Fig. 3. A. phagocytophilum colonizes I. ricinus cells by using core α1,3-fucosylation.
A. I. ricinus IRE/CTVM19 (2 × 105) cells were transfected with the α1,3-fucosyltransferase dsRNA and the silencing of α1,3-fucosyltransferase was examined in the presence (+) and absence (−) of A. phagocytophilum.
B. A. phagocytophilum load is shown 72 h post infection in mock and dsRNA-transfected I. ricinus cells.
C–E. Mock and dsRNA-transfected I. ricinus cells were visualized under the microscope and the presence of A. phagocytophilum is shown (arrows).
F. Protein extracts (1 µg) from I. ricinus cells (Iri) were electrophoresed and stained with Coomassie Blue R250 (lane A). Protein extracts were immunoblotted with the anti-HRP antibody (lane B) showing that anti-HRP antibodies recognized core α1,3-fucose residues in I. ricinus cells.
G. α1,3-Fucosyltransferase 7 (ft7) was amplified from I. ricinus cDNA originated from the IRE/CTVM19 cell line. A fragment of 110 bp was cloned into the plasmid pGEM-T Easy Vector and sequenced. This sequence was 99% identical to the conserved region (nucleotide position 452–561) of I. scapularis fucosyltransferase 7 (XM_023318). α1,3-Fucosyltransferase 2 (ft2) was amplified from I. ricinus cDNA originated from the IRE/CTVM19 cell line. A fragment of 189 bp was cloned into the plasmid pGEM-T Easy Vector and sequenced. This sequence shared 96% identity (nucleotide position 1–189) with the fucosyltransferase I. scapularis 2 (isft2) (XM_002401196). Cell culture experiments were repeated twice. Statistical analysis was performed using the Student’s t-test (P < 0.05).