Medical Microbiology and Infection Control, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1117, Amsterdam, Netherlands.
Department of Experimental Immunology, Amsterdam Infection & Immunity Institute, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, Netherlands.
Microb Cell Fact. 2019 Mar 6;18(1):44. doi: 10.1186/s12934-019-1093-1.
Mycobacterium bovis Bacille Calmette-Guérin (BCG) is not only used as a vaccine against tuberculosis but also protects against leprosy and is used as part of bladder cancer treatment to induce a protective immune response. However, protection by BCG vaccination is not optimal. To improve vaccine efficacy, recombinant BCG expressing heterologous antigens has been put forward to elicit antigen-specific cellular and humoral responses. Cell surface localized or secreted antigens induce better immune responses than their cytosolic counterparts. Optimizing secretion of heterologous proteins or protein fragments holds therefore unexplored potential for improving the efficacy of recombinant BCG vaccine candidates. Secretion of heterologous antigens requires crossing the mycobacterial inner and outer membrane. Mycobacteria have specialized ESX or type VII secretion systems that enable translocation of proteins across both membranes. Probing this secretion system could therefore be a valid approach to surface localize heterologous antigens.
We show that ESX-5 substrate LipY, a lipase, can be used as a carrier for heterologous secretion of an ovalbumin fragment (OVA). LipY contains a PE domain and a lipase domain, separated by a linker region. This linker domain is processed upon secretion. Fusion of the PE and linker domains of LipY to OVA enabled ESX-5-dependent secretion of the fusion construct LipY-OVA in M. marinum, albeit with low efficiency. Subsequent random mutagenesis of LipY-OVA and screening for increased secretion resulted in mutants with improved heterologous secretion. Detailed analysis identified two mutations in OVA that improved secretion, i.e. an L280P mutation and a protein-extending frameshift mutation. Finally, deletion of the linker domain of LipY enhanced secretion of LipY-OVA, although this mutation also reduced surface association. Further analysis in wild type LipY showed that the linker domain is required for surface association.
We show that the ESX-5 system can be used for heterologous secretion. Furthermore, minor mutations in the substrate can enhance secretion. Especially the C-terminal region seems to be important for this. The linker domain of LipY is involved in surface association. These findings show that non-biased screening approaches aid in optimization of heterologous secretion, which can contribute to heterologous vaccine development.
牛分枝杆菌卡介苗（BCG）不仅被用作预防结核病的疫苗，还可以预防麻风病，并被用作膀胱癌治疗的一部分，以诱导保护性免疫反应。然而，BCG 疫苗的保护效果并不理想。为了提高疫苗的效力，已经提出了表达异源抗原的重组 BCG，以引发抗原特异性的细胞和体液反应。细胞表面定位或分泌的抗原比其胞质对应物诱导更好的免疫反应。因此，优化异源蛋白或蛋白片段的分泌为提高重组 BCG 疫苗候选物的效力提供了尚未开发的潜力。异源抗原的分泌需要穿过分枝杆菌的内膜和外膜。分枝杆菌具有专门的 ESX 或 VII 型分泌系统，可使蛋白质跨双膜易位。因此，探测该分泌系统可能是表面定位异源抗原的有效方法。
我们表明，ESX-5 底物 LipY（一种脂肪酶）可作为异源分泌卵清蛋白片段（OVA）的载体。LipY 包含一个 PE 结构域和一个脂肪酶结构域，由一个连接区隔开。该连接区在分泌过程中被加工。将 LipY 的 PE 和连接结构域融合到 OVA 上，使融合构建体 LipY-OVA 能够在 M. marinum 中依赖 ESX-5 进行分泌，尽管效率较低。随后对 LipY-OVA 进行随机诱变和筛选以提高分泌效率，得到了具有改善异源分泌的突变体。详细分析确定了 OVA 中的两个突变可提高分泌效率，即 L280P 突变和蛋白质延伸移码突变。最后，删除 LipY 的连接区可增强 LipY-OVA 的分泌，但该突变也会降低表面结合。在野生型 LipY 中的进一步分析表明，连接区对于表面结合是必需的。
我们表明 ESX-5 系统可用于异源分泌。此外，底物中的微小突变可以增强分泌。特别是 C 末端区域似乎对此很重要。LipY 的连接区参与表面结合。这些发现表明，非定向筛选方法有助于优化异源分泌，这有助于异源疫苗的开发。
