Phase Variation in Mycoplasma Genitalium

生殖支原体的相变

• 批准号: 8770935
• 负责人: PATRICIA A TOTTEN
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-16 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8770935
• 关键词: Adherence; Agar; Amino Acid Sequence; Antibiotic Resistance; Antibiotics; Antibodies; Antibody Formation; Antigenic Variation; Appearance; Architecture; Archives; Bacteria; Bacterial Adhesins; Biological Assay; Biology; Cell Wall; Cells; Chromosomes; Chronic; Complement; Conserved Sequence; Detection; Development; Disease; Enhancing Antibodies; Event; Exhibits; Exposure to; Figs - dietary; Frequencies; Future; Genes; Genetic Recombination; Genital system; Genome; Hemadsorption; Human; Immune; Immune Sera; Immune response; Immunodominant Epitopes; Immunoglobulin Variable Region; In Vitro; Individual; Infection; Introns; Lead; Mediating; Membrane Proteins; Methods; Modeling; Molecular; Mycoplasma genitalium; Nature; Organism; Oryctolagus cuniculus; Pathogenesis; Phase; Phenotype; Prevention; Primates; Process; Proteins; Relative (related person); Resistance; Role; Serum; Site; Solid; Specimen; Surface; System; Techniques; Time; Treatment Protocols; Variant; Woman; base; design; genome sequencing; in vivo; innovation; interest; killings; men; mutant; novel; pathogen; protein profiling; public health relevance; reproductive; research study; screening

DESCRIPTION (provided by applicant): Mycoplasma genitalium (MG) is an emerging human pathogen increasingly recognized for its etiologic role in reproductive tract disease in men and women and its ability to persist for months and even years in vivo despite the induction of specific antibodies during infection. Antigenic variation of the immunodominant surface proteins, MgpB and MgpC, is thought to be critical for the ability of MG to evade the host immune response and persist. This RecA-mediated process is accomplished by reciprocal segmental recombination between the mgpBC expression site and architecturally distinct archived homeologous (partially homologous) truncated sequences (termed MgPars) distributed throughout the chromosome. In addition to antigenic variation, MgpB and MgpC can undergo phase variation, the focus of the current proposal. Phase variants are unable to adhere to host cells and can be easily detected by the hemadsorption negative [HA(-)] phenotype of colonies cultured on agar plates. Only two phase variants have been characterized to date; both contain large deletions in the mgpBC expression site, the adjacent MgPar site, and the intervening sequences, and thus are irreversible. Phase variants, in which the mgpBC expression site sequences were reciprocally exchanged with the MgPar sequences, were initially detected by PCR, but were not characterized further. However, the unusual architecture of the resulting sequences predicted that they could revert to wild type by the reverse of the recombination event that generated them, suggesting their relevance in pathogenesis. Several phase variants have now been isolated and sequenced, confirming their reciprocal and reversible nature. These findings, and the detection of phase variants at a frequency higher than that of antigenic variants, led to the hypotheses that phase variants are critical to the biology of MG and arise by a novel method of reversible reciprocal recombination dependent on the unusual differential architecture of the mgpBC and MgPar sites. The current study will explore the hypotheses that phase variants (1) arise via a mechanism not found in other bacteria, (2) are selected by antibodies targeting the conserved regions of MgpB, and (3) are important for the evasion of the immune response in vitro and in vivo. These hypotheses will be explored by (1) evaluating the architecture and reversibility of spontaneous phase variants obtained in vitro, (2) determining if rabbit antibodies targeting a conserved (nonvariable) immunodominant region of MgpB select for phase variants in vitro, and (3) assessing the in vivo significance of immune evasion by enumerating phase variants in longitudinally collected archived genital specimens from a primate model of MG infection and the ability of archived primate sera to select for phase variants in vitro. This study will employ innovative techniques to reveal the mechanisms responsible for this novel system of phase variation in an extremely fastidious pathogen with a very limited genome (580 kb) and few identified recombination genes. In addition, the biologic consequences of phase variation will be revealed for the first time and may lead to future studies of novel methods of prevention and treatment.

描述（由申请人提供）：生殖支原体（MG）是一种新兴的人类病原体，人们越来越认识到它在男性和女性生殖道疾病中的病因学作用，并且尽管在生殖过程中诱导了特异性抗体，但它能够在体内持续数月甚至数年。感染。免疫显性表面蛋白 MgpB 和 MgpC 的抗原变异被认为对于 MG 逃避宿主免疫反应并持续存在的能力至关重要。这种 RecA 介导的过程是通过 mgpBC 表达位点和分布在整个染色体上的结构上不同的归档同源（部分同源）截短序列（称为 MgPars）之间的相互片段重组来完成的。除了抗原变异之外，MgpB 和 MgpC 还可以发生相变，这是当前提案的重点。相变体无法粘附到宿主细胞上，并且可以通过琼脂平板上培养的菌落的血吸附阴性 [HA(-)] 表型轻松检测到。迄今为止，仅对两种相变体进行了表征；两者都在 mgpBC 表达位点、邻近的 MgPar 位点和插入序列中包含大量缺失，因此是不可逆的。相位变体，其中 mgpBC 表达位点序列与 MgPar 序列相互交换，最初通过 PCR 检测到，但没有进一步表征。然而，所得序列的不寻常结构预测它们可以通过逆转产生它们的重组事件而恢复为野生型，这表明它们在发病机制中的相关性。现在已经分离出几种相变体并进行测序，证实了它们的相互和可逆性质。这些发现以及以高于抗原变体的频率检测相变体，导致了这样的假设：相变体对于 MG 的生物学至关重要，并且是由依赖于不寻常的差异结构的可逆相互重组的新方法产生的。 mgpBC 和 MgPar 位点。目前的研究将探讨以下假设：相变体 (1) 通过其他细菌中未发现的机制产生，(2) 由针对 MgpB 保守区域的抗体选择，以及 (3) 对于逃避免疫反应很重要体外和体内。这些假设将通过以下方式进行探索：（1）评估体外获得的自发相变的结构和可逆性，（2）确定针对 MgpB 保守（非可变）免疫显性区域的兔抗体是否在体外选择相变，以及（3）通过枚举从 MG 感染的灵长类动物模型中纵向收集的存档生殖标本的相位变异以及存档灵长类动物血清选择的能力来评估免疫逃避的体内意义体外相变。这项研究将采用创新技术来揭示一种极其挑剔的病原体中这种新型相变系统的机制，该病原体的基因组非常有限（580 kb），并且几乎没有已识别的重组基因。此外，相变的生物学后果将首次被揭示，并可能导致未来对新的预防和治疗方法的研究。