Phase Variation in Mycoplasma Genitalium

描述（由申请方提供）：生殖支原体（MG）是一种新兴的人类病原体，由于其在男性和女性生殖道疾病中的病因作用以及尽管在感染期间诱导特异性抗体但其在体内持续数月甚至数年的能力而日益被认识。免疫显性表面蛋白MgpB和MgpC的抗原变异被认为是MG逃避宿主免疫应答并持续存在的关键。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存档同源（部分同源）截短序列（称为MgPars）之间的相互节段重组来完成的。除了抗原变异，MgpB和MgpC可以经历相位变异，这是当前提案的重点。相变体不能粘附于宿主细胞，并且可以通过琼脂平板上培养的菌落的血细胞吸附阴性[HA（-）]表型容易地检测到。到目前为止，只有两个相位变体被表征;两者在mgpBC表达位点、相邻的MgPar位点和间插序列中都含有大的缺失，因此是不可逆的。其中mgpBC表达位点序列与MgPar序列互换的相变体最初通过PCR检测，但未进一步表征。然而，所得到的序列的不寻常的结构预测，它们可以通过逆转产生它们的重组事件恢复到野生型，这表明它们在发病机制中的相关性。几个相位变体现已被分离和测序，确认其相互和可逆的性质。这些发现，以及检测相变体的频率高于抗原变体的频率，导致了相变体对MG的生物学至关重要的假设，并通过依赖于mgpBC和MgPar位点的不寻常的差异结构的可逆相互重组的新方法产生。目前的研究将探索以下假设：相变体（1）通过其他细菌中未发现的机制产生，（2）通过靶向MgpB保守区域的抗体选择，（3）对于逃避体外和体内免疫应答很重要。这些假设将通过以下方式进行探索：（1）评价体外获得的自发相变体的结构和可逆性，（2）确定靶向保守相的兔抗体是否能够抑制自发相的产生。MgpB的（非可变）免疫显性区域在体外选择相位变体，以及（3）通过在纵向收集的来自MG灵长类动物模型的存档生殖器样本中计数相变体来评估免疫逃避的体内意义感染和存档的灵长类动物血清在体外选择相变体的能力。这项研究将采用创新的技术来揭示这种新的系统的相位变化的机制，在一个非常挑剔的病原体具有非常有限的基因组（580 kb）和几个确定的重组基因。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.