Recombination in a Minimal Organism

最小生物体内的重组

基本信息

批准号：
8515926
负责人：
PATRICIA A TOTTEN
金额：
$ 18.15万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8515926
关键词：
Affect Amino Acid Sequence Anchored Polymerase Chain Reaction Animal Model Antibiotics Antibodies Antigenic Variation Archives Bacteria Biochemical Biological Assay Cell Communication Cell Wall Cells Cervical Cervicitis Cloning Complement Complex DNA Damage Development Disease Endometritis Enzymes Epidemiologic Studies Exonuclease Exudate Frequencies Funding Future Gene Conversion Gene Expression Regulation Genes Genetic Recombination Genital system Genome Genomics Heterogeneity Homologous Gene Hot Spot Human Immune response Immunoglobulin Variable Region In Vitro Individual Infection Infertility Inflammatory Response Intervention Iron Lead Mammalian Cell Measures Mediating Membrane Proteins Methods Modeling Molecular Molecular Biology Mutation Mycoplasma genitalium Organelles Organism Pathogenesis Pattern Pelvic Inflammatory Disease Peptide Sequence Determination Phase Population Premature Birth Primates Process Proteins Publishing Regulation Resistance Risk Role Sexual Transmission Site Surface Syndrome System Testing Urethritis Vagina Variant Virulence Woman base cell motility design enzyme activity experience helicase human disease in vitro Model in vivo innovation men mutant novel pathogen reproductive research study

项目摘要

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.

描述（由申请人提供）：支原体生殖器（MG）是男性尿道炎的原因，并且由于其在宫颈炎，子宫内膜炎，骨盆炎性疾病，管状因子不育症和女性早产而在宫颈炎，子宫内膜炎，骨盆炎性疾病，骨盆炎性疾病中的病因作用越来越受到认可。不幸的是，该细菌对细胞壁靶向抗生素具有抗性，以及用于治疗这些严重生殖道疾病综合征的许多抗生素。尽管在感染过程中诱导了炎症反应和特定抗体，但MG感染仍可能持续数月甚至数年。我们和其他人假设这种持久性是基于MG在其复杂且独特的末端细胞器中通过抗原变异MGPB和MGPC中抗原变异来逃避宿主免疫反应的能力。在支持这一假设的情况下，我们表明，编码这些蛋白质的相邻基因MGPB和MGPC的变异在体外和体内都广泛，颈椎/阴道渗出量持续感染的女性以及实验感染的灵长类动物。尽管在其580 kb基因组中鉴定出的一组推定重组基因，这是任何自我复制的细胞生物中最小的，但有4％的MG基因组专用于MGPB和MGPC的不完整拷贝（称为MGPAR）。我们已经表明，MGPBC序列与MGPAR位点之间的重组是通过相互分段的重组完成的，因此将该系统与其他细菌的系统区分开来，通过单向重组（也称为基因转化）来实现抗原变异的基因（也称为基因转换）。表面蛋白和存档供体序列。我们开发了衡量环境条件对MGPBC/MGPAR重组的影响的新方法，在MG中构建重组酶突变体的能力以及在研究该生物的分子生物学和发病机理方面的丰富经验，可以预测我们的项目成功完成建议的。我们假设：（1）通过对MGPBC变体的群体进行测序，我们将确定“热点”重组，首选MGPAR序列和与此过程相关的侧翼签名序列，（2）可能在VIVO中遇到的选择环境条件将提高重组率，（3）新的重组酶，以及与其他生物体一般重组所需的酶同源的重组酶，将调节MGPBC/MGPAR重组Mg。这些实验将补充我们的研究（由其他机制资助），评估重组的生物学影响，导致人类和我们新开发的灵长类动物感染模型中，导致抗原和相变。这项研究具有重要意义和创新性，因为对于极为挑剔的病原体，基因，抗原和相变的机制将被揭示，其重组基因很少，基因组非常有限。我们关注该研究细菌的分子生物学和发病机理的潜在影响很大，因为可以鉴定出新的干预和治疗目标。