Department of Bioengineering, Shanghai Institute of Technology, 100 Haiquan Rd, Fengxian District, Shanghai 201418, China.
Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Rd, Minhang District, Shanghai 200240, China.
Int J Food Microbiol. 2019 Jun 2;298:11-19. doi: 10.1016/j.ijfoodmicro.2019.03.008. Epub 2019 Mar 12.
Norovirus is a very contagious virus that causes acute gastroenteritis. Contaminated produce is a main vehicle for dissemination of human noroviruses (HuNoVs). As HuNoVs could bind to bacteria effectively, it is highly possible that produce could be contaminated by bacteria-HuNoVs complex. In this study, we used a bacterial-surface-display system to express genogroup I (GI) or genogroup II (GII) HuNoV capsid protein (P protein) on the surface of bacteria. The bacteria-P protein complex (BPC) was used to characterize the conditions for binding to Romaine lettuce extract and removal of the bound BPCs. We demonstrated both GI and GII BPCs could bind to extract from leaf (LE) and vein (VE) effectively. Carbohydrates in LE and VE were involved in GI BPCs binding, and both carbohydrates and proteins were involved in GII BPCs binding. Saliva from both type A and O secretors could completely block binding of both BPCs to LE and VE. Saliva from type B secretors only partially blocked binding of GII but not GI BPCs to LE and VE. However, LE- and VE-bound BPCs could not be reversely removed by washing solution containing free HBGAs from saliva. The binding of GI BPCs to LE and VE was enhanced when pH was below pI (6.1) of GI and reduced when pH was above pI of GI (p < 0.05). The optimal binding for GII BPCs to LE and VE occurred at pI (6.4) of GII. All LE- or VE-bound BPCs could be reversely removed by washing with low (3.0-5.0) or high (9.0-10.0) pH buffer. The effect of ionic strength (NaCl and MgCl, from zero to 100 g/L) on binding of BPCs to LE and VE was tested. The optimal ionic strength for binding of BPCs to LE and VE was 10.0 g/L (GI) and 5.0 g/L (GII) for NaCl, and 5.0 g/L for MgCl. LE- and VE-bound BPCs could be reversely removed by washing with high ionic solutions. All LE- or VE- bound BPCs could be released when washed with NaCl concentrations of above 75.0 g/L (GI) and 25.0 g/L (GII), or with MgCl concentrations of above 75.0 g/L (GI) and 50.0 g/L (GII). Binding of BPCs to LE and VE was inhibited in the presence of Tween-80 (nonionic surfactant) as low as 0.05% (v/v). All LE- and VE-bound BPCs could be reversed by Tween-80 concentrations over 0.1% (v/v). The study provided important parameters for BPCs binding to and removal from lettuce extract.
诺如病毒是一种非常具有传染性的病毒，会导致急性肠胃炎。受污染的农产品是人类诺如病毒（HuNoVs）传播的主要载体。由于 HuNoVs 可以有效地与细菌结合，因此农产品很有可能被细菌-HuNoVs 复合物污染。在这项研究中，我们使用细菌表面展示系统在细菌表面表达基因 I（GI）或基因 II（GII）HuNoV 衣壳蛋白（P 蛋白）。使用细菌-P 蛋白复合物（BPC）来描述与罗马生菜提取物结合和去除结合的 BPC 的条件。我们证明 GI 和 GII BPC 都可以有效地与叶（LE）和脉（VE）提取物结合。LE 和 VE 中的碳水化合物参与 GI BPC 结合，而碳水化合物和蛋白质都参与 GII BPC 结合。来自 A 型和 O 型分泌者的唾液可以完全阻止两种 BPC 与 LE 和 VE 的结合。来自 B 型分泌者的唾液仅部分阻断 GII 但不阻断 GI BPC 与 LE 和 VE 的结合。然而，含有来自唾液的游离 HBGAs 的洗涤溶液不能将 LE 和 VE 结合的 BPC 反向洗脱。当 GI 的 pH 值低于 pI（6.1）时，GI BPC 与 LE 和 VE 的结合增强，当 pH 值高于 GI 的 pI（p<0.05）时，结合减少。对于 GII BPC 与 LE 和 VE 的最佳结合发生在 GII 的 pI（6.4）时。用低（3.0-5.0）或高（9.0-10.0）pH 缓冲液洗涤可将所有 LE 或 VE 结合的 BPC 反向洗脱。还测试了离子强度（NaCl 和 MgCl，从零到 100g/L）对 BPC 与 LE 和 VE 结合的影响。BPC 与 LE 和 VE 结合的最佳离子强度为 10.0g/L（GI）和 5.0g/L（GII）用于 NaCl，以及 5.0g/L 用于 MgCl。高离子溶液可将 LE 和 VE 结合的 BPC 反向洗脱。当用 NaCl 浓度高于 75.0g/L（GI）和 25.0g/L（GII）或用 MgCl 浓度高于 75.0g/L（GI）和 50.0g/L（GII）洗涤时，可释放所有 LE 或 VE 结合的 BPC。当 Tween-80（非离子表面活性剂）的浓度低至 0.05%（v/v）时，BPC 与 LE 和 VE 的结合就会受到抑制。所有 LE 和 VE 结合的 BPC 都可以通过 Tween-80 浓度高于 0.1%（v/v）来逆转。该研究为 BPC 与生菜提取物的结合和去除提供了重要参数。
