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（由申请方提供）：肺炎支原体是年龄较大的儿童和年轻人肺炎的主要原因。支原体细胞和分子生物学的基本方面知之甚少，尽管支原体对公共卫生和农业的重大影响。更有效的预防和控制支原体感染的方法需要更详细地描述这些独特的无细胞壁原核生物的基本生物学过程。M.人类中的肺炎感染通过气溶胶传播，导致宿主呼吸道上皮在纤毛基部的定殖。M. pneumoniae细胞通过滑动运动而移动，这无疑有助于它们成功定位于营养优选部位的能力。因此，滑动运动可能构成一个毒力因子，但滑动对毒力的贡献尚未确定。滑行运动在一般细菌中，特别是在支原体中知之甚少。值得注意的是，在M.肺炎。最近的研究表明，M. pneumoniae细胞粘附突变体II-3也导致异常的细胞形态和滑动运动的丧失。然而，这种突变体的红细胞吸附回复突变体（称为II-3 R）仍然不活动，清楚地区分了P30在粘附和运动性方面的多种功能。运动性的丧失与回复突变体P30的一级序列在16个氨基酸区域上的差异相关。本研究的重点是P30在运动性背景下的结构-功能分析，在器官培养中评估运动性在仓鼠气管环毒力中的作用，以及鉴定和分析其他M. pneumoniae基因与滑行运动相关。将构建重组P30的衍生物，并评价其在P30背景下对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，并根据卫星生长的损失进行鉴定。将进一步表征保留细胞粘附能力的运动突变体。将通过测序和与基因组序列比较来鉴定插入失活的基因。将分离切除回复突变体，并通过转座子递送与重组野生型基因互补来拯救运动表型。将详细描述与滑行运动相关的蛋白质，包括确定亚细胞定位。将使用Boyden室或隔室培养皿评估趋化作用。将通过扫描电子显微镜测定细胞形态。