Mycoplasma penetrans tip structure and function

描述(由申请人提供)： 穿透性支原体是一种与免疫功能低下的患者相关的细菌，已被认为是艾滋病进展的潜在辅助因素。虽然它究竟如何损害宿主细胞尚不清楚，但像许多其他致病支原体一样，它通过一个极化的附件附着在宿主细胞上，附着细胞器(AO)也是细胞在滑动运动中的前端。干扰AO介导的过程，包括其组装、黏附和运动，是开发可缓解感染患者的治疗药物的潜在目标，其中包括相当数量的HIV阳性患者。此外，了解支原体是如何产生细胞极性的，对于试图理解世代的细胞生物学家通常是有用的。支原体一直是极简主义细胞的模型。 在更复杂的生物体中，从其他原核生物到人类细胞，细胞极性的变化。将穿透支原体基因组与较远的支原体物种如肺炎支原体和移动支原体进行比较，发现这些物种的AO蛋白没有同源基因。穿透支原体具有与其他物种不同的超微结构特征，包括其潜在的细胞骨架成分。此外，与移动分枝杆菌不同，穿透分枝杆菌的运动不直接依赖于三磷酸腺苷的水解。所有这些数据表明，穿透支原体在表面上只是与其他物种中类似的结构趋同，因此不能从这些物种的信息中推断穿透支原体AO组装和功能的分子基础。另一方面，穿透支原体是穿透支原体的近亲，它有一个超微结构相似的AO，其基因组中含有我们初步鉴定为穿透支原体AO蛋白的同源蛋白，包括一系列细胞骨架蛋白和一个候选粘附素，P42。虽然生化和细胞生物学研究已经开始揭示与穿透支原体有关的成分和机制，我们将继续使用它们，但这种有机体中缺乏遗传系统大大减缓了解决这些问题的进展。然而，我们的初步结果表明，与穿透分枝杆菌不同，穿透分枝杆菌能够被Tn4001衍生的转座子转化，四环素抗性可以作为一个可选择的标记。在这项建议中，我们描述了旨在将穿透支原体作为遗传系统来模拟穿透支原体的实验，研究绿色荧光蛋白融合到初步鉴定的AO组分的定位，以及在该蛋白的基因中产生零突变。我们还将通过抗体抑制研究来研究P42在穿透支原体黏附和运动中的作用。最后，我们将使用生化分离和质谱仪对穿透支原体的其他蛋白质进行鉴定。最后，这一领域的提案更新还将完成迈阿密大学本科生和研究生的培养。

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.