Mycoplasma penetrans tip structure and function

描述（由申请人提供）： 反式支原体是一种与免疫功能低下患者相关的细菌，并被认为是艾滋病进展的潜在辅助因子。虽然它究竟如何损害宿主细胞尚不清楚，但与许多其他致病性支原体一样，它通过极化附属物附着于宿主细胞，即附着细胞器（AO），这也是滑动运动期间细胞的前端。干扰AO介导的过程，包括其组装、粘附和运动性，是开发治疗剂的潜在目标，该治疗剂可以缓解感染患者，其中包括大量HIV阳性个体。此外，了解支原体，这已作为模型的最低限度的细胞，产生细胞极性，将一般提供信息的细胞生物学家试图了解代 在更复杂的生物体中，从其他原核生物到人类细胞。M.与更远相关的支原体种如肺炎支原体和移动的支原体的反式基因组揭示了这些种的AO蛋白的同源物的缺乏。分枝相对于这些其他物种，反式AO具有独特的超微结构特征，包括其潜在的细胞骨架元素。与M不同，移动的，M.反式运动不直接依赖于ATP水解。所有这些数据都表明M.反式AO仅在表面上与其他物种中的类似结构收敛，因此理解M的分子基础。不能从这些物种的信息推断出反式AO组装和功能。另一方面，衣阿华支原体是M.它的基因组中含有我们初步鉴定为M的蛋白质的同系物。P42是一系列细胞骨架蛋白和一种候选粘附素。虽然生化和细胞生物学研究已经开始揭示与M。我们将继续使用它们，但这种生物体中遗传系统的缺失显著减缓了解决这些问题的进展。然而，我们的初步结果表明，与M。M. Iowae能够用Tn4001衍生的转座子转化，四环素抗性可用作选择标记。在这个建议中，我们描述了旨在开发M的实验。iowae作为遗传系统来模拟M.反式AO，研究绿色荧光蛋白融合到初步鉴定的AO组分的定位，以及在该蛋白质的基因中产生无效突变体。我们还将研究P42在M.通过抗体抑制研究的抗血小板粘附和运动性。最后，我们将使用生化分离和质谱来鉴定M.反式AO。最后，这一地区的建议更新也将完成本科生和研究生在迈阿密大学的培训。

DESCRIPTION (provided by applicant): Mycoplasma penetrans is a bacterium associated with immunocompromised patients, and has been implicated as a potential cofactor in AIDS progression. Although exactly how it harms host cells is unclear, like many other pathogenic mycoplasmas it attaches to host cells by a polarized appendage, the attachment organelle (AO), which is also the leading end of the cell during gliding motility. Interference with AO-mediated processes, including its assembly, adherence, and motility, is a potential target for the development of therapeutic agents that could relieve infected patients, which include a substantial number of HIV-positive individuals. Furthermore, understanding how mycoplasmas, which have served as models for minimalistic cells, generate cell polarity, will be generally informative to cell biologists attempting to understand generation of cell polarity in more complex organisms, ranging from other prokaryotes to human cells. Comparison of the M. penetrans genome with more distantly related mycoplasma species like Mycoplasma pneumoniae and Mycoplasma mobile reveals the absence of homologs of AO proteins of these species. The M. penetrans AO has distinct ultrastructural features relative to these other species, including those of its underlying cytoskeletal elements. Furthermore, unlike M. mobile, M. penetrans motility does not directly depend on ATP hydrolysis. All these data indicate that the M. penetrans AO is only superficially convergent with analogous structures in other species, and therefore understanding the molecular basis for M. penetrans AO assembly and function cannot be inferred from information from these species. On the other hand, Mycoplasma iowae, a close relative of M. penetrans, has an ultrastructurally similar AO, and its genome contains homologs of proteins we have preliminarily identified as M. penetrans AO proteins, including a series of cytoskeletal proteins and a candidate adhesin, P42. Although biochemical and cell biological studies have begun to reveal the components and mechanisms associated with the M. penetrans AO, and we will continue to employ them, the absence of a genetic system in this organism has significantly slowed progress toward solving these problems. However, our preliminary results indicate that unlike M. penetrans, M. iowae is capable of being transformed with Tn4001- derived transposons, with tetracycline resistance available as a selectable marker. In this proposal we describe experiments aimed at developing M. iowae as a genetic system to model the M. penetrans AO, studying the localization of a green fluorescent protein fusion to a preliminarily identified AO component as well as generating a null mutant in the gene for that protein. We will also investigate the role of P42 in M. penetran adherence and motility through antibody inhibition studies. Finally, we will use biochemical fractionation and mass spectrometry to identify other proteins of the M. penetrans AO. Finally, this AREA proposal renewal will also accomplish the training of undergraduate and graduate students at Miami University.