Mycoplasma pneumoniae Gliding Motility

描述（由申请人提供）：肺炎支原体是大龄儿童和年轻人肺炎的主要原因，占所有社区获得性肺炎的20%。这种无细胞壁的原核生物通过滑动运动来移动，我们认为这有助于在呼吸道的传导气道中定植。人们对滑翔运动知之甚少，在肺炎支原体中没有发现已知的滑翔运动基因的同源物。在目前的项目期间，我们鉴定了一组不同的滑行相关基因，最终证明了仅末端细胞器是滑行马达，定义了滑行中几种末端细胞器成分的需求和功能，并产生了直接证据，证明了粘膜上皮的定植需要滑行。在此基础上提出的研究旨在进一步定义滑翔在发病机制中的作用，并探索滑翔的力学基础，包括三个具体目标。Aim 1研究滑翔机制，重点关注与滑翔相关的蛋白P65、P41和P24。终端细胞器随着P41的丧失而脱离细胞体，但仍保留着滑翔功能。P65基因的插入导致表面粘附素P30从末端细胞器拖到尾部，在那里它分离并在滑动的细胞后面留下痕迹。在缺乏P41的情况下，P24病灶似乎沿着细胞的长轴移动。在下一个项目期间，我们将应用生化和细胞成像方法，包括电子冷冻断层扫描，进一步探索P24， P41， P65和其他选定的蛋白质在滑翔中的作用。在Aim 2中，我们将详细分析某些滑翔突变体，这些突变体由于唯一注释的肺炎支原体蛋白磷酸酶和同源丝氨酸/苏氨酸蛋白激酶基因的破坏而表现出独特的草坪样生长。我们将确认这些突变的因果关系，并探讨蛋白激酶或磷酸酶功能的丧失对终端细胞器蛋白HMW1和HMW2磷酸化的影响。我们还将通过显微摄影详细检查这些突变体的细胞行为，以确定草坪样生长是如何实现的。在Aim 3中，我们将使用分化的正常人支气管上皮模型和野生型和滑翔缺陷支原体来探索滑翔运动如何在传导气道定植过程中特异性地促进粘膜纤毛防御的抵抗。肺炎支原体是大龄儿童和年轻人肺炎的主要病因，占所有社区获得性肺炎的20%。大多数感染会导致慢性和持续性呼吸道疾病，影响上学和工作效率，并可能导致永久性肺损伤。此外，越来越多的证据支持肺炎支原体在哮喘的发病和复发中发挥重要的促进作用。本文提出的研究将阐明滑行运动在导气管定植中的机制和作用。

DESCRIPTION (provided by applicant): Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults and accounts for 20% of all community-acquired pneumonia. This cell wall-less prokaryote moves by gliding motility, which we contend facilitates colonization of the conducting airways of the respiratory tract. Gliding motility is poorly understood, and no homologs of known motility genes, gliding or otherwise, are found in M. pneumoniae. In the current project period we identified a diverse set of gliding- associated genes, demonstrated conclusively that the terminal organelle alone is the gliding motor, defined the requirement and function of several terminal organelle components in gliding, and generated direct evidence that gliding is required for colonization of mucosal epithelium. The studies proposed here build upon that foundation to define further the role of gliding in pathogenesis and explore the mechanical basis for gliding, encompassing three specific aims. Aim 1 addresses the gliding mechanism, focusing on gliding-associated proteins P65, P41 and P24. The terminal organelle detaches from the cell body with the loss of P41 but retains gliding function. Insertions in the P65 gene result in the dragging of surface adhesin P30 from the terminal organelle to the trailing end, where it detaches to leave a trail behind the gliding cell. In the absence of P41, P24 foci appear to move along the long axis of the cell. In the next project period we will apply biochemical and cell imaging approaches including electron cryotomography to explore further the roles of P24, P41, P65, and other selected proteins in gliding. In Aim 2 we will analyze in detail certain gliding mutants exhibiting a distinctive lawn-like growth as a result of disruption of the genes for the only annotated M. pneumoniae protein phosphatase and cognate ser/thr protein kinase. We will confirm cause and effect for these mutants and explore the impact of loss of protine kinase or phosphatase function on the phosphorylation of terminal organelle proteins HMW1 and HMW2. We will also examine cell behavior of these mutants in detail by microcinematography to establish how lawn-like growth is achieved. In Aim 3 we will use a differentiated normal human bronchial epithelium model and wild-type and gliding-defective mycoplasmas to explore how gliding motility specifically contributes to resistance of mucociliary defenses in the colonization of conducting airways. Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults and accounts for 20% of all community-acquired pneumonia. Most infections result in respiratory disease that is chronic and protracted, impacting attendance at school and productivity in the workplace, and permanent lung damage can result. In addition, a growing body of evidence supports a significant, contributing role for M. pneumoniae in onset and recurrence of asthma. The studies proposed here will elucidate the mechanism and role of gliding motility in the colonization of the conducting airways.