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产生重大影响，但对支原体细胞和分子生物学的基本方面了解甚少。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、无细胞壁的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，导致宿主纤毛基部呼吸道上皮定植。肺炎支原体细胞通过滑动运动移动，这无疑有助于它们成功定位到营养首选位置的能力。因此，滑翔运动可能构成毒力因素，但滑翔对毒力的贡献尚未确定。一般来说，人们对细菌的滑动运动知之甚少，尤其是支原体。值得注意的是，在肺炎支原体的基因组序列中没有发现与已知运动基因同源的基因，无论是滑翔还是其他。最近的研究表明，肺炎支原体细胞粘附突变体II-3中蛋白P30的缺失也会导致细胞形态异常和滑行运动的丧失。然而，该突变体的血液吸附逆转录物II-3R仍然是非运动性的，这清楚地区分了P30在粘附性和运动性方面的多种功能。运动能力的丧失与一个16个氨基酸区域的可逆P30初级序列的差异有关。本研究将重点对P30在运动背景下的结构功能进行分析，评估运动在器官培养中对仓鼠气管环毒力的作用，以及鉴定和分析其他与滑翔运动相关的肺炎支原体基因。将构建重组P30衍生物，并评估其在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位产生，并在卫星生长损失的基础上识别。保留细胞粘附能力的运动性突变体将进一步表征。插入失活的基因将通过测序和与基因组序列的比较来鉴定。将分离出切除回复性基因，并通过转座子传递与重组野生型基因互补来挽救运动性表型。与滑翔运动相关的蛋白质将被详细描述，包括亚细胞定位的确定。化学运动将通过博伊登室或分区培养皿进行评估。细胞形态将通过扫描电子显微镜来确定。