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）、问号钩端螺旋体（Li）和齿垢密螺旋体（Td） 是螺旋体细菌，引起梅毒，莱姆病，钩端螺旋体病，并与牙周炎有关。 人类的疾病，分别。这些微生物在美国引起大量的发病率和死亡率， 国家和世界各地。由于莱姆病的流行和抗生素的出现， 我们的长期目标是开发特异性治疗疾病的新药 是由螺旋体引起的 螺旋体是高度侵袭性的细菌，其独特的运动模式在其能力中起着至关重要的作用。 侵入宿主组织和器官。螺旋体的鞭毛位于周质内， 从而避开免疫系统。细菌鞭毛的一个关键组成部分，称为钩， 鞭毛丝到膜嵌入的旋转马达。该钩由多个FlgE蛋白组成，并且在 与其它细菌鞭毛相反，螺旋体FlgE蛋白彼此共价交联。这 交联包括形成新的赖氨丙氨酸（Lal）氨基酸。中心假设是FlgE 蛋白质被共价交联以加强钩以获得最佳的运动性和毒力。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 导致开发抑制交联以治疗螺旋体疾病的药物。 具体目标1。研究FlgE交联对Bb感染性的影响。Td和Bb突变体 不能交联它们的钩蛋白的蛋白质也改变了形状并且缺乏翻译运动性。 为了确定交联对Bb毒力的重要性，我们将产生FlgE受损的强毒株 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形成的化学过程是 在生物学上是前所未有的。基于机理和结构研究，我们建立了交联 用来自Td和Bb的重组FlgE蛋白进行大规模抑制剂筛选的测定。通过这些测定， 已经发现了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点 化合物，并继续确定和表征其他类别的抑制剂用于研究 在宿主中的发病机制，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 hook.为了测试FlgE交联是否稳定钩以抵抗高机械应力， 在周质空间的经验，我们将分析交联和非交联的物理性质， 挂钩此外，交联的要求将通过化学恢复功能进行测试， 没有Lal。