The surface-layer (S-layer) protein of Lactobacillus acidophilus is a crystalline array of self-assembling subunits, non-covalently bound to the most outer cell wall envelope, which constitutes up to 20% of the total cell protein content. These attributes make S-layer proteins an excellent anchor for the development of microbial cell-surface display systems. In L. acidophilus, the S-layer is formed predominantly by the protein SlpA. We have previously shown that the C-terminal domain of SlpA is responsible for the cell wall anchorage on L. acidophilus ATCC 4356. In the present study, we evaluated the C-terminal domain of SlpA of L. acidophilus ATCC 4356 as a potential anchor domain to display functional proteins on the surface of non-genetically modified lactic acid bacteria (LAB). To this end, green fluorescent protein (GFP)-CTSlpA was firstly produced in Escherichia coli and the recombinant proteins were able to spontaneously bind to the cell wall of LAB in a binding assay. GFP was successfully displayed on the S-layer stripped surface of L. acidophilus. Both the binding stability and cell survival of L. acidophilus decorated with the recombinant protein were then studied in simulated gastrointestinal conditions. Furthermore, NaCl was tested as a safer alternative to LiCl for S-layer removal. This study presents the development of a protein delivery platform involving L. acidophilus, a microorganism generally regarded as safe, which utilizes the contiguous, non-covalently attached S-layer at the cell surface of the bacterium without introducing any genetic modification.

中文翻译：

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以嗜酸乳杆菌 SlpA 的 C 端结构域为锚在乳酸菌细胞表面展示异源蛋白的策略

嗜酸乳杆菌的表层（S 层）蛋白是自组装亚基的结晶阵列，非共价结合到最外层细胞壁包膜，占细胞总蛋白含量的 20%。这些属性使 S 层蛋白成为开发微生物细胞表面展示系统的极好锚。在嗜酸乳杆菌中，S 层主要由蛋白质 SlpA 形成。我们之前已经表明 SlpA 的 C 端结构域负责对嗜酸乳杆菌 ATCC 4356 的细胞壁锚定。在本研究中，我们评估了嗜酸乳杆菌 ATCC 4356 的 SlpA 的 C 端结构域作为潜在的锚在非转基因乳酸菌 (LAB) 表面展示功能性蛋白质的结构域。为此，绿色荧光蛋白 (GFP)-CTSlpA 首次在大肠杆菌中产生，在结合试验中，重组蛋白能够自发地与 LAB 的细胞壁结合。GFP 成功地展示在 L. acidophilus 的 S 层剥离表面上。然后在模拟胃肠条件下研究了用重组蛋白修饰的嗜酸乳杆菌的结合稳定性和细胞存活率。此外，测试了 NaCl 作为 LiCl 的更安全替代品，用于去除 S 层。本研究介绍了涉及嗜酸乳杆菌的蛋白质递送平台的开发，嗜酸乳杆菌是一种通常被认为是安全的微生物，它利用细菌细胞表面的连续、非共价连接的 S 层，而不引入任何遗传修饰。