Recombination in a Minimal Organism

最小生物体内的重组

• 批准号: 8385992
• 负责人: PATRICIA A TOTTEN
• 金额: $ 23.01万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8385992
• 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

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.

描述（由申请人提供）： 生殖支原体（MG）是男性尿道炎的一种病因，并且越来越多地认识到其在宫颈炎、尿道炎、盆腔炎、输卵管因素不孕症和女性早产中的病因作用。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，通过抗原变异，其表面蛋白，MgpB和MgpC位于其复杂和独特的终端细胞器。支持这一假设，我们已经表明，在mgpB和mgpC，编码这些蛋白质的相邻基因的变化，是广泛的在体外和体内之间的宫颈/阴道分泌物从持续感染的妇女和实验感染的灵长类动物。尽管在其580 kb的基因组中鉴定出了有限的推定重组基因，但MG基因组的4%致力于mgpB和mgpC的不完整拷贝（称为MgPars）。我们已经表明，mgpBC和MgPar位点的序列之间的重组是通过相互的节段性重组来完成的，从而将该系统与其他细菌的系统区分开来，其中抗原变异是通过编码其表面蛋白的基因与存档的供体序列之间的单向重组（也称为基因转换）来实现的。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，以及在该生物体的分子生物学和发病机制研究中的丰富经验，预测我们成功完成了所提出的项目。我们假设：（1）通过对mgpBC变体群体测序，我们将鉴定重组的“热点”、优选的MgPar序列和与该过程相关的侧翼标记序列，（2）可能在体内遇到的选定环境条件将提高重组率，和（3）新的重组酶，以及与其它生物体中一般重组所需的酶同源的那些酶，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动物感染模型中导致抗原和相位变异的重组的生物学影响。这项研究是有意义的和创新的，基因，抗原，和相位变化的机制将被揭示为一个非常挑剔的病原体，重组基因很少，基因组非常有限。我们关注这种未充分研究的细菌的分子生物学和发病机制的潜在影响是巨大的，因为可以确定新的干预和治疗靶点。 公共卫生关系：拟议的研究重点是生殖支原体，一种与男性和女性生殖道（生殖器）疾病相关的新兴病原体。这种微生物可以在受感染的个体中持续数月甚至数年，增加性传播和Ascension到上生殖道的风险，从而导致严重疾病。我们的研究将研究这种细菌用于改变其表面蛋白质序列的机制，使其能够逃避宿主免疫反应，从而在感染个体中引起持续性疾病。