Determining the role of lipooligosaccharide on the Opa structural ensemble and the formation of an Opa-CEACAM complex

确定脂寡糖对 Opa 结构整体和 Opa-CEACAM 复合物形成的作用

• 批准号: 10333374
• 负责人: Meagan Leigh Belcher Dufrisne
• 金额: $ 7.04万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10333374
• 关键词: Adhesions; Affect; Affinity; Antibiotic Resistance; Antibiotics; Antigens; Bacteria; Binding; Biochemical; Biological; Biology; Biophysics; Carcinoembryonic Antigen; Cell Adhesion; Cell Adhesion Molecules; Cells; Characteristics; Circular Dichroism; Communities; Complementarity Determining Regions; Complex; Development; Disease; Disease Progression; Electron Spin Resonance Spectroscopy; Electrons; Electrostatics; Environment; Epithelial Cells; Epitopes; Event; Family; Flow Cytometry; Foundations; Genetic Recombination; Gonorrhea; Gram-Negative Bacteria; Hand; Health; Heterogeneity; Human; Image; Immune system; In Vitro; Infection; Interferometry; Investigation; Knowledge; Laboratories; Left; Lipopolysaccharides; Liposomes; Maintenance; Mediating; Membrane; Meningitis; Meningococcal meningitis; Mentorship; Molecular; Molecular Conformation; Mucous Membrane; NMR Spectroscopy; Neisseria; Neisseria gonorrhoeae; Neisseria meningitidis; Pathogenicity; Pattern; Phagocytosis; Play; Population; Process; Property; Proteins; Protocols documentation; Research; Role; Sampling; Scientist; Sexually Transmitted Diseases; Solid; Specificity; Spin Labels; Structure; Techniques; Training; Universities; Vaccines; Virginia; base; biophysical techniques; experimental study; extracellular; flexibility; global health; human pathogen; lipooligosaccharide; member; mutant; nanodisk; novel vaccines; pathogen; pathogenic bacteria; prevent; protein complex; receptor; reconstitution; uptake; vaccine development

Project Summary Neisseria gonorrhoeae (Gc) and Neisseria meninigitidis (Nm) are pathogenic bacteria causing the sexually transmitted infection gonorrhea and meningococcal meningitis respectively. Gc is a seriously world-wide health concern, especially considering the rise of antibiotic resistance in Gc strains. Nm is a less severe threat, however development of vaccines and antibiotics to prevent and treat Nm infection is still needed. Understanding Neisseria infection and pathogen-host interactions at the basic molecular level becomes crucial for these essential tasks. Opacity-associated (Opa) proteins in Neisseria are known to trigger engulfment of the bacteria into human cells via their interaction with members of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM or CCM) family. The structure of Opa has been characterized by our lab, but attempts to characterize the Opa-CCM molecular complex have conflicted with the established knowledge of the field. There is evidence that lipooligosaccharide (LOS), a dominant component of the neisserial outer membrane, can bind Opa proteins on adjacent bacteria, and that changes in LOS structure can affect bacterial opacity, Opa expression, Opa-CCM interactions, and Neisseria survival. For these reasons, I hypothesize that lipooligosaccharide is an important factor in Neisseria for maintenance of Opa loop structure, for facilitating Opa-CCM complex formation and for triggering engulfment of Neisseria via Opa-CCM interaction, and may be an essential molecular piece we have been missing in our previous experimental characterization. I propose to build upon the lab’s expertise with Opa and CCM proteins to (i) determine the role of LOS on the Opa protein and the structural assembly of Opa’s characteristic dynamic and hypervariable loops (Aim 1) and (ii) to assess the effect of LOS on the affinity of the Opa-CCM complex formation and its ability to trigger engulfment in human cells in a CCM-dependent manner. I will take a biophysical approach to Aim 1, utilizing spectroscopic techniques to characterize any change in Opa’s conformational heterogeneity or secondary structure in the presence of LOS, which will be a large part of my training. In a world-class environment, I will be trained in electron paramagnetic resonance (EPR) spectroscopic techniques, circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. In Aim 2, I will combine biochemical and cell-based approaches to characterize the effect of LOS on Opa-CCM protein complex formation and cellular uptake. This experimental approach will supplement my biophysical training and help me become a well-rounded scientist who can tackle complex biological questions with an arsenal of techniques. By describing the molecular basis for Opa-CCM interaction, we will provide a more solid foundation for the development of novel vaccines and antibiotics to the broader community studying Neisseria infection.

项目摘要 淋病奈瑟菌（Gc）和脑膜炎奈瑟菌（Nm）是引起性传播疾病的病原菌， 传播感染淋病和脑膜炎球菌性脑膜炎。Gc是一个严重的世界性健康 特别是考虑到Gc菌株中抗生素耐药性的上升。Nm是一个不太严重的威胁， 然而，仍然需要开发预防和治疗Nm感染的疫苗和抗生素。 在基本分子水平上理解奈瑟菌感染和病原体-宿主相互作用变得至关重要 完成这些重要任务。已知奈瑟氏球菌中的不透明相关（Opa）蛋白触发噬菌体的吞噬。 通过与癌胚抗原相关细胞因子的相互作用， 粘附分子（CEACAM或CCM）家族。Opa的结构已被我们的实验室表征，但 试图表征Opa-CCM分子复合物的尝试与以下已建立的知识相冲突： 外地有证据表明，脂寡糖（LOS），一种主要成分的奈瑟球菌外 膜，可以将Opa蛋白结合在相邻的细菌上，并且LOS结构的变化可以影响细菌的 不透明度、Opa表达、Opa-CCM相互作用和奈瑟氏菌存活。基于这些原因，我假设 脂寡糖是奈瑟氏球菌中维持Opa环结构的重要因子， Opa-CCM复合物形成和通过Opa-CCM相互作用触发奈瑟氏球菌的吞噬，并且可以是 这是我们在之前的实验表征中遗漏的重要分子片段。 我建议建立在实验室的专业知识与Opa和CCM蛋白，以（i）确定的作用，LOS 对Opa蛋白和Opa的特征动态和高变环的结构组装（目的 1)以及（ii）评估LOS对Opa-CCM复合体形成的亲和力及其触发的能力的影响。 以CCM依赖性方式吞噬人类细胞。我将采用生物物理学方法来实现目标1， 光谱技术来表征Opa的构象异质性或次级构象异质性的任何变化。 在LOS存在的情况下，这将是我训练的一大部分。在世界一流的环境中，我会 在电子顺磁共振（EPR）光谱技术，圆二色性和核 磁共振（NMR）光谱。在目标2中，我将结合联合收割机和基于细胞的方法， 表征LOS对Opa-CCM蛋白复合物形成和细胞摄取的影响。该实验 这种方法将补充我的生物物理学训练，帮助我成为一个全面的科学家， 复杂的生物学问题和一系列的技术。通过描述Opa-CCM的分子基础， 我们将为开发新型疫苗和抗生素提供更坚实的基础， 更广泛的社区研究奈瑟菌感染。