Mycoplasma pneumoniae Gliding Motility

肺炎支原体滑行运动

• 批准号: 6723643
• 负责人: DUNCAN C KRAUSE
• 金额: $ 21.72万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723643
• 关键词: Mycoplasma pneumoniae; SDS polyacrylamide gel electrophoresis; antiserum; bacteria infection mechanism; cell adhesion; cell morphology; cell motility; cinemicrography; density gradient ultracentrifugation; hamsters; immunofluorescence technique; mutant; protein structure function; respiratory epithelium; restriction endonucleases; scanning electron microscopy; tissue /cell culture; trachea; transposon /insertion element; virulence

DESCRIPTION (provided by applicant): Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults. Fundamental aspects of mycoplasma cell and molecular biology are poorly understood, despite the significant impact of mycoplasmas on public health and agriculture. More effective means of prevention and control of mycoplasma infections requires that the basic biological processes of these unique, cell wall-less prokaryotes be characterized in more detail. M. pneumoniae infections in humans are transmitted by aerosol, leading to colonization of host respiratory epithelium at the base of the cilia. M. pneumoniae cells move by gliding motility, which undoubtedly contributes to their ability to localize successfully to a nutritionally preferred site. Therefore, gliding motility probably constitutes a virulence factor, but the contribution of gliding to virulence has not been determined. Gliding motility is poorly understood in bacteria in general and in mycoplasmas in particular. Remarkably, no homologs to known motility genes, either gliding or otherwise, have been identified in the genome sequence of M. pneumoniae. Recent studies revealed that the loss of protein P30 in the M. pneumoniae cytadherence mutant II-3 also results in an abnormal cell morphology and loss of gliding motility. However, the hemadsorbing revertant of this mutant, designated II-3R, remains non-motile, clearly distinguishing the multiple functions of P30 in adherence and motility. Loss of motility correlates with a difference in the primary sequence of the revertant P30 over a 16-amino acid region. This proposal focuses on structure-function analysis of P30 in the context of motility, assessment of the role of motility in virulence in hamster tracheal rings in organ culture, and identification and analysis of other M. pneumoniae genes associated with gliding motility. Derivatives of recombinant P30 will be constructed and evaluated for their impact on motility and adherence in a P30 background. In addition, other motility mutants will be generated by transposition and identified on the basis of loss of satellite growth. Motility mutants retaining the ability to cytadhere will be characterized further. The genes insertionally inactivated will be identified by sequencing and comparison to the genome sequence. Excision revertants will be isolated, and the motility phenotype will be rescued by complementation with the recombinant wild-type gene by transposon delivery. The proteins associated with gliding motility will be characterized in detail, including determination of subcellular localization. Chemokinesis will be assessed by using a Boyden chamber or compartmentalized petri plates. Cell morphology will be determined by scanning electron microscopy.

描述（由申请人提供）：支原体肺炎是大儿童和年轻人肺炎的主要原因。尽管支原体对公共卫生和农业产生了重大影响，但对支原体细胞和分子生物学的基本方面却鲜为人知。更有效的预防和控制支原体感染的手段要求这些独特的无细胞壁原核生物的基本生物学过程更详细地表征。人类肺炎支原体感染通过气溶胶传播，导致宿主呼吸道上皮在西里亚底部定植。肺炎支原体细胞通过滑行运动而移动，这无疑有助于其成功定位到营养优先的部位。因此，滑行运动可能构成毒力因子，但是尚未确定滑行对毒力的贡献。在细菌中，特别是在支原体中，滑行运动的理解很少。值得注意的是，在肺炎支原体的基因组序列中鉴定出对已知运动基因的同源物。最近的研究表明，肺炎支原体中的蛋白质P30损失II-3也导致异常的细胞形态和滑行运动的丧失。然而，该突变体的半标记恢复剂（指定为II-3R）仍然是非运动的，清楚地区分了P30在粘附和运动中的多重功能。运动丧失与16个氨基酸区域的恢复P30的一级序列的差异相关。该提案的重点是在运动性的背景下对P30的结构 - 功能分析，评估仓鼠气管在器官培养中毒力中的作用，以及对与滑行运动相关的其他肺炎支原体基因的鉴定和分析。重组P30的衍生物将在P30背景下对它们对运动性和依从性的影响进行构建和评估。此外，其他运动突变体将通过换位产生，并根据卫星生长的损失确定。保持Cytadhere能力的运动突变体将进一步表征。插入灭活的基因将通过测序和与基因组序列进行比较来鉴定。切除恢复体将被分离出来，并通过转座子递送与重组野生型基因互补来挽救运动表型。与滑行运动相关的蛋白质将进行详细表征，包括测定亚细胞定位。趋化因子将通过使用Boyden室或分室化培养皿进行评估。细胞形态将通过扫描电子显微镜确定。