Tuberculosis (TB) is one of the world’s most problematic infectious diseases. The pathogen Mycobacterium tuberculosis (Mtb) is contained by the immune system in people with latent TB infection (LTBI). No overt disease symptoms occur. The environmental and internal triggers leading to reactivation of TB are not well understood. Non-tuberculosis Mycobacteria (NTM) can also cause TB-like lung disease. Comparative analysis of blood plasma proteomes from subjects afflicted by these pathologies in an endemic setting may yield new differentiating biomarkers and insights into inflammatory and immunological responses to Mtb and NTM. Blood samples from 40 human subjects in a pastoral region of Ethiopia were treated with the ESAT-6/CFP-10 antigen cocktail to stimulate anti-Mtb and anti-NTM immune responses. In addition to those of active TB, LTBI, and NTM cohorts, samples from matched healthy control (HC) subjects were available. Following the generation of sample pools, proteomes were analyzed via LC-MS/MS. These experiments were also performed without antigen stimulation steps. Statistically significant differences using the Z-score method were determined and interpreted in the context of the proteins’ functions and their contributions to biological pathways. More than 200 proteins were identified from unstimulated and stimulated plasma samples (UPSs and SPSs, respectively). Thirty-four and 64 proteins were differentially abundant with statistical significance (P < 0.05; Benjamini-Hochberg correction with an FDR < 0.05) comparing UPS and SPS proteomic data of four groups, respectively. Bioinformatics analysis of such proteins via the Gene Ontology Resource was indicative of changes in cellular and metabolic processes, responses to stimuli, and biological regulations. The m7GpppN-mRNA hydrolase was increased in abundance in the LTBI group compared to HC subjects. Charged multivesicular body protein 4a and platelet factor-4 were increased in abundance in NTM as compared to HC and decreased in abundance in NTM as compared to active TB. C-reactive protein, α-1-acid glycoprotein 1, sialic acid-binding Ig-like lectin 16, and vitamin K-dependent protein S were also increased (P < 0.05; fold changes≥2) in SPSs and UPSs comparing active TB with LTBI and NTM cases. These three proteins, connected in a STRING functional network, contribute to the acute phase response and influence blood coagulation. Plasma proteomes are different comparing LTBI, TB, NTM and HC cohorts. The changes are augmented following prior blood immune cell stimulation with the ESAT-6/CFP-10 antigen cocktail. The results encourage larger-cohort studies to identify specific biomarkers to diagnose NTM infection, LTBI, and to predict the risk of TB reactivation.

中文翻译：

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有和没有 ESAT-6/CFP10 刺激的活动性结核病、潜伏性结核病和非结核分枝杆菌肺病患者血浆蛋白质组的差异


结核病（TB）是世界上最严重的传染病之一。病原体结核分枝杆菌 (Mtb) 被潜伏性结核感染 (LTBI) 患者的免疫系统所抑制。没有出现明显的疾病症状。导致结核病复发的环境和内部触发因素尚不清楚。非结核分枝杆菌 (NTM) 也可引起类似结核病的肺部疾病。对流行环境中患有这些疾病的受试者的血浆蛋白质组进行比较分析可能会产生新的差异化生物标志物，并深入了解 Mtb 和 NTM 的炎症和免疫反应。使用 ESAT-6/CFP-10 抗原混合物处理来自埃塞俄比亚牧区 40 名人类受试者的血液样本，以刺激抗 Mtb 和抗 NTM 免疫反应。除了活动性 TB、LTBI 和 NTM 队列的样本外，还可以获得来自匹配的健康对照 (HC) 受试者的样本。生成样品池后，通过 LC-MS/MS 分析蛋白质组。这些实验也是在没有抗原刺激步骤的情况下进行的。使用 Z 评分方法在蛋白质功能及其对生物途径的贡献的背景下确定和解释统计学上的显着差异。从未刺激和刺激血浆样本（分别为 UPS 和 SPS）中鉴定出 200 多种蛋白质。分别比较四组的 UPS 和 SPS 蛋白质组数据，34 种和 64 种蛋白质的丰度差异具有统计显着性（P < 0.05；Benjamini-Hochberg 校正，FDR < 0.05）。通过基因本体资源对此类蛋白质进行生物信息学分析表明细胞和代谢过程、对刺激的反应和生物调节的变化。 与 HC 受试者相比，LTBI 组的 m7GpppN-mRNA 水解酶丰度增加。与 HC 相比，NTM 中带电多泡体蛋白 4a 和血小板因子 4 的丰度增加，与活动性 TB 相比，NTM 中的丰度降低。与活动性结核病相比，SPS 和 UPS 中的 C 反应蛋白、α-1-酸性糖蛋白 1、唾液酸结合 Ig 样凝集素 16 和维生素 K 依赖性蛋白 S 也有所增加（P < 0.05；倍数变化≥2） LTBI 和 NTM 病例。这三种蛋白质连接在一个 STRING 功能网络中，有助于急性期反应并影响血液凝固。与 LTBI、TB、NTM 和 HC 队列相比，血浆蛋白质组有所不同。先前使用 ESAT-6/CFP-10 抗原混合物刺激血液免疫细胞后，这些变化会增强。研究结果鼓励进行更大规模的研究，以确定诊断 NTM 感染、LTBI 的特定生物标志物，并预测结核病复发的风险。