MRC Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London, London, SW7 2AZ, UK.
BMC Microbiol. 2018 Apr 12;18(1):30. doi: 10.1186/s12866-018-1165-0.
Resuscitation promoting factor proteins (Rpfs) are peptidoglycan glycosidases capable of resuscitating dormant mycobacteria, and have been found to play a role in the pathogenesis of tuberculosis. However, the specific roles and localisation of each of the 5 Rpfs in Mycobacterium tuberculosis remain mostly unknown. In this work our aim was to construct fluorescent fusions of M. tuberculosis Rpf proteins as tools to investigate their function.
We found that Rpf-fusions to the fluorescent protein mCherry are functional and able to promote cell growth under different conditions. However, fusions to Enhanced Green Fluorescent Protein (EGFP) were non-functional in the assays used and none were secreted into the extracellular medium, which suggests Rpfs may be secreted via the Sec pathway. No specific cellular localization was observed for either set of fusions using time-lapse video microscopy.
We present the validation and testing of five M. tuberculosis Rpfs fused to mCherry, which are functional in resuscitation assays, but do not show any specific cellular localisation under the conditions tested. Our results suggest that Rpfs are likely to be secreted via the Sec pathway. We propose that such mCherry fusions will be useful tools for the further study of Rpf localisation, individual expression, and function.
复苏促进因子蛋白（Rpfs）是能够复苏休眠分枝杆菌的肽聚糖糖苷酶，并且已被发现与结核病的发病机制有关。然而，分枝杆菌中的 5 种 Rpfs 中的每一种的具体作用和定位在很大程度上仍然未知。在这项工作中，我们的目的是构建分枝杆菌 Rpf 蛋白的荧光融合物，作为研究其功能的工具。
我们发现，与红色荧光蛋白 mCherry 融合的 Rpf 是功能性的，能够在不同条件下促进细胞生长。然而，与增强型绿色荧光蛋白（EGFP）融合的蛋白在所用的测定中是非功能性的，并且没有分泌到细胞外培养基中，这表明 Rpfs 可能通过 Sec 途径分泌。使用延时视频显微镜，没有观察到任何一组融合蛋白有特定的细胞定位。
我们提出了五种分枝杆菌 Rpfs 与 mCherry 融合的验证和测试，它们在复苏测定中是功能性的，但在测试条件下没有显示任何特定的细胞定位。我们的结果表明，Rpfs 可能通过 Sec 途径分泌。我们提出，这种 mCherry 融合将是进一步研究 Rpf 定位、个体表达和功能的有用工具。
