Phase Variation in Mycoplasma Genitalium

描述（由申请人提供）：生殖道支原体（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因学作用而日益得到认可，尽管在感染期间诱导了特异性抗体，但其在体内持续数月甚至数年的能力。免疫显性表面蛋白MgpB和MgpC的抗原变异被认为是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不同的存档同源（部分同源）截短序列（称为MgPars）之间的相互片段重组完成的。除了抗原性变异外，MgpB和MgpC还可能发生相变异，这是本研究的重点。期变异不能附着在宿主细胞上，可以很容易地通过琼脂板上培养的菌落的血吸附阴性[HA(-)]表型检测到。迄今为止，只有两种相变体被表征；两者都含有mgpBC表达位点、相邻MgPar位点和中间序列的大量缺失，因此是不可逆的。mgpBC表达位点序列与MgPar序列相互交换的相变异最初通过PCR检测到，但没有进一步表征。然而，结果序列的不寻常结构预测它们可以通过产生它们的重组事件的反向恢复到野生型，这表明它们在发病机制中具有相关性。现在已经分离出了几种相变体并对其进行了测序，证实了它们的互反和可逆性质。这些发现，以及相变异的检测频率高于抗原变异的频率，导致了相变异对MG生物学至关重要的假设，并且是由依赖于mgpBC和MgPar位点不寻常的差异结构的可逆互惠重组的新方法产生的。目前的研究将探索以下假设：(1)通过其他细菌中未发现的机制产生，(2)由靶向MgpB保守区域的抗体选择，以及(3)对于逃避体外和体内免疫反应很重要。这些假设将通过(1)评估体外获得的自发相变异的结构和可逆性来探索，(2)确定针对MgpB保守（非可变）免疫优势区域的兔抗体是否在体外选择相变异。(3)通过在纵向采集的灵长类动物MG感染模型的存档生殖器标本中枚举期变异来评估免疫逃避的体内意义，以及存档的灵长类动物血清在体外选择期变异的能力。这项研究将采用创新的技术来揭示这种具有非常有限的基因组（580 kb）和很少确定的重组基因的极其挑剔的病原体的新阶段变化系统的机制。此外，相位变化的生物学后果将首次被揭示，并可能导致未来研究新的预防和治疗方法。

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.