Toward novel therapies against Lyme disease through the inhibition of lysinoalaine cross-linking in the bacterial flagella.

通过抑制细菌鞭毛中的赖氨酸丙氨酸交联来开发针对莱姆病的新疗法。

• 批准号: 10470087
• 负责人: BRIAN R CRANE
• 金额: $ 59.35万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-16 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10470087
• 关键词: Affect; Affinity; Amino Acids; Animal Model; Bacteria; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biological Models; Borrelia burgdorferi; Cells; Cellular Morphology; Chemicals; Chemistry; Cryoelectron Microscopy; Crystallization; Development; Disease; Engineering; Filament; Flagella; Globus Pallidus; Goals; Growth; Human; Immune response; Immune system; Impairment; In Vitro; Infection; Invaded; Investigation; Joints; Knowledge; Lead; Leptospira interrogans; Leptospirosis; Lyme Disease; Mechanical Stress; Membrane; Modification; Morbidity - disease rate; Morphology; Motor; Movement; Mus; Mutation; Order Spirochaetales; Organ; Organelles; Organism; Pathogenesis; Penetration; Periodontal Diseases; Play; Polymers; Post-Translational Protein Processing; Prevalence; Prevention; Process; Proteins; Reaction; Recombinants; Role; Shapes; Structure; Structure-Activity Relationship; Swimming; Syphilis; Testing; Therapeutic Agents; Ticks; Tissues; Treponema denticola; Treponema pallidum; United States; Variant; Virulence; Virulent; Yaws; antimicrobial drug; base; cell motility; chemical synthesis; cofactor; crosslink; drug development; emerging antibiotic resistance; experience; experimental study; flexibility; functional restoration; genetic approach; high throughput screening; improved; in vivo; inhibitor; insight; laser tweezer; link protein; mechanical properties; microorganism; mortality; mutant; novel; novel therapeutics; pathogen; periplasm; physical property; resistant strain; single molecule; small molecule; small molecule inhibitor; small molecule libraries; structural biology; therapeutic lead compound; tool

Treponema pallidum (Tp), Borrelia burgdorferi (Bb), Leptospira interrogans (Li) and Treponema denticola (Td) are spirochete bacteria that cause syphilis, Lyme disease, leptospirosis, and are associated with periodontal diseases in humans, respectively. These organisms cause substantial morbidity and mortality in the United States and throughout the world. Owing to the prevalence of Lyme disease and emergence of antibiotic resistance in Tp and Td, our long-term goal is to develop novel drugs that specifically treat diseases caused by spirochetes. Spirochetes are highly invasive bacteria, and their unique mode of motility plays an essential role in their ability to penetrate and invade host tissues and organs. The flagella of spirochetes reside within the periplasm and are thereby shielded from the immune system. A key component of bacterial flagella termed the hook joins the flagella filament to the membrane-imbedded rotary motor. The hook consists of multiple FlgE proteins, and in contrast to other bacterial flagella, spirochete FlgE proteins are covalently cross-linked to one another. This cross-link involves formation of a novel lysinoalanine (Lal) amino acid. The central hypothesis is that the FlgE proteins are covalently cross-linked to strengthen the hook for optimal motility and virulence. It is proposed that understanding the structure of the cross-link, its chemical synthesis and its role in virulence will lead to the development of drugs that inhibit cross-linking for treating spirochetal diseases. Specific Aim 1. Investigate the effect of FlgE cross-linking on the infectivity of Bb. Mutants of Td and Bb that are unable to cross-link their hook proteins are also altered in shape and deficient in translational motility. To determine the importance of cross-linking for Bb virulence, we will produce a virulent strain impaired in FlgE cross-linking and evaluate its ability to swim and sustain infections in both mice and ticks. Specific Aim 2. Develop small molecule inhibitors of FlgE cross-linking. The chemistry of LA formation is biologically unprecedented. Based on mechanistic and structural studies we have established cross-linking assays with recombinant FlgE proteins from Td and Bb for large-scale inhibitor screens. With these assays we have discovered an inhibitor of FlgE cross-linking and Bb motility. We will further characterize the action of this compound and continue to identify and characterize additional classes of inhibitors to be used for studying pathogenesis in hosts and eventually as lead compounds for therapeutics. Specific Aim 3. Determine the effects of FlgE cross-linking on the structure and stability of the flagella hook. To test whether the FlgE cross-links stabilize the hook to resist the high mechanical stress it likely experiences in the periplasmic space, we will analyze the physical properties of cross-linked and non-cross- linked hooks. In addition, the requirement of cross-linking will be tested by chemically restoring function in absence of Lal.

梅毒螺旋体 (Tp)、伯氏疏螺旋体 (Bb)、询问钩端螺旋体 (Li) 和齿状螺旋体 (Td) 是螺旋体细菌，可引起梅毒、莱姆病、钩端螺旋体病，并与牙周病有关 分别是人类的疾病。这些微生物在美国造成大量发病率和死亡率 各国和世界各地。由于莱姆病的流行和抗生素的出现 Tp和Td耐药性，我们的长期目标是开发专门治疗疾病的新药 由螺旋体引起。 螺旋体是高度侵入性的细菌，其独特的运动方式对其能力起着至关重要的作用 穿透并侵入宿主组织和器官。螺旋体的鞭毛位于周质内， 从而免受免疫系统的侵害。细菌鞭毛的一个关键组成部分称为钩，它连接着 鞭毛丝连接到膜嵌入旋转电机。该钩子由多个 FlgE 蛋白组成，并且在 与其他细菌鞭毛不同，螺旋体 FlgE 蛋白彼此共价交联。这 交联涉及新型赖氨酸丙氨酸 (Lal) 氨基酸的形成。中心假设是 FlgE 蛋白质通过共价交联来增强钩子，从而实现最佳的运动性和毒力。这是 提出了解交联的结构、其化学合成及其在毒力中的作用将有助于 导致开发出抑制交联的药物来治疗螺旋体疾病。 具体目标 1. 研究 FlgE 交联对 Bb 感染性的影响。 Td 和 Bb 突变体 无法交联其钩蛋白的形状也会改变并且缺乏平移运动。 为了确定交联对 Bb 毒力的重要性，我们将产生 FlgE 受损的毒力菌株 交联并评估其在小鼠和蜱中游泳和维持感染的能力。 具体目标2.开发FlgE交联的小分子抑制剂。 LA 形成的化学方程式是 生物学上前所未有的。基于机械和结构研究，我们建立了交联 使用来自 Td 和 Bb 的重组 FlgE 蛋白进行测定，以进行大规模抑制剂筛选。通过这些分析，我们 发现了 FlgE 交联和 Bb 运动的抑制剂。我们将进一步描述这一行动的特征 化合物并继续鉴定和表征用于研究的其他类别的抑制剂 宿主的发病机制并最终作为治疗的先导化合物。 具体目标 3. 确定 FlgE 交联对鞭毛结构和稳定性的影响 钩。为了测试 FlgE 交联是否稳定钩子以抵抗高机械应力，它可能 周质空间的经验，我们将分析交联和非交联的物理性质 链接的钩子。此外，交联的要求将通过化学恢复功能来测试 拉尔缺席。