Fig. 6: Localization of the Th17Ag at the SFB surface.

Projection images from purified SFB a, b, e filaments (FIL) and c, d IOs stained using immunogold labeling with gold-conjugated protein A. Fixed SFB a, e filaments and c IOs were labeled with an anti-Th17Ag nanobody fused to the Fc region of human IgG1 (VHH-Fc). Fixed and denatured (Den) SFB b filaments and d IOs were labeled with a rabbit polyclonal antibody (Poly Ab) against Th17Ag. Imaging was performed at the SFB a–e(i) tip and a–e(ii) distal end. Close-ups (z) of the SFB tip and distal end are shown under or next to each panel. Examples of labeled regions without visible hair-like structures are shown by black arrow heads. e Examples of labeled regions with hair-like structures are shown by white arrow heads in a representative stage 4 filament. All projection images were acquired with a Tecnai F20 electron microscope equipped with a Falcon 2 camera with the exception of those included in (e). f Assessment of immunogold labeling for SFB imaged in the conditions described for a-d and after labeling of fixed and denatured SFB with a VHH-Fc anti-Th17Ag. The percentage of SFB labeled and labeled specifically at the tip are indicated on the corresponding bar. SFB were considered labeled if co-localization with at least 20 gold particles (black spheres) was observed, or at least 10 gold particles if labeling was restricted to the SFB tip. The number of SFB imaged per condition is included in the graph. Individual data points corresponding to the percentages shown are in the Supplementary Data 2 file. Source data are provided as a Source Data file. Immunofluorescence images of g fixed SFB incubated with a biotinylated VHH-Fc anti-Th17Ag and Streptavidin-Alexa568 and h fixed and denatured SFB incubated with a rabbit polyclonal antibody (Ab) against the Th17Ag and a secondary antibody anti-rabbit conjugated with Alexa 568. All SFB were additionally labeled with DAPI. An image of a labeled IO was included as an insert delimited by a white line (g). Close-ups of the tip of an SFB filament showing Th17Ag labeling (white arrow head) were included next to the main panel. Images showing the signal from bright field (BF), Alexa-568 and corresponding merged image are shown. A close-up of the IO shown in (h) was included as an insert delimited by a dashed white line. For all immunogold and immunofluorescence experiments, labeling was performed in two distinct days using two biological replicates for each experiment. i Localization of the nanobody binding epitope on the structure of the Th17Ag predicted by AlphaFold⁵⁹. Structure prediction with surface representations of the Th17Ag highlighting the protein regions (in red, orange and magenta) that form the conformational epitope recognized by the nanobody. Epitope location was determined by Hydrogen/Deuterium eXchange-Mass Spectrometry (HDX-MS). The Th17Ag sequence used to generate the AlphaFold model is provided in the Source Data file. Scale bars: a–e: 100 nm; g, h: 5 µm for main panel and 1 µm for inserts.