Fig 2: Influence of sialylated glycans on reovirus binding to JAM-A. a Binding of T3SA+ or T3SA- virions, or T3SA+ ISVPs to JAM-A was monitored following injection of 1 mM a-SA glycans (Neu5Ac [b, red] and LSTa [c, yellow]) or non-sialylated glycan (LNnT [d, green]). b–d DFS plots of interaction forces measured between T3SA+ and JAM-A after adding 1 mM glycan (Neu5Ac in b, LSTa in c, and LNnT in d). Grey dots represent the measured binding forces before injection. e DFS plot of the interaction forces between JAM-A and T3SA+ ISVPs, which display a more extended conformation of the s1 protein. Multivalent interactions are observed for T3SA+ ISVPs (blue) in comparison to T3SA+ virions (gray) without injection of free SA. f Example for analysis of the number of established bonds shown in panel g. Relative frequency of single and multiple bonds before and after adding free glycans was determined from areas under respective peaks (AI, AII, AIII; grey shaded) within force distribution histograms (red bars with cumulative peak, ATOT). g Number of bonds established between JAM-A and T3SA+ (left panel) or T3SA- (middle panel) virions or T3SA+ ISVPs (right panel), before and after injection of sialylated (Neu5Ac—red, LSTa—yellow) or non-sialylated (LNnT—green) glycans. Error bars indicate s.d. of the mean value. For all experiments, data are representative of at least n = 3 independent experiments. Source data are provided as a Source Data file

Fig 3: Probing reovirus binding to JAM-A on model surfaces. a Binding of single virions (T3SA+ or T3SA-) to JAM-A probed on a model surface. b DFS plot showing the force required to separate T3SA+ (upper panel) or T3SA- (lower panel) virions from JAM-A and fitted with the BE model. Error bars indicate s.d. of the mean value. For all experiments, data are representative of at least n = 5 independent experiments. c Box plot of the binding frequency of reovirus to JAM-A model surface. T3SA+ interactions are shown in grey, T3SA- in white, and hatched boxes represent injection of 10 µg/mL JAM-A antibody (Ab). The horizontal line within the box indicates the median, boundaries of the box indicate the 25th and 75th percentile, and the whiskers indicate the highest and lowest values of the results. The square in the box indicates the mean. ns, P > 0.05; ****P < 0.0001; determined by two-sample t-test in Origin. Source data are provided as a Source Data file

Fig 4: Confocal microscopic images of JAM1 in IHGE cells.(A) Schematic view of the JAM1 construct. The Myc-mCherry tag is indicated by the magenta box, and the HA-tag is indicated by the green box. The signal peptide is indicated by the blue box, and IG-LIKE and TMD domains are indicated by the gray boxes and orange box, respectively. (B) Confocal microscopic images of an IHGE cell expressing Myc-mCherry-JAM1, stained with anti-JAM1 (green) and anti-HA (cyan). mCherry is shown in magenta. Higher magnifications of the areas indicated by the white boxes in the left panels are shown at right. Scale bar, 5 µm. (C–F) IHGE cells were transiently transfected with plasmids encoding Myc-mCherry–tagged HA-inserted JAM1; in (C) and (D), the cells were also transfected with EGFP-SEC61ß (green). Following 48 h of incubation, cells were fixed, stained with anti-HA (D, magenta; F, green), and also stained with Alexa Fluor 488–conjugated phalloidin (E, green) or Alexa Fluor 633–conjugated phalloidin (F, magenta). The cells were then analyzed by immunofluorescence microscopy. Higher magnifications of the areas indicated by white boxes in the upper panels are shown on the lower side. Scale bars, 5 µm. See also S9A–S9D Fig.

Fig 5: P. gingivalis degrades JAM1 of gingival epithelium, causing penetration of LPS and PGN.(A, B) Schematic illustration of the three-dimensional culture (A) and confocal microscopic cross-sectional images (B) of the three-dimensional culture of IHGE cells. Gingival epithelial tissues (WT or overexpressing JAM1) were infected with P. gingivalis for 30 min. Tissues were then fixed, stained with anti-JAM1 (white) and Alexa Fluor 568–conjugated phalloidin (magenta), and analyzed by confocal microscopy. Scale bars, 30 μm. (C–G) Permeability to 40 kDa FITC-dextran (C), FITC–P. gingivalis LPS (D), FITC–P. gingivalis PGN (E), FITC–E. coli LPS (F), and FITC–S. aureus PGN (G) of gingival epithelial tissues (WT or overexpressing JAM1) infected with P. gingivalis. Three-dimensional tissues on culture inserts were infected with P. gingivalis and FITC-labeled tracer in the upper compartment. Following 30 min of incubation, the transmission of tracer from the upper compartment to the lower compartment was analyzed by spectrometry. Results are expressed as fold change relative to uninfected WT cells and are the means ± SD of seven technical replicates. *, p<0.05, one-tailed t test (closed testing procedure).