Chemical probes of mycobacterial growth and persistence

项目摘要/摘要 结核分枝杆菌(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 观察结果突出了了解耐多药结核病如何发展和持续的迫切但尚未得到满足的需要。此外， 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，从而限制 抵抗。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结核分枝杆菌生理学的工具。可以缓解这些问题的策略包括 早期诊断，了解细胞分裂比率的变化，以及对结核杆菌持久性的洞察。 使用传统的转录组学很难区分表型的异质性；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。我们已经研制出一种荧光探头 (QTF)，实时报告分枝杆菌细胞被膜组装。我将聘请QTF来识别和 描述棒状杆菌亚目细菌不同的空间和时间分布。因为QTF 是由真菌转移酶活性激活的，荧光动力学将用于诊断目的 在混合微生物群落中分离分枝杆菌和棒状杆菌。我还将评估QTF在一个 同基因单培养，正如我推测的那样，这个探针可以用来分离正常细胞和永存细胞。最后，我会 开发一种新的荧光探针，与QTF互补，将共价标记正在生长的细胞壁。我 EnVision QTF和该探针可以同时监测霉菌转移酶活性细胞膜结构 和改建。

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.