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Characterization of an amyloid-based antiphage defence system in Escherichia coli

Nature Microbiology volume 10, pages 2168–2178 (2025)Cite this article

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Abstract

Amyloids are β-sheet-rich protein polymers that can cause disease but also serve functional purposes in cells. In animals and fungi, functional amyloids have a role in regulated cell death as molecular switches activating key cell death effectors. Here we describe an amyloid-based abortive infection antiphage defence system in Escherichia coli. This system leads to death of phage-infected cells and involves two proteins, Bab and Agp, which share a common amyloid motif and are encoded by adjacent genes. Following infection, Agp activates Bab through amyloid signalling. Activation of Bab then causes membrane alterations and cell death. We determined the structure of the cell death execution domain of Bab, which is distantly related to pore-forming domains found in fungi, animals and plants. We show that Bab and HET-S, a fungal amyloid-controlled regulated cell death execution protein from Podospora anserina, are functionally interchangeable. These findings show that amyloid-mediated immune signalling is conserved across kingdoms.

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Fig. 1: Bab and Agp share an amyloid-forming motif with prion properties.
Fig. 2: Bab/Agp is an antiphage system.
Fig. 3: Agp-dependent BASS11Bab–GFP assembly after T5 infection.
Fig. 4: Bab/Agp-dependent Bab–GFP relocalization and Bab/Agp-dependent membrane alteration during T5 infection.
Fig. 5: Structure of the Babybell domain of Bab and mutagenesis of the ‘MEPIS’-motif region of Avast2.
Fig. 6: Functional redesigning of Bab and HET-S.

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Data availability

The protein structure reported in this Article has been deposited in the Protein Data Bank (PDB) under accession code 9H9V. Data supporting the findings of this study are available within the Article and Supplementary Information. Source data are provided with this paper. Any other relevant data supporting the findings of this study are available from the corresponding author on request.

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Acknowledgements

This work was funded by an Agence Nationale de la Recherche (ANR) grant to S.J.S. and A.L. (TransKingAmyloid, ANR-21-CE11-0007). We thank T. Dakhli, A. Grélard and E. Morvan for their help during protein expression and data collection. This work has benefited from the Biophysical and Structural Chemistry Platform at IECB, University of Bordeaux, CNRS UAR 3033, INSERM US001. This study received financial support from the French government in the framework of the University of Bordeaux’s France 2030 programme ‘Frontiers of Life’ (RRI) to S.J.S., A.L. and S. Deb. We thank F. Darfeuille and A. LeRhun for providing advice and phage strains.

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Authors and Affiliations

  1. IBGC, UMR 5095, CNRS-Université de Bordeaux, Bordeaux, France

    Léa Ibarlosa, Sonia Dheur, Alexandra Granger-Farbos, Virginie Coustou, Aurélie Massoni-Laporte, Bénédicte Salin, Corinne Blancard & Sven J. Saupe

  2. CBMN, UMR5248, IECB, Université de Bordeaux, CNRS, Bordeaux INP, Pessac, France

    Corinne Sanchez, Seamoon Deb, Mélanie Berbon, Claire Saragaglia, Birgit Habenstein & Antoine Loquet

  3. Bacteriophage Bacterium Host, Institut Pasteur, CNRS UMR 6047, Université Paris Cité, Paris, France

    Xavière Menatong Tene, Nicolas Dufour & Laurent Debarbieux

  4. Réanimation Médico-Chirurgicale, Hôpital NOVO—Site de Pontoise, Pontoise, France

    Nicolas Dufour

  5. Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France

    Ombeline Rossier

  6. UAR3033 INSERM, Université de Bordeaux, CNRS, IECB, Pessac, France

    Brice Kauffmann

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  1. Léa Ibarlosa
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Contributions

Conceptualization: S.J.S. Methodology: S.J.S., A.L., B.K., S. Dheur, L.I., B.H., L.D., B.S. and A.M.-L. Investigation: L.I., S. Dheur, C.S., S. Deb, A.G.-F., V.C., M.B., A.M.-L., B.S., C.B., C.S., X.M.T., N.D., O.R. and S.J.S. Funding acquisition: A.L. and S.J.S. Supervision: A.L. and S.J.S. Writing—original draft: S.J.S. Writing—review and editing: A.L. and S. Dheur.

