Mycoplasma pneumoniae P1 adhesin: association with the attachment organelle

肺炎支原体 P1 粘附素：与附着细胞器的关联

• 批准号: 10308107
• 负责人: MITCHELL F BALISH
• 金额: $ 7.23万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-25 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308107
• 关键词: Address; Adherence; Amino Acid Sequence; Amino Acids; Antibiotic Resistance; Antigenic Variation; Bacteria; Bacterial Adhesins; Binding; C-terminal; Cell surface; Cells; Cellular biology; Charge; Chimeric Proteins; Chronic; Complex; Data; Development; Disease; Elements; Engineering; Extracellular Domain; Failure; Fostering; Goals; Health; Health Care Costs; Hospitalization; Impairment; Infection; Infectious Agent; Integral Membrane Protein; Intervention; Lead; Mediating; Membrane; Mission; Molecular; Morbidity - disease rate; Mycoplasma; Mycoplasma pneumoniae; Organelles; Outcome; Patients; Process; Proteins; Public Health; Quality of life; Recombinant Fusion Proteins; Reporter; Research; Respiratory System; Role; Severities; Signal Transduction; Site; Structural Protein; Structure; Testing; Therapeutic; Therapeutic Agents; United States National Institutes of Health; Vaccines; Virulence; Work; antibiotic resistant infections; base; combat; cost; human pathogen; immunogenicity; improved; infection rate; innovation; mutant; new therapeutic target; prevent; recruit; reduce symptoms; screening; targeted treatment; therapeutic development; vaccine access; yeast two hybrid system

The bacterium Mycoplasma pneumoniae is a human pathogen, chronically infecting both the respiratory tract and extrapulmonary sites, causing high rates of morbidity and considerable expense. Antibiotic resistance is in- creasing rapidly and no vaccines are available to combat this prevalent infectious agent. Adherence to host cells, which is critical for the virulence of M. pneumoniae, is primarily mediated by the immunodominant but anti- genically variable P1 adhesin protein, which must be clustered at a membrane protrusion called the attachment organelle (AO) to function. Although preliminary data indicate that its intracellular C-terminal region is required for localization, neither the mechanism by which P1 is localized to the AO nor the interactions its C-terminal region has with other AO proteins are known. Continuing not to know how P1 is localized to and maintained within the AO would prevent a complete understanding of M. pneumoniae virulence and impede development of therapeutics aimed at this process. The long-term goal of our research is to understand construction and function of the mycoplasma AO to develop it as a novel therapeutic target. The overall objective of this applica- tion is to identify the mechanism by which the primary adhesin of M. pneumoniae, P1, reaches and interacts with the AO, enabling adherence to host cells. The underlying working hypothesis being addressed is that P1 locali- zation is conferred by interactions between the positively charged sequence in the intracellular C-terminal region of P1 and negatively charged proteins on the AO interior. The rationale underlying the proposed research is that understanding the molecular basis for AO assembly and function is expected to lead to development of critically important therapeutic agents that prevent or reduce the severity of infection with M. pneumoniae, potentially by impairing AO function or engineering non-variable proteins to cluster at the AO, increasing their immunogenic- ity and resulting in successful vaccines. The central hypothesis of this proposal will be objectively tested, thereby attaining the goal of this application, by pursuing the specific aims of determining the AO localization signal within the C-terminal regions of the P1 adhesin protein and screening for binding partners, including AO struc- tural proteins. Identification of the AO targeting regions will be achieved by testing their ability to drive a chi- meric reporter protein to the AO, a classic, powerful, and appropriate cell biology approach, followed by testing of mutants in potential key amino acids within targeting regions. Testing for interactions with AO structural proteins will be done by both a bacterial two-hybrid approach and probing for interactions between a recombi- nant fusion protein and proteins from M. pneumoniae cells. This contribution will be significant because it is expected to direct future research efforts toward development of therapeutic intervention strategies targeting AO-mediated processes during M. pneumoniae infection and disease. The proposed research is innovative be- cause it represents a substantive departure from the status quo by focusing on a critical stage of AO development that has never been examined, namely the mechanism by which P1 is recruited to and maintained within the AO.

肺炎支原体（Mycoplasma pneumoniae）是一种人类病原体， 和肺外部位，导致高发病率和相当大的费用。抗生素耐药性- 并且没有疫苗可用于对抗这种流行的传染性病原体。对宿主细胞的粘附， 这对M.肺炎，主要由免疫显性但抗- 基因可变的P1粘附素蛋白，其必须聚集在称为附着的膜突起处 细胞器（AO）的功能。虽然初步数据表明其细胞内C-末端区域是必需的， 对于定位，P1定位于AO的机制和其C-末端的相互作用都不是 与其他AO蛋白的区域是已知的。继续不知道P1是如何定位和维护的 在AO内将阻止对M的完全理解。肺炎的毒力和阻碍发展 针对这一过程的治疗方法。我们研究的长期目标是了解建筑， 支原体AO的功能，将其开发为新的治疗靶点。本申请的总体目标是- 目的是确定M. pneumoniae，P1，到达并与 AO，使其能够粘附于宿主细胞。研究的基本假设是，P1局部- 通过细胞内C-末端区域中的带正电荷的序列之间的相互作用来赋予 P1和带负电荷的蛋白质在AO内部。拟议研究的基本原理是， 了解AO组装和功能的分子基础有望导致关键的 预防或降低M感染严重性的重要治疗剂。肺炎，可能通过 削弱AO功能或将非可变蛋白质工程化以聚集在AO，增加它们的免疫原性， 成功的疫苗。这一建议的核心假设将得到客观的检验，从而 通过追求确定AO定位信号的特定目的来实现本申请的目的 在P1粘附素蛋白的C-末端区域内，并筛选结合伴侣，包括AO结构， 天然蛋白质AO靶向区域的识别将通过测试其驱动chi的能力来实现。 梅里克报告蛋白的AO，一个经典的，强大的，和适当的细胞生物学方法，然后测试 靶向区域内潜在关键氨基酸的突变体。测试与AO结构的相互作用 蛋白质将通过细菌双杂交方法和探测重组蛋白之间的相互作用来完成， nant融合蛋白和M.肺炎细胞。这一贡献将是重大的，因为它是 预计将指导未来的研究工作，以发展治疗干预策略， AO介导的过程在M.肺炎感染和疾病。这项研究是创新的，因为- 因为它代表了对现状的实质性偏离，它关注的是AO发展的关键阶段 这一点从未被研究过，即P1被招募到AO并在AO内维持的机制。