Bacterial Pathogenesis Laboratory, Infectious Diseases and Immunology Group, Translational Health Science and Technology Institute, NCR Biotech Science Cluster.
Bacterial Pathogenesis Laboratory, Infectious Diseases and Immunology Group, Translational Health Science and Technology Institute, NCR Biotech Science Cluster; Clinical Microbiology Division, CSIR-Indian Institute of Integrative Medicine.
J Vis Exp. 2023 Dec 8(202). doi: 10.3791/65138.
Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.
大多数细菌，包括分枝杆菌，都会产生细胞外囊泡（EVs）。由于细菌 EVs（bEVs）包含细胞成分的一部分，包括代谢物、脂质、蛋白质和核酸，因此有几个小组评估了 bEVs 的天然或重组版本作为亚单位疫苗候选物的保护效力。与天然 EVs 不同，重组 EVs 经过分子工程设计，以包含一种或多种感兴趣的免疫原。在过去的十年中，不同的小组探索了多种生成重组 bEVs 的方法。然而，在这里，我们报告了在分枝杆菌中设计、构建和富集重组分枝杆菌 EVs（mEVs）的方法。为此，我们使用了非致病性土壤分枝杆菌分枝杆菌 smegmatis（Msm）作为模型系统。我们首先描述了 Msm 天然 EVs 的生成和富集。然后，我们描述了重组 mEVs 的设计和构建，这些 mEVs 包含红色荧光报告蛋白 mCherry 或结核分枝杆菌的主要免疫原 EsxA（Esat-6）。我们通过将 mCherry 和 EsxA N 端分别与小 Msm 蛋白 Cfp-29 的 C 端融合来实现这一点。Cfp-29 是 MsmEVs 中少数大量存在的蛋白质之一。从 Msm 生成和富集重组 mEVs 的方案与从 Msm 生成和富集天然 EVs 的方案相同。
