Mycoplasma – Structural Characterization of Host Cell Interaction Determinants

支原体是能够自我复制的最小和最简单的原核生物之一，其基因组可能小至600kb。它们是重要的病原体，可能会引起临床相关的表现。在之前的资助期间，我们使用冷冻电子显微镜解决了肺炎支原体和生殖支原体的主要黏附复合体Nap的结构，Nap负责病原体与宿主细胞的黏附，是表现感染所必需的。此外，我们还利用冷冻电子断层扫描技术研究了大量的生殖支原体突变体，从而解释了粘附性和运动性的机制。在这里，我们申请资金，通过两种途径继续这项研究：(I)使用单粒子冷冻电子显微镜来解决NAP细胞内部分的结构(NAPIC)，我们有证据表明它负责触发黏附释放机制，以及进一步的免疫优势和/或丰富的支原体表面蛋白(特别是P116/MG075和MPN474/MG328)。这些结构将补充我们以前的主要粘附素的结构，并应该为我们提供对粘附性的完整机械理解。从机理上理解粘连有可能促进针对这些病原体的新疗法的开发，这些病原体是出了名的难以用目前的抗生素控制。(Ii)使用冷冻电子断层扫描和聚焦离子束方法，我们希望研究支原体与宿主细胞的直接相互作用，以了解感染是如何表现出来的。特别是，我们还希望看到支原体与上皮纤毛的粘连，这显然会在感染过程中导致纤毛功能障碍。对于上述两个途径，我们已经做了大量的前期工作，无论是独立的还是作为充满活力的协作社区的成员。最后，最近的研究加强了我们工作的动机，这些研究报告了肺炎支原体对呼吸道病毒感染的临床结果的关键作用。

Mycoplasmas are among the smallest and simplest prokaryotes capable of self-replication, with a genome which may be as small as 600 kbases. They are significant pathogens that may induce clinically relevant manifestations. In the previous funding period we used cryo- electron microscopy to solve the structure of the main adhesion complex of Mycoplasma pneumoniae and Mycoplasma genitalium, the Nap, that is responsible for the adhesion of the pathogens to host cells and indispensable for manifesting the infection. In addition, we studied numerous mutants of M. genitalium by cryo-electron tomography, which allowed for explaining the mechanism of adhesion and motility. Here we apply for funding to continue this study using two avenues: (i) Using single-particle cryo-electron microscopy to solve the structure of the intracellular part of the Nap (Napic), for which we have evidence that it is responsible for triggering the adhesion release mechanism, and of further immunodominant and/or abundant Mycoplasma surface proteins (specifically P116/MG075 and MPN474/MG328). The structures will complement our previous structures of the main adhesin and should provide us with a complete mechanistic understanding of the adhesion. Mechanistic understanding of the adhesion has the potential for facilitating development of new therapeutics against these pathogens that are notoriously difficult to control with current antibiotics. (ii) Using cryo- electron tomography and a focused-ion-beam approach, we would like to investigate the direct interaction of the Mycoplasmas with host cells to understand how the infection manifests. In particular, we would also like to visualize the adhesion of Mycoplasmas to epithelial cilia, which apparently causes ciliary dysfunction during the infection. For both of the above avenues we have already done significant prior work, both independently and as members of a vibrant collaborative community. Finally, the motivation for our work is reinforced by recent studies that have reported a critical role of Mycoplasma pneumoniae for the clinical outcome of viral infections of the respiratory tract.