Additional file 2 of Systemic antibody responses against gut microbiota flagellins implicate shared and divergent immune reactivity in Crohn’s disease and chronic fatigue syndrome
Creators: name: Bourgonje, Arno R.
name: Hörstke, Nicolai V.
name: Fehringer, Michaela
name: Innocenti, Gabriel
name: Vogl, Thomas
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Supplementary Material 2: Supplemental Figure S1. Sequences of antibody-bound flagellins overrepresented in CD and ME/CFS resemble both stimulator- and silent flagellins while C-terminal antibody-bound flagellin sequences exhibit higher resemblance to stimulator flagellins in CD. (A-B) Boxplots showing the degree of statistical significance (e-values) for full-length antibody-bound flagellin peptides towards reference sequences of stimulator-, silent-, and evader-type flagellin peptides (panel A: CD; panel B: ME/CFS). (C-D) Boxplots demonstrating the degree of statistical significance (e-values) between full-length antibody-bound flagellin peptide sequences and reference motifs for the C-terminal D0 domain (containing the allosteric binding site) (panel C: CD; panel D: ME/CFS). (E-F) Boxplots demonstrating the degree of significance (e-values) between N-terminal domains of antibody-bound flagellin peptides and N-terminal domains of reference sequences for stimulator-, silent-, and evader-type flagellin in patients with CD (left panel) and ME/CFS (right panel). (G-H) Boxplots demonstrating the degree of significance (e-values) between C-terminal domains of antibody-bound flagellin peptides and C-terminal domains of reference sequences for stimulator-, silent-, and evader-type flagellin in patients with CD (left panel) and ME/CFS (right panel). Supplemental Figure S2. Peptide sequences of antibody-bound flagellins overrepresented in CD and ME/CFS resemble full-length stimulator- and silent flagellins and highly (&gt;2.5 fold) overrepresented full-length antibody-bound flagellins overrepresented in CD and ME/CFS resemble both stimulator- and silent flagellins. (A-B) Boxplots showing the degree of sequence similarity (identity%, left panel) and statistical significance (e-values, right panel) for peptide-length antibody-bound flagellins in CD towards reference sequences of stimulator-, silent-, and evader-type flagellin peptides. (C-D) Boxplots showing the degree of sequence similarity (identity %, left panel) and statistical significance (e-values, right panel) for peptide-length antibody-bound flagellins in ME/CFS towards full-length reference sequences of stimulator-, silent-, and evader-type flagellin peptides. (E-F) Boxplots showing the degree of sequence similarity (identity %, left panel) and statistical significance (e-values, right panel) for highly (&gt;2.5 fold) overrepresented full-length antibody-bound flagellins in CD towards reference sequences of stimulator-, silent-, and evader-type flagellin peptides. (G-H) Boxplots showing the degree of sequence similarity (identity %, left panel) and statistical significance (e-values, right panel) for highly (&gt;2.5 fold) overrepresented full-length antibody-bound flagellins in ME/CFS towards full-length reference sequences of stimulator-, silent-, and evader-type flagellin peptides. Supplementary Figure S3. Full segment alignments of antibody-bound bacterial flagellins overrepresented in patients with CD and ME/CFS sharing distinct N- and C-terminal sequence motifs. (A) Full alignment of subset of antibody-bound flagellin peptides overrepresented in both patients with CD and ME/CFS; the location of the hinge epitope (nD0-1) and TLR5 motif (nD1) are highlighted and show shared motifs putatively bound by antibody responses. (B) Full alignment of antibody-bound flagellin peptides that were&gt;25 fold overrepresented in patients with CD which all share a common C-terminal motif putatively bound by antibody responses. Alignments were generated using MEGA software using the multiple sequence comparison by log-expectation (MUSCLE) algorithm in default settings (see Methods). Supplementary Figure S4. Full alignments of antibody-bound bacterial flagellins overrepresented in patients with CD showing both N- and C-terminal sequence motifs. Shown is the full alignment of antibody-bound flagellin peptides overrepresented in patients with CD in which both the N-terminal motifs shared with patients with ME/CFS