SBP of the ABC Type Fucose Catabolism System Hijacks Host Fucose to Drive Streptococcus suis Respiratory Colonization

Abstract

Figure S1. Determination of NAC treatment concentration and subcellular localization analysis of SBP. Survival rates of Hep-2 (A) cell after 2 h treatment with different concentrations of NAC, with untreated cells as controls. Western blot analysis of MUC5AC expression in Hep-2 (B) cell after 2 h treatment with serial concentrations of NAC, with tubulin as internal control. (C) Western blot analysis of secreted, cell wall, and cytoplasmic protein fractions isolated from S. suis WT and Δsbp using anti-SBP antibody. Recombinant SBP (rSBP) was included as a positive control, with the corresponding western blot and SDS-PAGE analysis shown on the right. Protein markers (M) indicate molecular weights (Vazyme, MP102). Figure S2. Neuraminidase treatment of Hep-2 cell fails to affect S. suis adhesion. Relative adhesion rates of WT (A), Δsbp (B), ΔxtrSs (C) and CΔsbp (D) to Hep-2 cell with or without 3 h of pretreatment with 100 mU/mL α2,3,6,8,9-neuraminidase A, normalized to the untreated control group. Data are presented as mean ± SD from three independent experiments (ns: p ≥ 0.05). Figure S3. SBPM Construction and conservation analysis of SBP among Streptococcus species. (A) Schematic diagram of the amino acid sequence of the SBPM, where the six key fucose-binding residues (Val113, Ile118, Gln145, Tyr153, Ser392, and Val393) are replaced with alanine (A) and highlighted in red. (B) Purification and immunodetection of SBPM. Left panel: SDS-PAGE analysis of the SBPM purification process. The lane "Inclusion body" shows improperly folded protein retained in inclusion bodies, while "Purification" shows soluble protein correctly folded and purified from the supernatant. Right panel: Western blot analysis of purified rSBPM using anti-rSBP serum. Multiple sequence alignment (C) and three-dimensional structural models (D) of SBP homologs from various respiratory Streptococcus species. Key residues highlighted in red boxes correspond to the hydrophobic pocket region (highlighted in red in the structural model), which serves as the critical binding site for fucose residues. Figure S4. Bacterial Loads, Histopathological Changes, and Cytokine Expression in Organs. Bacterial load in spleen (A), liver (B), and brain (C) of WT, Δsbp, and CΔsbp mice at 6 h post-infection, with or without NAC pretreatment. (D) H&E staining of brain and spleen sections. Brain sections show neuronal atrophy and blurred nuclear-cytoplasmic boundaries (black arrows), while spleen sections reveal lymphocyte necrosis and nuclear fragmentation (black arrows). Scale bar: 50 μm. (E) Relative transcriptional levels of TNF-α, IL-1β, IL-2, and IL-6 in brain and spleen of mice infected with WT, Δsbp, and CΔsbp, with or without NAC pretreatment. (F) Relative MUC5AC transcriptional levels in Hep-2 cell treated with SBPM (200 ng/μL), normalized to untreated control cells (cultured in DMEM medium only). (G) Western blot analysis of MUC5AC expression in Hep-2 cell treated with serial concentrations of SBPM (0, 100, 200 ng/μL) for 4 h. Tubulin was used as a loading control. All data are presented as mean ± SD from at least three independent experiments (ns p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).Table S1 Bacterial strains and plasmids used in this studyTable S2 Primers used in this study