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）：支原体生殖器（MG）是一种新兴的人类病原体，由于其在男性和女性的生殖道疾病中的病因学作用越来越多，尽管在感染过程中诱导了特定的抗体，但在体内持续数月甚至数年的持续数月甚至数年的能力。免疫主导表面蛋白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。通过MGPBC表达位点与分布在整个染色体的架构上不同的同源（部分同源）截断序列（称为MGPARS）之间的互相分段重组来完成此介导的过程。除抗原变异外，MGPB和MGPC还可以进行相位变化，这是当前建议的重点。相变的相变无法粘附在宿主细胞上，可以通过在琼脂平板上培养的菌落的半吸附阴性[HA（ - ）]表型轻松检测。迄今为止，仅表征了两个相变。两者都在MGPBC表达位点，相邻的MGPAR位点和中间序列中包含大删除，因此是不可逆的。最初通过PCR检测到MGPBC表达位点序列与MGPAR序列相互交换的相变变量，但未进一步表征。但是，由此产生的序列的异常结构预测，它们可以通过产生它们的重组事件的逆转来恢复为野生类型，这表明它们在发病机理中的相关性。现在已经隔离和测序了几种相变，证实了它们的相互和可逆性。这些发现，以及比抗原变异频率高的相变的相变的检测，导致假说，相变为MG的生物学至关重要，并通过一种新型的可逆互惠重组方法而产生的，取决于MGPBC和MGPAR位置的异常差异化结构。当前的研究将探讨以下假设：相位变异（1）通过其他细菌中未发现的机制产生，（2）是通过针对MGPB保守区域的抗体选择的（2），并且（3）对于逃避体外和体内的免疫反应。 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来自MG感染灵长类动物模型的存档生殖器标本以及存档灵长类动物血清在体外选择相变的能力。这项研究将采用创新技术来揭示导致这种新型相位变异系统的机制，其基因组非常有限（580 kb）且几乎没有鉴定出的重组基因。此外，将首次揭示相变的生物学后果，并可能导致对新型预防和治疗方法的未来研究。