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Correspondence to Sven J. Saupe.

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Extended data

Extended Data Fig. 1 Sequence analysis of Bab/Agp and structural characterization of BASS11Bab.

a, An alignment of the sequence of the N-terminal region of selected bacterial Bab and Bell domain proteins, fungal Hell domain proteins and Avast2, bNACHT02, 11 and 23 NLRs involved in antiphage defence is given together with the domain and gene architecture in which the sequences occur. The color code is as in Fig. 1a, with amyloid forming sequences in blue and predicted N-terminal hydrophobic helix in red. Genbank entries for all mentioned gene products are given in Supplementary Table 1. b, Domain associations of BASS11-motifs. An alignment of BASS11-motifs occurring in different domain architectures is given. Corresponding domain architectures and the motif position (in blue) are given for each protein. Sequences grouped with a bracket accolade are encoded by adjacent genes. c, AlphaFold3 model of Agp. The TPR, GreA/B-C and PIN domains are given in cyan, blue and gold respectively. d, Structural characterization of Bab (50-109) fibrils by solid-state NMR spectroscopy. Top panel, excerpts of 2D 13C-13C correlation experiments recorded at 800 MHz 1H frequency, using mixing times of 50 ms (in black) and 150 ms (in red). Sequential assignment of the I87-G88-S89-V90 stretch is highlighted. Middle panel, excerpt of the aromatic region of the 2D 1H-13C INEPT, detecting disordered residues. Bottom panel, secondary chemical shifts for the stretch T85-V90. Positive or negative values correspond to α-helical or β-strand conformation, respectively. e, AlphaFold3 model of Bab(82-109) in multimeric form (with a sequence input of 6 monomers, ipTM=0.82, pTM=0.84), the positions of predicted β-strands are given in blue in the amino acid sequence. A consensus sequence logo of the BASS11 motif based on the alignment of Extended Data Fig. 1b is given. Position of conserved residues underlined in the sequence are given on the structural model, glycine residues interrupting the β-strands are given in grey and marked by asterisks. The boxed region corresponds to residues with spectral assignment in ssNMR for which chemical shift and thus secondary structure have been determined (Extended Data Fig. 1d). The model is consistent with chemical shifts experimentally determined by ssNMR. AF3 predictions for the BASS11Agp region were also performed but were of very low confidence.

Extended Data Fig. 2 Aggregation properties of the Agp and Bab BASS11 amyloid motifs.

