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.

肺炎支原体细菌是一种人类病原体，长期感染呼吸道 和肺外部位，导致高发病率和相当大的费用。抗生素耐药性存在于—— 迅速增加，并且没有疫苗可以对抗这种流行的传染原。粘附宿主细胞， 这对于肺炎支原体的毒力至关重要，主要由免疫显性但抗- 基因可变的 P1 粘附素蛋白，必须聚集在称为附件的膜突出处 细胞器（AO）发挥作用。尽管初步数据表明其胞内C端区域是必需的 对于定位，既不包括 P1 定位到 AO 的机制，也不包括其 C 端的相互作用 区域与其他 AO 蛋白是已知的。仍然不知道 P1 是如何本地化和维护的 AO 内将妨碍对肺炎支原体毒力的完全了解并阻碍发展 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和 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 的目的是确定肺炎支原体的初级粘附素 P1 到达并相互作用的机制。 AO，能够粘附宿主细胞。正在解决的基本工作假设是 P1 locali- 化是由细胞内 C 末端区域带正电荷的序列之间的相互作用赋予的 P1 和 AO 内部带负电的蛋白质。拟议研究的基本原理是 了解 AO 组装和功能的分子基础预计将导致关键技术的发展 预防或减轻肺炎支原体感染严重程度的重要治疗药物，可能通过 损害 AO 功能或将非可变蛋白改造为聚集在 AO 处，增加其免疫原性 性并导致成功的疫苗。该提案的中心假设将得到客观检验，从而 通过追求确定 AO 定位信号的特定目标来实现本应用的目标 在 P1 粘附素蛋白的 C 端区域内，筛选结合伙伴，包括 AO 结构 天然蛋白质。 AO 目标区域的识别将通过测试其驱动芯片的能力来实现。 将 meric 报告蛋白连接到 AO，这是一种经典、强大且合适的细胞生物学方法，然后进行测试 目标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体之间的相互作用来完成 南特融合蛋白和来自肺炎支原体细胞的蛋白质。这一贡献将是巨大的，因为它 预计将未来的研究工作转向针对目标的治疗干预策略的开发 肺炎支原体感染和疾病期间 AO 介导的过程。拟议的研究具有创新性 因为它通过关注 AO 发展的关键阶段，代表了对现状的实质性偏离 从未被研究过，即 P1 被招募到 AO 并在其内部维持的机制。