Journal: bioRxiv

Article Title: Bacterial exo-α-sialidases subvert the complement system through desialylation

doi: 10.64898/2026.02.05.703967

Figure Lengend Snippet: (a) SDS-PAGE analysis of six purified recombinant sialidases, including Td -NanH, Cp -NanI, Cp -NanH, Bf -NanH2, Tf -NanH, and Vc -NanH. Cp -NanI was purchased from Sigma-Aldrich, N2133 and the remaining five sialidases were expressed in E. coli and purified using FPLC. (b) Kinetic analysis of six GH33 sialidases. This assay was carried out as described in the Methods section using 4-methylumbelliferyl-α-D-N-acetylneuraminic acid (4-MUNANA) as a substrate. Saturation curves were fitted to Michaelis-Menten kinetics using GraphPad Prism ; and K m and V max were calculated (see details in Supplementary Table 5). (c-e) GH33 sialidases desialylate human serum, C4 and FH proteins. For this study, normal human serum (NHS), C4 or FH proteins were incubated with six recombinant sialidases at 37°C for 1 or 3 hours, followed by SDS-PAGE and lectin blots using SNA. Abbreviations: Td, Td- NanH; Cp-I, Cp- NanI; Cp-H, Cp- NanH; Bf, Bf- NanH2; Tf, Tf- NanH; and Vc, Vc- NanH.

Article Snippet: Briefly, 50 nM purified bacterial sialidases were incubated with increasing concentrations (15.625 - 1250 μM) of either 2′-(4-methylumbelliferyl)-α-D- N -acetylneuraminic acid (4-MUNANA; GoldBio, St. Louis, MO) or 4-methylumbelliferyl α- N -glycolylneuraminic acid (4-MUNGNA; Sussex Research, Ontario, Canada) in the appropriate reaction buffer.

Techniques: SDS Page, Purification, Recombinant, Incubation

Journal: bioRxiv

Article Title: Bacterial exo-α-sialidases subvert the complement system through desialylation

doi: 10.64898/2026.02.05.703967

Figure Lengend Snippet: (a) Schematic illustration of the domain architecture of T. denticola NanH ( Td -NanH). Annotated features include the signal peptide, the conserved “RIP” motif, and three Asp-box motifs with their respective amino acid positions. InlB: Listeria monocytogenes Internalin B. (b-c) Enzymatic kinetics of Td -NanH toward the fluorogenic substrates 4-MUNANA and 4-MUNAGc. Assays were performed as described in using wild-type Td -NanH and three point mutants including Td -NanH R140A , Td -NanH P142A , and Td -NanH R140AP142A ; and as C-terminal fragment of Td -NanH ( Td-C ) (d) Serum bactericidal assays. T. denticola wild-type, the Td -NanH deletion mutant (Δ Tde471 ), and the isogenic complemented strain ( CTde471 ) were incubated with 25% normal human serum (NHS) or heat-inactivated serum (HIS) for 30 min at 37 °C. Viable bacteria were quantified using a Petroff– Hausser counting chamber. Survival was calculated as the ratio of viable cells in NHS relative to HIS. Data are presented as mean ± SEM and analyzed by one-way ANOVA with Tukey’s multiple comparisons test ( P < 0 . 01 ). (e) Td -NanH restores serum resistance in the Δ Tde471 mutant. NHS (25%) was pre-treated with recombinant Td -NanH or the catalytically inactive mutant Td -NanH * for 1-3 h at 37 °C prior to serum killing assays with the Δ Tde471 strain using the same protocol described in (d). (f) GH33 sialidases broadly inhibit serum bactericidal activity. NHS (25%) was pre-treated with six GH33 sialidases for 1 h at 37 °C and subsequently used in serum killing assays with the Δ Tde471 mutant, following the protocol in (d). (g) Complement deposition on Δ Tde471 cells. A total of 5 × 10□ Δ Tde471 cells were incubated with 25% NHS for 15 min at 37 °C under three conditions: untreated NHS, Td -NanH-treated NHS, or Td -NanH * -treated NHS. Cell-associated complement components were analyzed by SDS-PAGE and immunoblotting using antibodies against C3 and C9. A T. denticola specific antibody served as a loading control.

Article Snippet: Briefly, 50 nM purified bacterial sialidases were incubated with increasing concentrations (15.625 - 1250 μM) of either 2′-(4-methylumbelliferyl)-α-D- N -acetylneuraminic acid (4-MUNANA; GoldBio, St. Louis, MO) or 4-methylumbelliferyl α- N -glycolylneuraminic acid (4-MUNGNA; Sussex Research, Ontario, Canada) in the appropriate reaction buffer.

Techniques: Mutagenesis, Incubation, Bacteria, Recombinant, Activity Assay, SDS Page, Western Blot, Control