Characterization of conditions for bacteria-human norovirus capsid P protein complex (BPC) binding to and removal from Romaine lettuce extract

Authors: RONG, SHAOFENG - SHANGHAI INSTITUTE OF TECHNOLOGY; ZHOU, YUE - SHANGHAI INSTITUTE OF TECHNOLOGY; WANG, MING - SHANGHAI INSTITUTE OF TECHNOLOGY; GUAN, SHIMIN - SHANGHAI INSTITUTE OF TECHNOLOGY; ZHANG, SHUO - SHANGHAI INSTITUTE OF TECHNOLOGY; CAI, BAOGUO - SHANGHAI INSTITUTE OF TECHNOLOGY; WANG, DAPENG - SHANGHAI JIAOTONG UNIVERSITY; Tian, Peng; LI, QIANQIAN - SHANGHAI INSTITUTE OF TECHNOLOGY

Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/11/2019
Publication Date: 3/12/2019
Citation: Rong, S., Zhou, Y., Wang, M., Guan, S., Zhang, S., Cai, B., Wang, D., Tian, P., Li, Q. 2019. Characterization of conditions for bacteria-human norovirus capsid P protein complex (BPC) binding to and removal from Romaine lettuce extract. International Journal of Food Microbiology. 298:11-19. https://doi.org/10.1016/j.ijfoodmicro.2019.03.008.
DOI: https://doi.org/10.1016/j.ijfoodmicro.2019.03.008

Interpretive Summary: Human noroviruses (HuNoVs) are the major cause of outbreaks of acute non-bacterial gastroenteritis. The faecal-oral route is recognized as the main mode of transmission. The outbreaks of HuNoV are often associated with consumption of food contaminated during production, harvesting, or processing. A CDC survey estimated that 58.3% of all food-borne disease outbreaks associated with leafy greens were caused by HuNoV. It has been demonstrated that both GI and GII HuNoV or viral like particles could bind to the surface of Romaine Lettuce. Although some knowledge on candidate receptors on lettuce have been obtained from these studies, the kinetic of viral associate (binding) and disassociate (binding reverse) in lettuce has never been studied. In addition, most previous studies were only performed with purified viral like particles or viruses in the absence of bacteria. However, human stools from sick and recovered patients contain high titers of infectious viruses as well as bacteria. Recent studies demonstrated that HuNoV could bind to bacteria isolated from human gut. The naturally occurring strains of bacteria isolated from human stools could rapidly bind to both GI and GII HuNoVs. Currently, HuNoV-bacteria interaction is an emerging topic. Although certain bacteria could express histo-blood group antigens (HBGAs) on their surface and bind to HuNoV, non-HBGA expressing bacteria also bind to HuNoV and surrogates such as Tulane virus (TV) and MNV. Previously, we demonstrated that expression of P proteins from HuNoV on the surface of bacteria could form a bacteria-P-protein complex (BPC). BPC could bind to candidate viral receptors in Romaine lettuce extract and human saliva. In this study, we used this system to determine conditions required for GI and GII BPCs binding to and removal from lettuce. We demonstrated that both GI and GII BPCs could bind to extract from Romaine leaves (LE) and veins (VE) effectively. The results also suggested that carbohydrates in LE and VE were involved in GI BPC binding, and both carbohydrates and proteins in LE and VE were involved for GII BCP binding. Binding of BPC to LE or VE were not significantly affected by variation of pH tested ranging from pH 5.1 to 7.6. However, LE- and VE-bound BPC could be reversely removed by washing with low pH buffer (3-5) or high pH buffer (9-10). An ionic strength-dependent binding of BPC to LE and VE was observed in the range of zero to 10 g/L of NaCl and from zero to 5 g/L of MgCl2. The optimal ionic strength for binding of BPC to LE and VE was 10 g/L for NaCl and 5 g/L for MgCl2. A higher ionic strength could reduce binding of BPC to LE or VE. The binding was abolished when the ionic strength was above 75 g/L. LE- and VE-bound BPC could not be reversely removed by washing with low ionic strength solutions. However, the bound BPC could be reversely removed by washing with high ionic solutions. The effect of nonionic surfactant (Tween 80) on binding and binding-reverse was also tested. The binding of BPCs to LE and VE was inhibited dose-dependently by Tween 80. LE- and VE-bound BPCs could be reversed by Tween 80.

Technical Abstract: Human noroviruses (HuNoVs) are the major cause of outbreaks of foodborne gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. As HuNoV could bind to bacteria in human gut, it is highly possible that produce could be contaminated by bacteria-HuNoV complex. In this study, we used a bacterial-surface-displace system to express capsid protein (P protein) from genetype I (GI) and II (GII) of HuNoV on the surface of bacteria. The bacteria-P protein complex (BPC) was used to characterize the conditions for binding of BPC to Romaine lettuce extract and, more importantly, the conditions for removal of the bound BPC. We demonstrated that both GI and GII BPCs could bind to extract from Romaine leaves (LE) and veins (VE) effectively. The components in LE and VE for binding of GI and GII BPC were further explored. The ability of binding of BPC to LE or VE was either abolished (GII) or significantly (80%) reduced (GI) when LE and VE were oxidized by NaIO4. The ability of binding of BPC to LE or VE were unaffected (GI) or partially affected when LE or VE were denatured by heat. The results suggested that carbohydrates in LE and VE were involved in GI BPC binding, and both carbohydrates and proteins in LE and VE were involved for GII BCP binding. Saliva from both type A and type O secretors could completely block binding of both BPCs to LE and VE. Saliva from type B secretors could only partially block binding of GII but not GI BPC to LE and VE as expected. However, LE- and VE-bound BPCs could not be reversely removed by using washing solution containing free HBGA from saliva or pig stomach mucin. Binding of BPC to LE or VE were not significantly affected by variation of pH tested ranging from pH 5.1 to 7.6. However, LE- and VE-bound BPC could be reversely removed by washing with low pH buffer (3-5) or high pH buffer (9-10). An ionic strength-dependent binding of BPC to LE and VE was observed in the range of zero to 10 g/L of NaCl and from zero to 5 g/L of MgCl2. The optimal ionic strength for binding of BPC to LE and VE was 10 g/L for NaCl and 5 g/L for MgCl2. A higher ionic strength could reduce binding of BPC to LE or VE. The binding was abolished when the ionic strength was above 75 g/L. LE- and VE-bound BPC could not be reversely removed by washing with low ionic strength solutions. However, the bound BPC could be reversely removed by washing with high ionic solutions. The effect of nonionic surfactant (Tween 80) on binding and binding-reverse was also tested. The binding of BPCs to LE and VE was inhibited dose-dependently by Tween 80. LE- and VE-bound BPCs could be reversed by Tween 80.