Understanding bacterial localization and protein dynamics as indicators during tuberculosis treatment efficacy

项目摘要 为了补充目前基于痰液的结核病（TB）诊断和治疗方法， 监测，需要新的检测方法来检测活结核分枝杆菌（Mtb）杆菌的存在， 患者结核分枝杆菌感染者释放的外泌体中结核分枝杆菌特异性蛋白包封的发现 巨噬细胞培养是探索血清外泌体部分中的现象的动力; 应用靶向质谱法来确认在外来体中鉴定出十几种结核分枝杆菌抗原 从确诊的结核病患者身上分离出来的。外泌体是从有核细胞释放的小囊泡， 细胞，是生物标志物的丰富来源。补充和扩展血清外泌体作为标志物的效用 治疗效果，该提案侧重于使用无偏质量分析超纯外泌体 质谱（MS）用于发现Mtb蛋白及其在25名TB患者中的动力学反应，超过4个点 在抗分枝杆菌治疗期间。靶向MS测定（MRM）将用于确认蛋白质组学 差异将使用统计建模来确定与以下清除一致的蛋白质组特征： 将使用训练集（n = 256，基线/治疗对）和验证集（n = 256，基线/治疗对） 血清外泌体样品组（n = 64，基线/治疗对）。最终，血清中的结核分枝杆菌蛋白质特征 外泌体可与痰液诊断结合使用或当痰液不可用时（在儿童和 治疗期间，当排痰/咳嗽消退时），并将反映 痰为了将发现转化为与临床治疗监测相关的监测测定， 方法必须被简化为独立于质谱的平台，然而为了预分析的目的， 临床动物研究MRM-MS仍然是新药和治疗方案功效试验的可行选择。

Project Summary In order to complement the current sputum based methods of tuberculosis (TB) diagnosis and treatment monitoring, novel assays are required to detect the presence of live Mycobacterium tuberculosis (Mtb) bacilli in patients. The discovery of Mtb-specific protein encapsulation in exosomes released from Mtb-infected macrophage culture was the impetus for exploration of the phenomenon in the exosome fraction of serum; targeted mass spectrometry was applied to confirm the identification of over a dozen Mtb-antigens in exosomes isolated from sputum-confirmed tuberculosis patients. Exosomes are small vesicles released from nucleated cells and are a rich-source of biomarkers. To complement and extent the utility of serum exosomes as markers of treatment efficacy, this proposal focuses on the analysis of ultrapure exosomes using unbiased mass spectrometry (MS) for the discovery of Mtb proteins and their kinetic response in 25 TB patients over 4 points during anti-mycobacterial treatment. Targeted MS assays (MRM) will be used to confirm the proteomic differences. Statistical modeling will be used to determine a proteomic signature consistent with clearance of Mtb from the sputum and will be performed with a training set (n = 256, baseline/treatment pairs) and a validation set (n = 64, baseline/treatment pairs) of serum exosome samples. Ultimately, an Mtb protein signature in serum exosomes can be used in conjunction with sputum diagnostics or when sputum is not available (in children and during treatment when production/cough resolves) and will reflect the elimination of transmissible bacilli in the sputum. In order to translate discoveries to a monitoring assay relevant to treatment monitoring in the clinic our methods must be simplified to a platform independent of mass spectrometry, however for the purpose of pre- clinical animal research MRM-MS remains a feasible option for novel drug and treatment regimen efficacy trials.