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.

肺炎支原体是一种人类病原体，慢性感染两种呼吸道。 和肺外部位，造成高发病率和相当大的费用。抗生素耐药性在- 这种传染病迅速蔓延，而且没有疫苗可用来对抗这种流行的传染病病原体。依附于宿主细胞， 它对肺炎支原体的毒力至关重要，主要由免疫显性但抗- 遗传可变的P1粘附素蛋白，它必须聚集在称为附着的膜突起上 细胞器(AO)起作用。虽然初步数据表明它的细胞内C-末端区域是必需的 对于定位，无论是P1定位到AO的机制还是其C末端的相互作用 该区域与其他已知的AO蛋白有关联。继续不知道P1是如何本地化和维护的 会阻碍对肺炎支原体毒力的全面了解，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的功能使其成为新的治疗靶点。本申请的总体目标是- 确定肺炎支原体的初级粘附素P1到达并与之相互作用的机制 AO，能够与宿主细胞黏附。正在讨论的潜在工作假说是P1位置- 糖化是由细胞内C-末端带正电的序列之间的相互作用引起的 P_1和带负电荷的蛋白质在AO内侧。这项拟议的研究的基本原理是 了解AO组装和功能的分子基础有望导致关键的 预防或减轻肺炎支原体感染严重程度的重要治疗药物，可能通过以下方式 损害AO功能或设计非可变蛋白聚集在AO上，增强其免疫原性- 并产生了成功的疫苗。这项提议的中心假设将受到客观检验，从而 通过追求确定AO定位信号的特定目的来实现该应用的目标 在P1粘附素蛋白的C-末端区域内，筛选结合伙伴，包括AO结构- 结构蛋白。通过测试它们的驾驶能力来实现对AO目标区域的识别- Meric Report Protein to the AO，一种经典的、强大的、合适的细胞生物学方法，随后进行检测 靶区内潜在关键氨基酸的突变体数量。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针之间的重组之间的相互作用 NANT融合蛋白和肺炎支原体细胞中的蛋白。这一贡献将是重大的，因为它是 预期将指导未来的研究努力，以开发有针对性的治疗干预策略 肺炎支原体感染和疾病过程中的Ao介导过程。建议的研究是创新的，因为- 因为它代表着对AO发展的关键阶段的关注，实质上偏离了现状 这一点从未被研究过，也就是将P1招募到行政办公室并在其内部维持的机制。