Materials and Methods
VLPs and antibodies used in the virus overlays, Ab3912 and NS14 were provided courtesy of Dr. Robert Atmar (Baylor College of Medicine, Houston, TX, USA). Primary antibodies used in Western blots were obtained from Abcam and corresponded to blood groups AB (ab24223), B (ab24224), H (ab24213), Lewis a (ab2967), Lewis b (ab3968), and Lewis y (ab3359). Additional materials included biotinylated H type 1, A, B HBGAs (GlycoTech) and biotinylated lectins isolated from: Bandeiraea simplicifolia (Sigma-Aldrich), Dolichos biflorus (Bio-world), and Ulex europaeus agglutinin (Vector Laboratories).
Seven different bacterial strains were used in this study. Two reference strains were supplied by the American Type Culture Collection: Staphylococcus aureus (ATCC 25235) and Enterobacter cloacae (ATCC 13047). The remaining five strains (Klebsiella spp., Bacillus spp., Enterococcus faecium, Citrobacter spp., and Hafnia alvei) were previously isolated from GI.6 norovirus positive stool [3]. For all studies, bacterial strains were grown aerobically at 37°C overnight in 40 ml of half-strength tryptic soy broth (TSB) [6].
Overnight cultures were centrifuged and resuspended in 4ml of 1X phosphate buffered saline (PBS; pH 7.2). To disrupt cellular integrity, the resuspensions were chilled on ice and sonicated using a Branson Digital Sonifier 450 at an amplitude setting of 60% for 7 rounds of 10 s followed by 20 s on ice. Sonicates were mixed 1:1 with 2X Laemmli sample buffer (Bio-Rad Laboratories) supplemented with β-mercaptoethanol and boiled in screw-top microcentrifuge tubes for 5 min. They were then loaded in 25µl aliquots into 12% mini-PROTEAN TGX precast gels (Bio-Rad Laboratories) with a spectra multicolor broad range protein ladder (Thermo Fisher Scientific). Gels were run for 35 min at 200 V in a Tris-glycine buffer.
Western blots were performed at room temperature using both HBGA primary antibodies and lectins. In preparation for blots, SDS-PAGE protein gels were transferred to 0.45μm nitrocellulose membranes using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad Laboratories) and blocked at 4°C overnight in SuperBlock blocking buffer (Thermo Fisher Scientific). To determine HBGA activity, the membranes were exposed to PBS containing 0.5% skim milk/0.05% Tween 20 and supplemented with a 1:500 ratio of the appropriate primary antibody. Membranes were incubated for 1 h with shaking and washed three times in PBS-0.5% Tween (PBS-T). They were then exposed for 2 h to secondary antibody (Anti-mouse IgG-alkaline phosphatase; Sigma-Aldrich) diluted 1:5000 in PBS with 5% skim milk-0.5% Tween. Membranes were again washed thrice in PBS-T and developed with a 5-bromo-4-chloro-3-indoyl-phosphate/p-nitroblue tetrazolium chloride (BCIP/NBT) solution (MP Biomedicals). Biotinylated HBGAs (A, B or H; Glycotech) were included as positive controls, while growth media was included as a negative control.
The protocol for Western blots probed using lectins was similar to that above except that 10μg of biotinylated lectin was used in place of primary antibody; and streptavidin-conjugated horseradish peroxidase (Invitrogen) at a 1:5000 dilution followed by addition of the One-Step TMB-Blotting Substrate Solution (Thermo Fisher) was used for signal development. Controls were HBGAs containing (positive) or missing (negative) the sugar of interest.
The virus overlay protocol was adapted from Kikkert et al. [7]. The nitrocellulose membrane was washed three times in binding buffer (25 mM Tris-HCl [pH 7.5], 50mM NaCl, 2mM dithioretitol [DTT], 2 mM EDTA, 0.25% Tween 20), then washed four times in renaturation buffer (25 mM Tris-HCl [pH 7.5], 50mM NaCl, 2mM DTT, 2 mM EDTA), and incubated overnight in fresh renaturation buffer. The blot was washed twice in 5% skim milk-0.05% Tween 20, followed by 30 min incubation in overlay buffer (5% skim milk-0.05% Tween 20, 2% polyvinylpyrrolidone). Diluted VLPs (2 μg/ml) were added to 10ml of overlay buffer and incubated with the blot for 2 h followed by washing three times in 0.5% skim milk-0.05% Tween. This was followed by exposure to primary antibody [Ab3912 (GI.1, GI.6, GI.7) and NS14 (GII.1, GII.4 Sydney, GII.17)] and development as described. As a positive control, 10μl of 1:1000 diluted norovirus antibody was included in each blot for viral adherence. Growth media was included as a negative control. An overlay was completed with the norovirus surrogate MS-2 as an additional negative control.