Evaluating the Role of L,D-Transpeptidases in Mycobacterial Pathogenesis

项目摘要 结核病在2017年导致130万人死亡，是世界上最致命的传染病。 由药物敏感的结核分枝杆菌(Mtb)引起的感染可以治愈。但是治疗 治疗方案很长，需要至少6个月的多种药物治疗。结核病已经变得越来越多 由于耐药菌株的全球传播而难以治疗。这类菌株至少占5% 感染，这些患者的治愈率很低。迫切需要新药来治疗耐药性 但药物开发是一个缓慢而昂贵的过程。 但是，如果临床批准的β-内酰胺类抗生素可以被重新用于治疗结核病和耐药结核病，情况会怎样？ 这将大大加快患者获得新疗法的速度。自20世纪90年代以来，一直有 偶尔会有结核感染对β-内酰胺类抗生素有反应的报告。例如，阿莫西林/克拉维酸 美罗培南/克拉维酸已成功用于治疗耐药结核病患者。β-内酰胺 抗生素针对细胞壁中的酶。靶标包括青霉素结合蛋白(PBPs)和 新近发现的L，D-转肽酶(LDT)。这两种酶对生存都至关重要，因为它们 在细菌细胞壁中保持肽聚糖的结构和刚性。 我们建议开发新的分子探针来确定和验证β-内酰胺类药物的敏感性 分枝杆菌。我们的方法便于同时监控多个PBP和LDT，从而实现 对这些酶进行全面检查。我们将检测蛋白质凝胶裂解物中的酶 源于β-内酰胺类药物的活性探针(ABP)。用活性识别药物 对抗休眠的分枝杆菌是当务之急。因此，在目标1中，我们将使用ABPS来分析 PBPs和LDT在休眠、复活和活跃复制的Mtb中的调节。在目标2中，我们将 确定Mtb、PBPs和LDT在接触后的药物选择性和抑制剂分布 临床批准的β-内酰胺类抗生素。在目标3中，我们将研究 LDTS。在目标4中，我们将把我们的发现扩展到第二种高度优先的分枝杆菌病原体：脓肿分支杆菌。 在这个项目成功完成后，我们将有一个有效的方法来评估 分枝杆菌菌株对β-内酰胺类药物及联合用药的敏感性。我们期待着 我们的发现将对选择有效的治疗结核分枝杆菌或结核分枝杆菌感染患者产生积极影响 脓肿支原体。

Project Summary Tuberculosis (TB) killed 1.3 million people in 2017 and is the most deadly infectious disease in the world. Infections caused by drug-susceptible Mycobacterium tuberculosis (Mtb) can be cured. But treatment regimens are long, requiring at least 6 months of therapy with multiple drugs. TB has become increasingly difficult to treat due to the global spread of drug resistant strains. Such strains account for at least 5% of infections, and the cure rate for those patients is low. New drugs are urgently needed to treat drug-resistant strains, but drug development is a slow and costly process. But what if clinically-approved β-lactam antibiotics could be repurposed to treat TB and drug resistant TB? This would significantly speed up patient access to new therapies. Since the 1990s, there have been occasional reports of TB infections responding to β-lactam antibiotics. For example, amoxicillin/clavulanate and meropenem/clavulanate have been used successfully to treat patients with drug resistant TB. β-lactam antibiotics target enzymes in the cells wall. Targets include penicillin-binding proteins (PBPs) and the recently discovered L,D-transpeptidases (LDTs). Both classes of enzyme are critical to survival because they maintain the structure and rigidity of peptidoglycan in the bacterial cell wall. We propose to develop new molecular probes to identify and validate β-lactam drug susceptibility in mycobacteria. Our approach facilitates monitoring multiple PBPs and LDTs at once, enabling a comprehensive examination of these enzymes. We will detect enzymes in protein gel-resolved lysates using activity-based probes (ABPs) derived from the major classes of β-lactam drugs. Identifying drugs with activity against dormant mycobacteria is a high-priority. Therefore, in Aim 1, we will use ABPs to profile the regulation of PBPs and LDTs in dormant, reactivating, and actively-replicating Mtb. In Aim 2, we will determine the drug selectivity and inhibitor profiles for the Mtb PBPs and LDTs following exposure to clinically-approved β-lactam antibiotics. In Aim 3, we will investigate the spatio-temporal regulation of the LDTs. In Aim 4, we will extend our findings to a second high-priority mycobacterial pathogen: M. abscessus. Upon successful completion of this project, we will have an effective approach for assessing the susceptibility of mycobacterial strains to treatment with β-lactams and combination therapies. We anticipate that our findings will have a positive impact on selecting effective therapies for patients infected with Mtb or M. abscessus.