Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms

Abstract

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

Fig. 1. CryoEM structure of pili surface variation.

a Illustrative Cryo-EM micrograph of type IV pili (SB sequence type, GATDH, G3P). Filament segments were selected (white circles). 2D classes averages (inset) already show secondary structure features that allow to identify monomers (vertical white bars). b CryoEM maps of SB and SA sequence types; left, cryoEM map at 2.99 Å resolution of the SB sequence type pilus, pilin monomers appear in different colors, G3P in red and GATDH in yellow; middle, cryoEM map at 3.15 Å resolution of the pilus made of the SA sequence type pilin (GATDH, G3P); right, pilus surface showing variable regions, amino acid changes between SA and SB sequence types (green), G3P (red) and GATDH (yellow). c Individual SB pilin subunit with G3P and GATDH, the hypervariable loop appears in dark blue. Close-ups of the phosphoglycerol moiety on S69 and on the GATDH moiety present on S63 are indicated. The dashed line represents a potential hydrogen bond. d Overlay of the SB (blue) and SA (orange) sequence type structures. Side chains of amino acids that are different between the two sequence types appear as sticks. Close-ups of regions of interest are presented; top, amino acid changes in strand 1 of the beta sheet head; bottom, comparison of the hypervariable loop of SB and SA sequence types.

Fig. 2. Isolation of a set of nanobodies reacting with type IV pili.

a Illustration of the procedure, pilus isolation, alpaca immunization and nanobody domain cloning. Created with BioRender.com. b Labeling of type IV pili expressed on the surface of bacteria adhering to endothelial cells with the 20D9 monoclonal antibody. Top panel, DNA staining labeled in blue shows the cell nucleus and two bacterial aggregates. Bottom panel, labeled pili on the same infected cell appear in red; the position of bacterial aggregates identified with the DNA stain were added with a dotted line and the nucleus with a gray shape with the letter N. The same representation of bacterial colony and nucleus was used in (c) and (d). c Testing of the ability of 6 representative nanobodies to react with pili. For comparison purposes the same settings were used for pilus image acquisition and display for the different nanobodies. d Determination of the recognition site of the F10 and C24 nanobodies using mutants affecting different variable surface structures, glycosylation (pglD), phosphorylation (pptB) and amino acid sequence (SA). The scale bars on (b–d) correspond to 5 µm. Each of the immunofluorescence experiments were performed 3 times.

Fig. 3. Characterization of the F10 and C24 nanobodies.

a ELISA assay using the F10-mFc fusion on the wild type and indicated strain immobilized on the bottom of the ELISA plate. b Same as in A using the C24-mFc fusion. For (a, b), graphs represent the result of 3 independent experiments each done in triplicate. Data represent the mean ± SEM. c Impact of the indicated amount of F10 (gray) or C24 (green) on the ability of meningococci to form type IV pili-dependent aggerates. A nanobody directed against the Tau protein was used as a negative control at a concentration of 5 µg/ml (black). Results from 3 independent experiments each done in triplicate are presented. Data represent the mean ± SEM. d Impact of nanobodies fused to mouse antibody Fc regions on the survival of bacteria in the circulation. Fc-fused nanobodies were injected at 1 h post-infection and the bacteria in the blood collected at indicted times. A nanobody directed against the SARS-Cov2 nucleoprotein (Fc-αNP) was used as a control. A total of 8 mice per group were used in two independent experiments. Data represent the mean ± SEM. After checking for normality using a Shapiro-Wilkinson test, a two-way ANOVA statistical test was performed. Source data are provided as a Source Data file.

Fig. 4. Overall structure of F10 and C24 nanobodies complexed with the pilus (SB sequence type).

a Illustrative Cryo-EM image of type IV pili alone or complexed with the F10 and C24 nanobodies. Scale bar corresponds to 5 nm. b Color coded final Cryo-EM map of the SB-GATDH pilus structure (blue) complexed with the F10 nanobody (gray) at 2.92 Å resolution. c Cryo-EM map of the pilus structure (blue) complexed with the C24 nanobody (green) at 2.90 Å resolution. d Ribbon structure of the F10 nanobody complexed with the pilin monomer. e Ribbon structure of the C24 nanobody complexed with the pilin monomer. f Binding sites of the F10 and C24 nanobodies on the pilin surface.

Fig. 5. Detailed structures of the interaction of nanobodies with posttranslational modifications and amino acid sequence variants.

a Surface representation of F10 complexed with PilE with GATDH represented as sticks. b Interaction between F10 and the GATDH. c Surface representation of the F10 nanobody with the pilin. The hypervariable loop is indicated in blue. d Interactions between F10 and the PilE hypervariable loop. e Surface representation of C24 complexed with PilE with GATDH represented as sticks. f Interaction between C24 and the GATDH moiety. g Surface representation of C24 complexed with PilE with the phosphoglycerol represented as sticks. h Interactions between C24 and the the phosphoglycerol.

Fig. 6. Impact of amino acid changes on nanobody binding.

a Close-up of the F10/pilin binding region involving the hypervariable loop of the SB sequence type pilin (PilE aa 120-154). b Identified amino acid clashes of SA-variant mutations in the hypervariable loop preventing the binding of the F10 nanobody. c Close-up of the C24/SB pilin binding region involving the β1 pilin beta-sheet strand of the SB sequence type and the phosphoglycerol PTM. d Clashes preventing C24 binding to the SA sequence type.

Fig. 7. Impact of sugar variants on nanobody binding.

a Sharpened cryo-EM map of SB_DATDH (left) and the same structure complexed with the C24 nanobody (right). The PilE monomer is pink, DATDH is blue and G3P is red. b Close up of DATDH within the EM density. c Nanobody staining of HUVECs infected with the pglB1 strain displaying DATDH, with F10 (left) and C24 (right). d Close-up of PilE and DATDH interactions with C24_R103. e The GATDH-aligned double sugar (GATDH-GLA) attached to S63 modeled in the SB-GATDH-C24 structure reveals clashes with S112 and S114. f Type IV pili detection on HUVEC cells infected with the pglA_ON* strain expressing GATDH-GLA double sugar with the C24 and C24_S112-114G nanobodies. g Type IV pili detection on HUVEC cells infected with the GATDH expressing wild type strain. The same parameters were used for image acquisition and display. For (c, f, g), immunofluorence assays were done in 3 independent experiments. h ELISA assay characterization of the C24-mFc_S112-114G mutant binding to the pglA_ON* strain which expresses the SB sequence type with a GATDH-Gal glycosylation. i ELISA assay characterization of the C24-mFc_S112-114G mutant binding to the pglAOFF strain which, as the WT strain, expresses the SB sequence type with a GATDH. For (h, i), graphs represent the result of 3 independent experiments each done in triplicate. Data represent the mean ± SEM. Source data are provided as a Source Data file.