Mycoplasma pneumoniae Gliding Motility

肺炎支原体滑行运动

• 批准号: 6640091
• 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/6640091
• 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）：肺炎支原体是大龄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具有重大影响，但人们对支原体细胞和分子生物学的基本原理知之甚少。预防和控制支原体感染的更有效方法需要更详细地表征这些独特的无细胞壁原核生物的基本生物学过程。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纤毛基部定植。肺炎支原体细胞通过滑动运动来移动，这无疑有助于它们成功定位到营养偏好位点的能力。因此，滑行运动可能构成毒力因子，但滑行对毒力的贡献尚未确定。对于一般细菌，尤其是支原体，人们对滑动运动知之甚少。值得注意的是，在肺炎支原体的基因组序列中尚未鉴定出与已知运动基因（无论是滑动基因还是其他基因）的同源物。最近的研究表明，肺炎支原体细胞粘附突变体 II-3 中 P30 蛋白的缺失也会导致细胞形态异常和滑动运动丧失。然而，该突变体的血吸附回复体（命名为 II-3R）仍然不运动，清楚地区分了 P30 在粘附和运动方面的多种功能。运动性丧失与回复体 P30 一级序列在 16 个氨基酸区域的差异相关。该提案的重点是在运动性背景下对 P30 进行结构功能分析，评估运动性在器官培养中仓鼠气管环毒力中的作用，以及与滑动运动相关的其他肺炎支原体基因的鉴定和分析。将构建重组 P30 的衍生物并评估其对 P30 背景中运动性和粘附性的影响。此外，其他运动突变体将通过转座产生并根据卫星生长损失进行鉴定。保留细胞粘附能力的运动突变体将被进一步表征。插入失活的基因将通过测序并与基因组序列比较来鉴定。将分离切除回复体，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。与滑动运动相关的蛋白质将被详细表征，包括亚细胞定位的确定。将使用博伊登室或分隔培养皿评估趋化作用。细胞形态将通过扫描电子显微镜测定。