Targeting the LpqY-SugABC transporter system for synergistic anti-TB compounds

摘要 结核分枝杆菌(Mtb)是一种非常成功的人类病原体，仍然是世界上引起结核病的主要原因之一。 疾病和死亡。结核分枝杆菌多重和广泛耐药菌株的出现引起了人们对 无法治疗的结核病(TB)，并要求开发非传统疗法来缩短结核病 治疗并有效根除耐药菌亚群。MTB需要几天时间才能死亡 在体外和体内与致死浓度的化合物孵育后。我们的实验室有 确定了在暴露于每一类现有抗结核药物期间结核分枝杆菌的全球代谢重塑 发现的“逃逸”途径和酶，同时属于多种代谢 途径，允许病原体改变转录反应并存活更长时间，即使在存在 杀菌化合物。在我们的研究中确定的一个协同靶点是LpqY-SugABC转运蛋白 参与二糖海藻糖吸收和循环的系统，海藻糖是分枝杆菌的一种成分 细胞壁。LpqY系统的转运蛋白基因对结核分枝杆菌的体外生长不是必需的，然而，它们是 对于宿主细胞在缺乏营养的微环境中的生存和体内的生长至关重要。我们的预赛 数据表明，LpqY系统可以作为有前途的药物靶点，与药物具有协同作用 现有的抗结核药物，在体外和巨噬细胞中杀死结核杆菌的速度更快。这个项目的总体目标是 确定抑制LpqY-SugABC功能的抗菌剂。而LpqY的Mtb基因敲除克隆- SugABC在体外和感染的巨噬细胞中无法内化和处理细胞外海藻糖，使用 互补克隆，我们可以证明海藻糖探针的特异性掺入是通过 LpqY系统在体外以及在THP-1巨噬细胞感染过程中。因此，在目标1a中，我们将 利用荧光素标记的海藻糖实现高通量的表型筛选， 确定可以阻止Mtb代谢标记的活性化合物，从而阻止Mtb的功能 LpqY转运蛋白系统直接在感染的人巨噬细胞内。通过利用番茄红蛋白- 在目标1a中表达结核分枝杆菌菌株，我们将同时区分所鉴定的化合物是否具有 对胞内结核分枝杆菌生长也有抑制作用。目标1b将建立已识别的 化合物在体外，并将测试与一线抗结核药物在感染期间对结核分枝杆菌的疗效和协同作用 巨噬细胞。在目标1c中，我们将确定LpqY-SugABC系统的哪个组件为目标 通过这项研究中确定的每一种化合物。该提案使用理性发现的目标，这有助于 结核分枝杆菌的内在耐药性和耐药表型促进宿主巨噬细胞内细菌存活。 同样重要的因素是，LpqY系统的失活通过以下方式协同加速结核分枝杆菌的杀伤 目前的抗结核化合物，因此，拟议的筛选有可能导致新的药物，可以 缩短治疗时间，减少耐药和持久性机制的发展机会。

ABSTRACT M. tuberculosis (Mtb) is a highly successful human pathogen that remains one of the world's major causes of illness and death. The emergence of multi and extensively drug resistant strains of Mtb raises concern about untreatable tuberculosis (TB) and demands the development of unconventional therapies to shorten the TB treatment and to effectively eradicate subpopulation of the drug-tolerant bacteria. Mtb takes several days to die following incubation with lethal concentrations of compounds in vitro and in vivo. Our laboratory have identified the global metabolic remodeling of Mtb during exposure to each class of available anti-TB drugs and discovered “escape” pathways and enzymes, simultaneously belonging to number of diverse metabolic pathways, allowing the pathogen to shift the transcriptional response and survive longer, even in presence of bactericidal compounds. One of the synergistic targets identified in our study is the LpqY-SugABC transporter system involved in the uptake and recycling of the disaccharide trehalose, a constituent of the mycobacterial cell wall. The transporter genes of LpqY system are non-essential for Mtb growth in vitro, however, they are vital for survival in nutrient-deprived microenvironment of host cells and for growth in vivo. Our preliminary data shows that the LpqY system can serve as the promising drug-target that has a synergistic effect with existing anti-TB drugs, killing Mtb faster in vitro and in macrophages. The overall goal of this project is to identify antimicrobials that inhibit the function of LpqY-SugABC. While the Mtb knockout clone of the LpqY- SugABC fails to internalize and process the extracellular trehalose in vitro and in infected macrophages, using the complemented clone, we can demonstrate that the specific incorporation of trehalose probe occurs via the LpqY system in vitro as well as during infection of THP-1 macrophages. Therefore, in the Aim 1a, we will achieve the high-throughput phenotypic screening by utilizing the fluorescein-labeled trehalose, and will identify active compounds that can block metabolic labeling of Mtb and, consequently, the function of the LpqY transporter system directly within infected human macrophages. By utilizing the tomato red protein- expressing Mtb strain in the Aim 1a, we will simultaneously distinguish if identified compounds have an inhibitory effect on intracellular Mtb growth as well. The Aim 1b will establish the killing kinetics of identified compounds in vitro, and will test efficacy and synergy with frontline anti-TB drugs against Mtb during infection of macrophages. In the Aim 1c, we will determine which component of the LpqY-SugABC system is targeted by each compound identified in this study. The proposal uses the rationally discovered target that contributes to intrinsic resistance and drug-tolerant phenotype of Mtb promoting bacterial survival within host macrophages. Equally significant factor is that inactivation of the LpqY system synergistically accelerates Mtb killing by current anti-TB compounds and, thus, the proposed screening has the potential to lead to novel drugs that can shorter therapy time and decrease chances for development of the drug resistance and persistence mechanisms.