Impact of Sterilizing and Non-sterilizing Antibiotics on M. tuberculosis rRNA Synthesis

结核病是结核病流行地区退伍军人和服役人员的重要健康问题。 国家。迫切需要一种新的短期结核病治疗方法。预防所需的治疗时间长短 复发取决于药物在随后漫长的“消毒”治疗阶段的效果。 早期的杀菌杀戮。不同的组织微环境中含有结核分枝杆菌(Mtb)种群 不同的复制率对药物治疗的反应不同。例如，药物更有效 抗结核分枝杆菌(Mtb)在呼吸道和充气细胞肺浸润物比在干酪低氧核心 坏死性肉芽肿。我们首创了一套新颖的分析方法，使我们能够探索基本的 首次发现了结核分枝杆菌在组织中的生理状态特征。这些化验提供了一个前所未有的视角 以此来重新审视组织微环境对药物影响的历史假设 回应。这些新的分析方法也使我们能够测试药物治疗缩短的新的替代范例。 (消毒)活动。具体地说，我们新构造是在灭菌阶段减少结核分枝杆菌的负担 是两个不同过程的综合结果：(1)杀死和(2)抑制复制。第一个过程-- 杀戮--长期以来一直是结核病药物开发的主要重点。我们假设第二个过程- 消除结核分枝杆菌复制-是一个重要的，但以前没有被认识到的药物灭菌活性的贡献者。 我们认为，在治疗的消毒阶段抑制药物复制可以缩短 持久、不复发的治疗方法。我们将在C3HeB/FEJ(Kramnik)小鼠身上进行测试，这是一种发育良好的品系- 包裹性坏死性肉芽肿，内氧分压低。这个模型使我们能够研究结核杆菌。 模拟人类结核病病理的微环境光谱。我们将测量结核分枝杆菌rRNA的合成 在三个阶段(基线、杀菌和消毒阶段)：(1)呼吸道、(2)细胞病变、(3)淋巴细胞 肉芽肿的外袖和(4)坏死性肉芽肿的缺氧性干酪。使用几种互补的方法 为了量化结核分枝杆菌的负担，我们将具体确定哪里清除了结核分枝杆菌，哪里继续存在分枝杆菌。测试我们的 假设抑制复制有助于缩短治疗时间，我们将把这些方案与 不同的消毒效力：标准处理(异烟肼、利福平、吡津酰胺、乙胺丁醇[HRZE])和 治愈结核病的新方案(贝达奎林、前列甘露、利奈唑胺、吡津酰胺[BPaLZ])。目标 1将在治疗前测试组织微环境条件和结核分枝杆菌复制之间的关联。 我们假设了一系列mtb rrna的合成和复制，从呼吸道中最快的、中间的 在细胞性病变和淋巴细胞性袖套中以干酪层最慢。目标2将测试结核分枝杆菌与 在杀菌阶段的复制和药物杀灭。我们假设药物会优先杀死结核分枝杆菌 结核分枝杆菌快速复制的微环境(即呼吸道、淋巴细胞袖带和细胞病变)。那里 在杀菌阶段对干酪中结核分枝杆菌的杀灭作用最小。目标3将对灭菌阶段进行评估。 目标3将验证我们的假设，即高度有效的消毒方案(BPaLZ)将更有效地清除结核分枝杆菌 并比标准方案(HRZE)更深刻地抑制Mtb rRNA的合成和复制。这部作品 将有助于开发一种实用的杀菌活性生物标志物。杀菌活动的标志将是 通过改进临床前药物评估和提高治疗的准确性和效率来缩短治疗时间 药物试验。最终，这项计划还将导致临床生物标志物的常规监测，以指导持续时间 在接受结核病治疗的退伍军人中，这是一种有效的治疗方法。我们的方法可以进一步扩展到许多其他慢性疾病 对退伍军人来说很重要的细菌感染。

Tuberculosis (TB) is an important health concern for veterans and service members who serve in TB-endemic countries. There is an urgent need for new shorter TB treatment. The length of treatment required to prevent relapse is determined by the effectiveness of drugs during the long “sterilizing” phase of treatment that follows early bactericidal killing. Different tissue micro-environments harbor M. tuberculosis (Mtb) populations with different replication rates that respond differently to drug treatment. For example, drugs are more effective against M. tuberculosis (Mtb) in airway and aerated cellular lung infiltrates than in the caseous hypoxic core of necrotic granulomas. We have pioneered a suite of novel assays that will enable us to probe fundamental characteristics of Mtb physiologic state in tissues for the first time. These assays provide an unprecedented lens with which to re-examine historical assumptions about the influence of tissue micro-environment on drug response. These novel assays also enable us to test a novel alternative paradigm for drugs' treatment shortening (sterilizing) activity. Specifically, our novel construct is that reduction in Mtb burden during the sterilizing phase is the combined result of two distinct processes: (1) killing and (2) inhibition of replication. The first process – killing – has long been the primary focus of TB drug development. We hypothesize that the second process – abrogation of Mtb replication – is an important but previously unrecognized contributor to drug sterilizing activity. We believe that drug inhibition of replication in the sterilizing phase of treatment shortens the time needed for durable, non-relapsing cure. We will test this in the C3HeB/FeJ (Kramnik) mouse, a strain that develops well- encapsulated necrotic granulomas with low internal oxygen tension. This model enables us to study Mtb in a spectrum of micro-environments that mimic the pathology of human TB. We will measure Mtb rRNA synthesis at three points (baseline, bactericidal and sterilizing phases) in: (1) airway, (2) cellular lesions, (3) lymphocytic outer cuff of granulomas and (4) hypoxic caseum of necrotic granulomas. Using several complementary methods to quantify Mtb burden, we will determine specifically where Mtb is cleared and where Mtb persists. To test our hypothesis that inhibition of replication contributes to treatment shortening, we will compare regimens with different sterilizing potency: standard treatment (isoniazid, rifampin, pyrazinamide, ethambutol [HRZE]) and a novel regimen that cures TB substantially faster (bedaquiline, pretomanid, linezolid, pyrazinamide [BPaLZ]). Aim 1 will test the association between tissue micro-environmental conditions and Mtb replication before treatment. We hypothesize a spectrum of Mtb rRNA synthesis and replication, ranging from fastest in airway, intermediate in cellular lesions and lymphocytic cuff to slowest in the caseum. Aim 2 will test the association between Mtb replication and drug killing during the bactericidal phase. We hypothesize that drugs will preferentially kill Mtb in micro-environments where Mtb is replicating rapidly (i.e., airway, lymphocytic cuff and cellular lesions). There will be minimal killing of Mtb in caseum during the bactericidal phase. Aim 3 will evaluate the sterilizing phase. Aim 3 will test our hypothesis that a highly potent sterilizing regimen (BPaLZ) will both clear Mtb more effectively and suppress Mtb rRNA synthesis and replication more profoundly than the standard regimen (HRZE). This work will contribute to development of a practical biomarker of sterilizing activity. A marker of sterilizing activity will lead to shorter treatment by improving preclinical drug assessment and improving the accuracy and efficiency of drug trials. Ultimately, this program will also lead to a clinical biomarker for routine monitoring to guide duration of treatment among veterans treated for TB. Our approach could be further extended to many other chronic bacterial infections that are important for veterans.