a, Time course of BASS11 synthetic peptide amyloid aggregation followed by ThT fluorescence. Agp(2-25) (upper panel) or Bab(86-108) (lower panel) peptides were incubated at the given concentration with 50 µM ThT. Results are given in triplicates in different shades of red, magenta and blue for peptide concentrations of 30, 15 and 7.5 µM respectively. The buffer alone controls are given in shades of grey. b, Bathochromic shift in the Congo Red absorbance spectrum in the presence of Agp(2-25) (upper panel) or Bab(86-108) (lower panel) measured at 25 µM Congo Red and 90 µM of synthetic peptide, absorbance are normalized to the absorbance value at the maximal absorbance wavelength. Results are given as means +/−s.d. for triplicates. The absorbance spectra were recorded 15 sec, 10 min and 50 min after diluting the peptides (dissolved in 8 M GuHCl 250 mM acetic acid 1 mM DTT) into 100 mM Tris-HCl pH 8 150 mM NaCl 25 µM Congo Red. The absorbance spectrum of the buffer alone control is given in grey. A picture of the Agp(2-25), Bab(86-108) samples and buffer alone control (-) is given at the right. c, Time course of aggregation of recombinant full-length Bab-6his followed by ThT fluorescence at 7.5 µM (in triplicates) is given in different shades of red. The time course of Bab(86-108) synthetic peptide at the same concentration (in triplicates) is given in shades of blue. The buffer alone controls (in triplicates) is given in shades of grey. d, Representative example of electron micrograph of full length Bab-6his fibrils. Scale bar is 200 nm. e, The graph gives the proportion of cells containing GFP foci at different time points after transformation for the listed strains expressing wild-type or mutant versions of the BASS11 motif of Bab or Agp fused to GFP (BASS11-GFP). Results are given as means +/− s.d. of three technical replicates. P-values are calculated from one-way ANOVA and are Tukey’s multiple comparison tests comparing the values for the mutants (N12P or N100P) to the corresponding wild-type. f, Insolubility of BASS11-GFP in cells with foci. Anti-GFP western-blot of crude and fractionated E. coli extracts. Cells were harvested 5h or 24h after transformation and crude extracts (tot.) were centrifuged for 1h at 17,000g to yield the supernatant (sup.) and pellet (pel.) fractions. Size of molecular weight markers is given in kDa.

Source data

Extended Data Fig. 3 Formation of dot-like foci by BASS11Bab-GFP in E. coli.

a, Examples of colocalization of BASS11Bab-GFP foci with refringent inclusion bodies at the cell poles, scale bar is 2 µm. Two examples of representative fields are shown. b, Examples of cells with BASS11Bab-GFP cable-like structures, scale bar is 4 µm. Cables are observed in 3.3%±0.6 of the cells (means +/− s.d. of three technical replicates). c, Representative examples of colonies obtained after plating liquid cultures containing cells with dots and cells with diffuse BASS11Bab-GFP fluorescence: from top to bottom, a colony homogeneously displaying cells with dots, a colony homogeneously displaying cells with diffuse fluorescence and a chimeric sectored colony with a dot and a diffuse fluorescence sector. Colonies were imaged after 6 h of growth at 37° on solid LB medium. Scale bar is 10 µm. d, Correlation between the proportions of dot cells in starting liquid cultures and the proportion of dot colonies formed after plating. The upper graph gives the proportion of cells with dots at four different time points after transformation of MG1655 E. coli cells with a plasmid expressing BASS11Bab-GFP (t1 to t4 are 8h, 12h, 15h and 18h respectively). The two lower graphs give the proportion of dot and chimeric sectored colonies obtained after plating the liquid cultures from the time points t1 to t4. Results are means +/− s.d. of technical replicates (the same culture plated in triplicate). Pearson correlation coefficient between the proportion of cells with dots at t1, t2, t3 and t4 and proportion of colonies with dots at the same time points is r=0.9874 (two tailed p-value=0.0126, CI for r (0.5143; 0.9997)).

Source data

Extended Data Fig. 4 Bab/Agp confers resistance to T5 and induces Abi.

