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

梅毒螺旋体(TP)、伯氏疏螺旋体(BB)、问号钩端螺旋体(Li)和齿密螺旋体(TD) 是引起梅毒、莱姆病、钩端螺旋体病的螺旋体细菌，并与牙周有关 分别是人类的疾病。这些微生物在美国造成了相当大的发病率和死亡率 美国和全世界都是如此。由于莱姆病的流行和抗生素的出现 在TP和TD的耐药性方面，我们的长期目标是开发专门治疗疾病的新药 由螺旋体引起的。 螺旋体是一种高度侵袭的细菌，它们独特的运动方式对它们的能力起着至关重要的作用 侵入并侵入宿主组织和器官。螺旋体的鞭毛位于周质内， 从而对免疫系统起到保护作用。细菌鞭毛的一个关键成分，称为钩子，连接到 鞭毛细丝到埋膜的旋转马达。钩子由多个FlgE蛋白组成，在 与其他细菌鞭毛不同，螺旋体FlgE蛋白是相互共价交联的。这 交联会形成一种新的赖氨酸丙氨酸(LAL)氨基酸。中心假设是FlgE 蛋白质是共价交联的，以加强钩子的最佳运动性和毒力。它是 提出了解交联链的结构、化学合成及其在毒力中的作用 导致用于治疗螺旋体疾病的抑制交叉连接的药物的开发。 具体目的1.研究FlgE交联物对BB感染性的影响。Td和Bb的突变体 不能使其挂钩蛋白交联者也会改变其形状，缺乏翻译动力。 为了确定交联对BB毒力的重要性，我们将产生一株在FlgE中受损的毒株 交联剂，并评估其游泳能力和在小鼠和扁虱中持续感染的能力。 具体目的2.开发FlgE交联的小分子抑制剂。LA形成的化学成分是 在生物学上史无前例。基于机理和结构研究，我们已经建立了交联剂 利用TD和BB重组FlgE蛋白进行大规模抑制剂筛选。通过这些化验，我们 已经发现了一种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，并继续确定和表征用于研究的更多类别的抑制剂 宿主的致病机制，并最终作为治疗学的先导化合物。 特定目的3.测定FlgE交联物对鞭毛结构和稳定性的影响 胡克。为了测试FlgE交联物是否稳定钩子以抵抗可能的高机械应力 在周质空间的经验，我们将分析交联物和非交联物的物理性质。 连接的挂钩。此外，将通过化学修复功能来测试交联剂的要求 拉尔的缺席。

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.