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。

基本信息

批准号：
10663966
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
项目类别：
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/10663966
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 Development Disease Engineering Filament Flagella Goals Growth Human Immune Evasion Immune response Immune system Impairment In Vitro Infection Invaded Investigation Joints Knowledge Lead Leptospira interrogans Leptospirosis Link 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 Rotation Shapes Structure Structure-Activity Relationship Swimming Syphilis Testing Therapeutic Agents Ticks Tissues Treponema denticola Treponema pallidum Treponema pallidum subspecies pallidum United States Variant Virulence Virulent Yaws antimicrobial drug cell motility chemical synthesis cofactor crosslink design drug development emerging antibiotic resistance experience experimental study flexibility functional restoration genetic approach high throughput screening improved in vivo inhibitor insight laser tweezer mechanical properties microorganism mortality mutant novel novel therapeutics optic tweezer pathogen periplasm physical property protein crosslink resistant strain single molecule small molecule small molecule inhibitor small molecule libraries structural biology therapeutic lead compound tool

项目摘要

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.

Treponema Pallidum（TP），Borrelia Burgdorferi（BB），Leptospira Excentogans（Li）和Treponema Denticola（TD）是引起梅毒，莱姆病，钩端螺旋体病并且与牙周有关的螺旋体细菌人类的疾病分别是人类的疾病。这些生物在曼联导致大量发病率和死亡率国家和全世界。由于莱姆病的患病率和抗生素的出现 TP和TD的抗性，我们的长期目标是开发专门治疗疾病的新型药物由螺旋体引起。螺旋体是高度侵入性的细菌，其独特的运动模式在其能力中起着至关重要的作用穿透并入侵宿主组织和器官。螺旋体的鞭毛位于周期内，是从而避开免疫系统。细菌鞭毛的关键组成部分称为钩子鞭毛丝膜成膜的旋转电动机。钩子由多种FLGE蛋白组成，在与其他细菌鞭毛形成鲜明对比的是，螺旋体Flge蛋白是共价交联的。这交联涉及形成一种新型的硅氨酸（LAL）氨基酸。中心假设是Flge 蛋白质是共价交联的，以增强钩子的最佳运动性和毒力。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导致抑制交联治疗螺旋疾病的药物的发展。具体目标1。研究FLGE交联对BB感染性的影响。 TD和BB的突变体无法交联的钩子蛋白的形状也会改变，并且不足于翻译运动。为了确定交联对BB毒力的重要性，我们将在Flge中产生毒性障碍交联并评估其游泳和维持小鼠和壁虱感染的能力。特定目标2。发展FLGE交联的小分子抑制剂。洛杉矶形成的化学是生物学上的前所未有。基于机械和结构研究，我们建立了交联大规模抑制剂筛选的TD和BB重组FLGE蛋白的测定。在这些测定中我们已经发现了FLGE交联和BB运动的抑制剂。我们将进一步描述这一行动复合并继续识别和表征用于研究的其他类别的抑制剂宿主的发病机理，最终作为治疗剂的铅化合物。具体目标3。确定FLGE交联对鞭毛结构和稳定性的影响钩。测试FLGE交联是否稳定钩子以抵抗高机械应力我们将在周质空间中的经验，我们将分析交联和非交联的物理特性链接的钩子。另外，交联的要求将通过化学恢复功能测试没有LAL。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A chemosensory-like histidine kinase is dispensable for chemotaxis in vitro but regulates the virulence of Borrelia burgdorferi through modulating the stability of RpoS.

DOI：
10.1371/journal.ppat.1011752
发表时间：
2023-11
期刊：
PLoS pathogens
影响因子：
6.7
作者：
通讯作者：

Lysinoalanine cross-linking is a conserved post-translational modification in the spirochete flagellar hook.

DOI：
10.1093/pnasnexus/pgad349
发表时间：
2023-12
期刊：
PNAS NEXUS
影响因子：
0
作者：
Lynch, Michael J.;Deshpande, Maithili;Kurniyati, Kurni;Zhang, Kai;James, Milinda;Miller, Michael;Zhang, Sheng;Passalia, Felipe J.;Wunder Jr, Elsio A.;Charon, Nyles W.;Li, Chunhao;Crane, Brian R.
通讯作者：
Crane, Brian R.

A new class of protein sensor links spirochete pleomorphism, persistence, and chemotaxis.