FIG 4 .

PUL9 gene products allow mucin degradation and deglycosylation. (A) Schematic representation of the PUL9 locus. (B) Schematic drawing of the PNA lectin target oligosaccharides. (C) PNA lectin staining of human saliva after incubation in the presence of wt C. canimorsus 5 and the 13 PUL knockout strains. The upper band represents mucin, while the lower band represents sIgA. (D) Saliva PNA staining after incubation with wt bacteria, PUL9, and Ccan_17430 knockout bacteria. (E) Saliva PNA staining after incubation with wt bacteria, PUL9, or PUL9 bacteria expressing the only Ccan_17430 protein. (F) Western blot analysis of human mucin 7 in saliva after incubation with wt bacteria, PUL9, or PUL9 bacteria expressing the only Ccan_17430 protein. See also Fig. S3 in the supplemental material. (G) Growth after 23 h of wt (C. canimorsus 5), PUL5 (ΔPUL5), PUL9 (ΔPUL9), and PUL5/PUL9 (ΔPUL5 ΔPUL9) mutant bacteria in the presence of HEK293 cells with or without bovine submaxillary gland mucin (1 mg/ml) or GlcNAc (0.005%) (MOI, 0.005). The averages from three independent experiments are shown. Error bars represent 1 standard deviation from the mean. (H) Bright-field microscopy picture of PUL5 (ΔPUL5) mutant bacteria grown for 23 h on HEK293 cells in the presence of bovine mucin (1 mg/ml) (MOI, 0.05). (I) Functional model of mucin degradation and deglycosylation. Mucin is bound at the bacterial surface by the MucCDEG surface protein complex. The MucG protease cleaves mucin into glycosylated peptides that are imported into the periplasm through the MucC pore. Terminal sialic acid is cleaved by sialidase (SiaC), and terminal sulfate groups are removed by the sulfatase. The glycan is further processed by the sequential activity of several periplasmic exoglycosidases that allow the liberation of monosaccharides.