Phase Variation in Mycoplasma Genitalium

描述(申请人提供)：生殖支原体(MG)是一种新兴的人类病原体，因其在男性和女性生殖道疾病中的病原学作用而日益得到认可，并且尽管在感染期间诱导出特定的抗体，它仍能在体内持续数月甚至数年。免疫优势表面蛋白mgpB和mgpC的抗原性变异被认为是MG逃避宿主免疫反应并持续存在的关键。这个由RecA介导的过程是通过mgpBC表达位点和结构上不同的存档同源(部分同源)截断序列(称为mGPars)之间的相互片段重组完成的，这些序列分布在整个染色体上。除了抗原变异外，mGpB和mGpC还可以经历时相变异，这是目前提案的重点。相变体不能附着在宿主细胞上，可以通过琼脂平板上培养的菌落的血吸附阴性[HA(-)]表型很容易地检测到。到目前为止，只有两个阶段的变体被鉴定；两个阶段的变体都在mgpBC表达位点、相邻的mgPar位点和中间序列中包含大量缺失，因此是不可逆的。阶段变体，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，产生的序列的不寻常的结构预示着它们可以通过产生它们的重组事件的逆转而恢复到野生型，这表明它们在发病机制中具有相关性。现在已经分离和测序了几个相变体，证实了它们的可逆性和可逆性。这些发现，以及检测到的相变异体频率高于抗原变异体，导致了一种假说，即相变异体对MG的生物学至关重要，并由一种新的可逆互惠重组方法产生，该方法依赖于mgpBC和mgPar位点的不寻常差异结构。目前的研究将探索以下假设：(1)通过在其他细菌中没有发现的机制产生阶段变体，(2)由针对mgpB保守区的抗体选择，以及(3)在体外和体内对免疫反应的逃避是重要的。这些假说将通过(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的保守(非可变)免疫优势区域的兔抗体是否在体外选择相变，以及(3)通过列举从MG感染的灵长类动物模型纵向收集的生殖器标本中的相变以及存档灵长类血清在体外选择相变的能力来评估免疫逃避的体内意义。这项研究将使用创新的技术来揭示在一种极其挑剔的病原体中导致这种新的相变系统的机制，这种病原体的基因组非常有限(580kb)，几乎没有识别的重组基因。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。

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.