Recombination in a Minimal Organism

描述(申请人提供)：生殖支原体(MG)是男性尿道炎的一种病因，在女性的子宫颈炎症、子宫内膜炎、盆腔炎、输卵管因素不孕和早产中的病因学作用越来越得到认可。不幸的是，这种细菌对针对细胞壁的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抗药性。尽管在感染过程中会诱导炎症反应和特异性抗体，但MG感染可能会在人类体内持续数月甚至数年。我们和其他人假设，这种持久性是基于MG通过其复杂而独特的末端细胞器中的两种表面蛋白--MgpB和MgpC的抗原变异来逃避宿主免疫反应的能力。支持这一假设的是，我们已经证明，编码这些蛋白的相邻基因mgpB和mgpC在体外和体内都在持续感染的女性的宫颈/阴道分泌物和实验感染的灵长类动物的分泌物中存在广泛的变异。尽管在其580kb的基因组中确定的重组基因有限，但MG基因组的4%用于mgpB和mgpC的不完整拷贝(称为mgpC)。我们已经证明了mgpBC和mgPar位点之间的重组是通过相互的片段重组完成的，从而区别于其他细菌的系统，在其他细菌中，抗原变异是通过编码其表面蛋白的基因和存档的供体序列之间的单向重组(也称为基因转换)实现的。我们开发了新的方法来测量环境条件对mgpBC/mgPar重组的影响，在MG中构建重组酶突变体的能力，以及在这种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)通过对一组mgpBC变异体进行测序，我们将确定与这一过程相关的“重组热点”、首选的mgPar序列和侧翼签名序列，(2)体内可能遇到的特定环境条件将提高重组率，(3)新的重组酶，以及那些与其他生物中普遍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)，评估重组导致抗原和阶段变化的生物影响，在人类和我们新开发的灵长类感染模型中。这项研究具有重要的意义和创新之处，因为对于一种极其挑剔的病原菌，重组基因很少，基因组非常有限，其基因、抗原和时相变化的机制将被揭示。我们对这种未被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，因为可能会确定干预和治疗的新靶点。

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.