Recombination in a Minimal Organism

描述（由申请人提供）：生殖道支原体（MG）是男性尿道炎的一种病因，其在宫颈炎、子宫内膜炎、盆腔炎、输卵管性不孕和女性早产中的病因作用越来越被认识。不幸的是，这种细菌对细胞壁靶向抗生素和目前用于治疗这些严重生殖道疾病综合征的许多抗生素具有耐药性。MG感染可能持续数月，甚至数年，尽管在感染期间会诱导炎症反应和特异性抗体。我们和其他人假设，这种持久性是基于MG逃避宿主免疫反应的能力，通过其复杂和独特的末端细胞器中两种表面蛋白MgpB和MgpC的抗原变异。为了支持这一假设，我们已经证明，在体外和体内，在持续感染的女性和实验感染的灵长类动物的宫颈/阴道渗出液中，编码这些蛋白质的邻近基因mgpB和mgpC的变异是广泛的。尽管在其580 kb的基因组（任何自我复制的细胞生物中最小的基因组）中鉴定的假定重组基因集有限，但MG基因组的4%致力于mgpB和mgpC的不完整拷贝（称为MgPars）。我们已经证明，mgpBC和MgPar位点序列之间的重组是通过反向片段重组完成的，从而将该系统与其他细菌区分开来，在这些细菌中，抗原变异是通过编码其表面蛋白的基因与已归档的供体序列之间的单向重组（也称为基因转换）实现的。我们开发了测量环境条件对mgpBC/MgPar重组影响的新方法，在MG中构建重组酶突变体的能力，以及在该生物的分子生物学和发病机制研究方面的丰富经验，预示着我们将成功完成所提出的项目。我们假设：(1)通过对mgpBC变异群体进行测序，我们将确定重组的“热点”，优选的MgPar序列，以及与此过程相关的侧翼特征序列；(2)在体内可能遇到的特定环境条件将提高重组率；(3)新型重组酶，以及与其他生物中一般重组所需的酶同源的酶，将调节MG中的mgpBC/MgPar重组。这些实验将补充我们的研究（由其他机制资助），评估在人类和我们新开发的灵长类感染模型中重组导致抗原和期变异的生物学影响。这项研究具有重要的创新意义，因为它将揭示一种极其挑剔的病原体的基因、抗原和期变异机制，这种病原体具有很少的重组基因和非常有限的基因组。我们对这种未被充分研究的细菌的分子生物学和发病机制的关注的潜在影响是巨大的，因为可能会确定新的干预和治疗靶点。

DESCRIPTION (provided by applicant): Mycoplasma genitalium (MG) is a cause of urethritis in men and is becoming increasingly recognized for its etiologic role in cervicitis, endometritis, pelvic inflammatory disease, tubal factor infertility, and preterm birth in women. Unfortunately, this bacterium is resistant to cell wall-targeting antibiotics and to many of the antibiotics curretly used to treat these serious reproductive tract disease syndromes. MG infection may persist for months, and even years, in humans despite the induction of an inflammatory response and specific antibodies during infection. We and others have hypothesized that this persistence is based on the ability of MG to evade the host immune response by antigenic variation in two of its surface proteins, MgpB and MgpC located in its complex and unique terminal organelle. Supporting this hypothesis, we have shown that variation in mgpB and mgpC, the adjacent genes encoding these proteins, is extensive both in vitro and in vivo among cervical/vaginal exudates from persistently infected women and in experimentally infected primates. Despite the limited set of putative recombination genes identified in its 580 kb genome, the smallest of any self-replicating cellular organism, 4% of the MG genome is devoted to incomplete copies (termed MgPars) of mgpB and mgpC. We have shown that recombination between the sequences of mgpBC and the MgPar sites is accomplished by reciprocal segmental recombination, thus distinguishing this system from those of other bacteria in which antigenic variation is achieved by unidirectional recombination (also termed gene conversion) between the genes encoding their surface proteins and archived donor sequences. Our development of novel methods to measure the effect of environmental conditions on mgpBC/MgPar recombination, ability to construct recombination enzyme mutants in MG, and extensive experience in the study of the molecular biology and pathogenesis of this organism, predicts our successful completion of the projects proposed. We hypothesize that: (1) by sequencing a population of mgpBC variants, we will identify "hot spots" for recombination, preferred MgPar sequences, and flanking signature sequences associated with this process, (2) selected environmental conditions likely to be encountered in vivo will enhance the recombination rate, and (3) novel recombination enzymes, as well as those homologous to enzymes required for general recombination in other organisms, will modulate mgpBC/MgPar recombination in MG. These experiments will complement our studies (funded by other mechanisms) assessing the biologic repercussions of recombination leading to antigenic and phase variation, both in humans and in our newly developed primate model of infection. This study is significant and innovative in that the mechanisms of gene, antigenic, and phase variation will be revealed for an extremely fastidious pathogen with few recombination genes and a very limited genome. The potential impact of our focus on the molecular biology and pathogenesis of this understudied bacterium is great in that novel targets for intervention and treatment may be identified. PUBLIC HEALTH RELEVANCE: The proposed studies are focused on Mycoplasma genitalium, an emerging pathogen associated with reproductive tract (genital) disease in men and women. This organism can persist for months and even years in infected individuals, increasing the risk of sexual transmission and ascension into the upper reproductive tract to cause serious disease. Our study will examine the mechanisms used by this bacterium to vary the sequences of the proteins on its surface, allowing it to evade the host immune response to cause persistent disease in infected individuals.