 抗原）。这些拟议的实验是非常重要的，因为它们填补了优先考虑的研究空白， NIAID赞助的小组，即动物模型的开发，MG在 严重的女性上生殖道疾病，以及MG血清学试验的发展。

PROJECT SUMMARY Mycoplasma genitalium (MG), a sexually transmitted bacterial pathogen, is increasingly recognized as a significant public health concern. The prevalence of MG ranges from 1-4% in population-based studies to more than 20% in patients at high risk of acquiring sexually transmitted infections. The disease spectrum of MG is similar to Neisseria gonorrhoeae and Chlamydia trachomatis, and includes urethritis in men and cervicitis in women. Of particular concern, MG infection is associated with serious upper reproductive tract sequelae in women including pelvic inflammatory disease, infertility, preterm birth, and spontaneous abortion. Alarmingly, antimicrobial resistance in MG is increasing: 40-100% of strains are completely resistant to azithromycin and some infections are totally untreatable with US approved therapies. The recent FDA approval of two MG diagnostic tests will certainly increase public awareness of MG and demands for improved treatment. An animal model is urgently needed to understand the naturally history of MG infection including mechanisms of persistence and immune evasion, studies that are difficult in patients given the imperative to treat symptomatic infection. We have optimized our pig-tailed macaque model of persistent genital tract infection and now propose to use this model to study the role of antigenic variation in immune avoidance. Extending our previous work defining mechanisms of antigenic and phase variation of the immunodominant MgpB and MgpC adhesin proteins, we will determine if variation is required for persistence in the genital tract. First, using whole genome sequencing we will correlate the appearance of variants during 18 weeks of infection with the appearance of antibodies specific to MgpB and MgpC in three MG-infected primates and archived specimens from prior primate experiments. Second, the ability of the identical vs variant strain to re-infect animals that clear genital tract infection will be assessed in order to understand strain specific immunity. Third, a non-variable, “locked” MG strain that is unable to undergo antigenic variation will be constructed and characterized in vitro. Three primates will be inoculated cervically with a mixed inoculum of wild type and “locked” MG to compare the persistence and upper tract ascension of the two strains simultaneously over the 18 weeks of our model. All infected primates will undergo necropsy to examine the cervix, uterus, and Fallopian tubes for gross pathology and histology, and to assess the presence of MG in the upper reproductive tract. These experiments will not only determine if gene variation is required for persistence but will also provide additional observations in individual primates of the natural history of lower tract persistence, upper tract ascension, and immune response (including cytokines, cellular infiltrates and antibodies specific for conserved and variable MG antigens). These proposed experiments are highly significant in that they fill research gaps prioritized by NIAID-sponsored panels, namely the development of an animal model, the exploration of the role of MG in serious upper reproductive tract disease in women, and the development of an MG serologic test.