Division of Toxicology and Experimental Medicine, CSIR-Central Drug Research Institute, Lucknow 226031, India; Academy of Scientific and Innovative Research, New Delhi 110025, India.
Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow 226031, India.
Life Sci. 2019 Mar 1;220:21-31. doi: 10.1016/j.lfs.2019.01.035. Epub 2019 Jan 24.
β casein fragment peptide (54-59) downregulates Basic Transcription factor 3a (BTF3a) in macrophages and exhibits enhanced clearance of M. bovis BCG and several other intracellular pathogens. However, the direct effect of BTF3a downregulation on Mycobacterium tuberculosis (Mtb) survival and the probable pathways involved have not yet been studied. Therefore, the present study was undertaken to deduce the antimycobacterial significance of BTF3a in human macrophages.
CRISPR/Cas 9 gRNA was designed to downregulate BTF3a in THP1 derived macrophages. Fold change in BTF3a, p62 and Lamp 1 expression was evaluated through immune blot analysis. CFU assay was done to enumerate the intracellular burden of Mtb H37Rv. LC3B-II turnover and Lamp 1 expression was checked through immunoblotting and also visualized through confocal microscopy. Colocalization of Mtb H37Rv with LC3B, Lysotracker and Rab 7 was visualized through confocal microscopy.
The current study identifies BTF3a as a critical host factor assisting intracellular survival of Mtb. In THP1 derived macrophages, infection with Mtb H37Rv resulted in upregulation of BTF3a and targeted depletion of BTF3a resulted in augmented Mtb clearance. Furthermore, BTF3a knockdown demonstrated increased autophagy flux and ameliorated the lysosomal targeting of Mtb containing autophagosomes for lysosomal degradation.
Deep understanding of macrophage-Mtb interactions and their roles in the pathogenesis can offer exciting new therapeutic targets for alternative host-specific adjunct therapies in tuberculosis treatment. The present study highlights a novel and significant role of BTF3a in curbing the intracellular survival of Mtb through modulation of autophagy and lysosome biogenesis.
β酪蛋白片段肽（54-59）下调巨噬细胞中的基本转录因子 3a（BTF3a），并表现出增强的牛分枝杆菌 BCG 和其他几种细胞内病原体的清除能力。然而，BTF3a 下调对结核分枝杆菌（Mtb）存活的直接影响以及可能涉及的途径尚未研究。因此，本研究旨在推断 BTF3a 在人巨噬细胞中对分枝杆菌的抗菌意义。
设计 CRISPR/Cas 9 gRNA 下调 THP1 衍生巨噬细胞中的 BTF3a。通过免疫印迹分析评估 BTF3a、p62 和 Lamp 1 的表达变化。通过 CFU 测定来计数 Mtb H37Rv 的细胞内负担。通过免疫印迹检查 LC3B-II 周转和 Lamp 1 的表达，并通过共聚焦显微镜进行可视化。通过共聚焦显微镜可视化 Mtb H37Rv 与 LC3B、Lysotracker 和 Rab 7 的共定位。
本研究确定 BTF3a 是一种关键的宿主因子，有助于 Mtb 的细胞内存活。在 THP1 衍生的巨噬细胞中，感染 Mtb H37Rv 导致 BTF3a 上调，而靶向敲除 BTF3a 导致 Mtb 清除增加。此外，BTF3a 敲低显示增加的自噬通量，并改善含有自噬体的 Mtb 的溶酶体靶向，以进行溶酶体降解。
深入了解巨噬细胞-Mtb 相互作用及其在发病机制中的作用，可以为结核病治疗中的替代宿主特异性辅助治疗提供令人兴奋的新治疗靶点。本研究强调了 BTF3a 通过调节自噬和溶酶体生物发生来抑制 Mtb 细胞内存活的新的和重要作用。
