Chemical probes of mycobacterial growth and persistence

项目总结/摘要 结核分枝杆菌（Mycobacterium tuberculosis，Mtb）是人类结核病的病原体。尽管 结核病死亡率稳步下降，耐多药结核病和复发病例有所增加。这些 观察结果突出表明，迫切需要了解耐多药结核病如何发展和持续存在。此外，委员会认为， 诊断和治疗结核分枝杆菌感染的新策略可以限制疾病的传播， 阻力尽管有这些需要，结核分枝杆菌逃避治疗的机制仍然没有得到充分的界定， 部分原因是缺乏可用于探测Mtb生理学的工具。可以缓解这些问题的策略包括 早期诊断，了解细胞间分裂速率的变化，并深入了解结核分枝杆菌的持久性。 使用传统的转录组学很难区分表型异质性;化学生物学可以产生 检测分枝杆菌和监测细胞分裂动态的工具。我们开发了一种荧光探针 (QTF)它能真实的报告分枝杆菌细胞包膜的组装情况。我将雇用QTF来识别和 表征来自棒杆菌亚目的细菌的不同空间和时间概况。因为QTF 是由分枝菌酰转移酶活性激活的，荧光动力学将用于诊断目的，以确定 并分离混合微生物群落中的分枝杆菌和棒状杆菌。我还将在一个 同基因的单一培养，因为我假设这个探针可以用来分离正常和持久的细胞。最后，我将 开发一种新的荧光探针，与QTF互补，将共价标记生长的细胞壁。我 EnvisionQTF和该探针可以同时监测分枝菌酰转移酶活性细胞包膜结构 和重塑。

PROJECT SUMMARY/ABSTRACT The bacillus Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB) in humans. Despite a steady decline in TB mortality rates, multidrug resistant TB (MDR-TB) and relapse cases have increased. These observations highlight the urgent yet unmet need to understand how MDR-TB develops and persists. Moreover, new strategies to diagnose and treat Mtb infection can limit the spread of the disease and therefore the spread of resistance. Despite these needs, the mechanisms by which Mtb evades treatment remain insufficiently defined, in part due to a lack of tools available to probe Mtb physiology. Strategies that could mitigate these problems include early diagnosis, an understanding of variation in division rate between cells, and insight into Mtb persistence. Distinguishing phenotypic heterogeneity is difficult using traditional transcriptomics; chemical biology can yield tools to detect mycobacteria and monitor the dynamics of cell division. We have developed a fluorogenic probe (QTF) that reports on mycobacterial cell envelope assembly in real time. I shall employ QTF to identify and characterize distinct spatial and temporal profiles of bacteria from the Corynebacterineae suborder. Because QTF is activated by mycolyltransferase activity, fluorescence kinetics will be used for diagnostic purposes to identify and separate mycobacteria and corynebacteria in mixed microbial communities. I will also evaluate QTF in an isogenic monoculture, as I postulate this probe can be used to separate normal from persister cells. Finally, I shall develop a new fluorogenic probe, complementary to QTF, that will covalently label the growing cell wall. I envision QTF and this probe can simultaneously monitor mycolyltransferase activity cell envelope construction and remodeling.