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 淋病奈瑟氏菌（GC）和奈瑟氏菌（NM）（NM）是致病性细菌 分别传播感染淋病和脑膜炎球菌脑膜炎。 GC是全球严重的健康 关心，特别是考虑到GC菌株中抗生素抗性的兴起。 NM是一个不太严重的威胁， 但是，仍然需要开发疫苗和抗生素以预防和治疗NM感染。 了解基本分子水平的奈瑟氏菌感染和病原体宿主相互作用变得至关重要 对于这些基本任务。已知奈瑟氏菌中的不透明度相关（OPA）蛋白会引发吞噬 细菌通过与癌症抗原相关细胞成员的相互作用进入人类细胞 粘附分子（CECAM或CCM）家族。 OPA的结构以我们的实验室为特征，但是 尝试表征OPA-CCM分子络合物的尝试与已建立的知识相抵触 领域。有证据表明lipoligosacachide（LOS）是邻居外部的主要成分 膜，可以在相邻细菌上结合OPA蛋白，并且LOS结构的变化会影响细菌 不透明度，OPA表达，OPA-CCM相互作用和奈瑟氏菌生存。由于这些原因，我假设 Lipoligosacachide是维持OPA环结构的奈瑟氏菌的重要因素，以促进 OPA-CCM复合物的形成和通过OPA-CCM相互作用触发Neisseria的吞噬，可能是 我们先前的实验表征中缺少的一个必不可少的分子。 我建议以OPA和CCM蛋白质的专业知识为基础（i）确定LOS的作用 在OPA蛋白以及OPA特征性动态和高变量环的结构组件上（AIM 1）和（ii）评估LOS对OPA-CCM复合形成的亲和力及其触发能力的影响 以CCM依赖性方式吞噬人类细胞。我将采用一种生物物理方法来实现目标1，并使用 光谱技术以表征OPA构象异质性或次要的任何变化 在LOS面前的结构，这将是我训练的很大一部分。在世界一流的环境中，我会 接受电子顺磁共振（EPR）光谱技术，圆形二色性和核训练 磁共振（NMR）光谱。在AIM 2中，我将结合生化和基于细胞的方法 表征LOS对OPA-CCM蛋白复合物形成和细胞摄取的影响。这个实验 方法将补充我的生物物理培训，并帮助我成为一名全面的科学家 具有技术库的复杂生物学问题。通过描述OPA-CCM的分子基础 相互作用，我们将为开发新型疫苗和抗生素提供更坚实的基础 更广泛的研究奈瑟氏症感染的社区。