is observed (albeit in a different order, lower red rectangle) as well as the CD-specific C-terminal motif (upper red rectangle). Alignments were generated using MEGA software using the multiple sequence comparison by log-expectation (MUSCLE) algorithm in default settings (see Methods). Supplementary Figure S5. Sequences of antibody-bound flagellins overrepresented in CD and ME/CFS resemble both stimulator- and silent flagellins also using alternative domains for alignments. (A) Histogram showing the distributions of sequence lengths of extracted and annotated N- and C-terminal domains of antibody-bound flagellin peptides when using the silent domain rather than the stimulator one as reference for the N-termini (see Methods). (B-C) Boxplots demonstrating the degree of sequence identity (%) between N-terminal domains of antibody-bound flagellin peptides and N-terminal domains of reference sequences for stimulator-, silent-, and evader-type flagellin in patients with CD and ME/CFS, yielding similar results as shown in Fig. 4, Fig. S1, and Fig. S2, demonstrating that the selection of the reference domain for alignments does not strongly impact outcomes. (D-E) Boxplots demonstrating the degree of sequence identity (%) between C-terminal domains of antibody-bound flagellin peptides and C-terminal domains of reference sequences for stimulator-, silent-, and evader-type flagellin in patients with CD and ME/CFS. (F-G) Boxplots demonstrating the degree of significance (e-values) between N-terminal domains of antibody-bound flagellin peptides and N-terminal domains of reference sequences for stimulator-, silent-, and evader-type flagellin in patients with CD (left panel) and ME/CFS (right panel). (H-I) Boxplots demonstrating the degree of significance (e-values) between C-terminal domains of antibody-bound flagellin peptides and C-terminal domains of reference sequences for stimulator-, silent-, and evader-type flagellin in patients with CD (left panel) and ME/CFS (right panel). Supplementary Figure S6. In contrast to unbound flagellins, the antibody bound flagellins in disease- and healthy control cohorts overlap in occurrence and resemble the stimulator and silent subgroups of 44 reference flagellins. (A) Venn diagrams illustrating overlaps of the antibody bound flagellins in CD, ME/CFS, HC-NL and HC-UK. (B) Bar plot showing the degree of sequence identity (%) for full length antibody-bound flagellin peptides in CD towards each of the 44 reference sequences (Table S4). (C) Boxplots showing the summarized degree of sequence identity (%) for full length antibody-bound flagellin peptides in CD, ME/CFS, and the HCs as well as unbound flagellins towards the 44 reference sequences (Table S4). P-values for comparison of sequence identity (%) were derived from Kruskal-Wallis tests followed by Dunn’s post hoc tests with Bonferroni P-value adjustment. Supplementary Figure S7. N- and C-terminal domain-sequences of antibody-bound flagellins overrepresented in CD and ME/CFS as well as domain sequences of flagellins antibody-bound in HCs resemble both stimulator- and silent flagellin group of 44 references while C-terminal antibody-bound flagellin sequences exhibit higher resemblance to stimulator flagellins in CD, ME/CFS and the HCs. (A) Histogram showing the distributions of the relative start and end positions of the N- and C-terminal domains of antibody-bound flagellins in CD. The brackets describe the ratio of all domain sequences in contained the marked bins. (B) Boxplots demonstrating the summarized degree of sequence identity (%) between N- and C-terminal domains of antibody-bound flagellin peptides and N- and C-terminal domains of 44 reference sequences (Table S4) in patients with CD. (C) Boxplots demonstrating the summarized degree of sequence identity (%) between N- and C-terminal domains of antibody-bound flagellin peptides and N- and C-terminal domains of 44 reference sequences (Table S4). Al in patients with ME/CFS. (D) Boxplots demonstrating the summarized degree of sequence identity (%) between N- and C-terminal domains of antibody-bound flagellin peptides and N- and C-terminal domains of 44 reference sequences (Table S4) in the healthy control cohorts. P-values for comparison of sequence identity (%) were derived from Kruskal-Wallis tests followed by Dunn’s post hoc tests with Bonferroni P-value adjustment.