Mycoplasma pneumoniae Gliding Motility

肺炎支原体滑行运动

• 批准号: 6884626
• 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 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6884626
• 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(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。尽管支原体对公众健康和农业有重大影响，但人们对支原体细胞和分子生物学的基本方面知之甚少。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。肺炎支原体在人类中的感染是通过气溶胶传播的，导致宿主呼吸道上皮在纤毛底部定植。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地定位到营养优先的位置。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。一般的细菌，尤其是支原体，对滑行运动知之甚少。值得注意的是，在肺炎支原体的基因组序列中还没有发现与已知的运动基因同源的基因，无论是滑行基因还是其他基因。最近的研究表明，肺炎支原体细胞黏附突变体II-3中P30蛋白的丢失也导致了细胞形态的异常和滑行运动的丧失。然而，这个突变体的吸血突变，命名为II-3R，仍然不能运动，清楚地区分了P30在黏附和运动方面的多重功能。运动能力的丧失与回复突变体P30在16个氨基酸区域的初级序列的差异有关。这项建议侧重于运动性背景下P30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，以及鉴定和分析与滑动运动相关的其他肺炎支原体基因。将构建重组P30的衍生物，并评估它们在P30背景下对运动性和粘附性的影响。此外，还将通过转座产生其他运动性突变体，并在卫星生长丧失的基础上进行鉴定。保持细胞黏附能力的运动性突变体将被进一步鉴定。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。将分离切除逆转株，并通过转座子传递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被详细描述，包括亚细胞定位的确定。化学运动将通过使用Boyden小室或分隔式培养板进行评估。细胞形态将通过扫描电子显微镜来确定。