a, Comparison of plaque formation in MG1655 and MG1655::Bab/Agp strains infected with T5 (three biological replicates are shown for each strain). b, Counting of colony forming units after infection with T5 of MG1655 (white bars) or MG1655::Bab/Agp (blue bars) cells at MOI 0.01 (open symbols, black for MG1655, blue for MG1655::Bab/Agp) or MOI 10 (filled symbols, black for MG1655, blue for MG1655::Bab/Agp), cultures were infected at OD600nm of 0.1 and plated 45 min or 200 min after infection. Results are means +/− s.d. of three technical replicates and have been normalized to the number of colony forming units obtained with non-infected controls of the same strain (NI) (mean of three technical replicates). P-values are calculated from one-way ANOVA and are Tukey’s multiple comparison tests comparing the values for MG1655::Bab/Agp to the MG1655 control. All differences were non-significant except where marked. c, Phage titer in supernatant of liquid cultures of MG1655 (white bar) or MG1655::Bab/Agp (blue bar) cells inoculated with T5 at MOI 0.05 and cultivated for 8 h at 37°. Cells were infected at OD600nm 0.1. The grey bar corresponds to a control with medium alone inoculated with the same phage titer and incubated similarly for 8 h at 37 °C. The phage titer in the culture supernatant was then measured in spot assays on MG1655 cells. Results are means +/− s.d. of three technical replicates (the phage titer of the same culture supernatant was measured in triplicates). P-values are calculated from one-way ANOVA and are Tukey’s multiple comparison tests comparing the values to the medium-only control. d, T5-infected MG1655::Bab/Agp cells die but do not lyse. Photographs of culture tubes of MG1655 and MG1655::Bab/Agp cells infected with T5 at MOI 10 (or non-infected, NI) at OD600nm 0.45 and incubated an additional hour at 37 °C with agitation. For each culture OD600nm is given. e, Counting of colony forming units in the cultures shown in d. obtained after plating on LB medium. Results are means +/− s.d. of three technical replicates (the same culture was plated in triplicate). P-values are calculated from one-way ANOVA and are Tukey’s multiple comparison tests comparing the values for infected cells to the corresponding non-infected control. f,. Microscopic examination of the cultures shown in d (NI; non-infected control). Scale bar is 4 µm.

Extended Data Fig. 5 Effect of mutations in Bab and Agp on resistance to T5 and Agp(2-25) induced aggregation of Bab-GFP.

a, Spot assay of E. coli cells of the given genotype infected with 10-fold serial dilutions of T5 in triplicates. Each spot is 4 µL and dilutions are 102 to 108 of the phage stock. Dilutions are from top to bottom and left to right (as depicted in Fig. 2b). b, Western-blot of crude extracts of E. coli MG1655 cells expressing V5-tagged version of Agp and Agp mutants from an arabinose-inducible pBAD vector with an antibody to the V5 tag. As a loading control, the same blot was probed with an antibody to the β’ subunit of E. coli RNA polymerase (αRNAP). c, Western-blot of E. coli MG1655 extracts expressing Bab-GFP or Bab N100P-GFP incubated with various amounts of Agp(2-25) peptide in amyloid conformation and fractionated by centrifugation at 17,000g for 30 min. Agp(2-25) peptide was at 2 mg.mL−1 and was added to 50 µL of crude extract (corresponding to 109 cells). Size of molecular weight markers (lane M) are given in kDa. d, The GFP tag affects antiphage activity of Bab. Spot assay of E. coli cells of the given genotype infected with 10-fold serial dilutions of T5 in triplicates. Each spot is 4 µL and dilutions are 102 to 108 of the phage stock. Dilutions are from top to bottom and left to right (as depicted in Fig. 2b).

Extended Data Fig. 6 Relocalization of Bab-GFP.

a, Three additional representative examples of MG1655::Bab/Agp cells expressing Bab-GFP infected with T5 at MOI 10, labelled with Nile Red and imaged 25 min after infection. The image gives a series of Z-stacks for Nile red and GFP overlay. On the right, an orthogonal view of the Nile Red/GFP overlay is given and orthogonal views progress from left to right. White arrowheads point to examples of puncta located at the cell periphery. Scale bar is 5 µm. b, E. coli cells expressing Bab-GFP or Bab N100P-GFP and with the given genotype were infected with T5 at MOI 10 and imaged 25 min after infection (NI, non-infected). White arrowheads point to examples of Bab-GFP puncta at the cell periphery. Scale bar is 5 µm. Three representative fields are shown for each condition.

Extended Data Fig. 7 Structural comparison of Bab and related Bell bacterial proteins.

The experimental Bab structure and predicted AlphaFold3 structures of the Bell domain belonging to different clusters (as given in Supplementary Fig. 1) are presented with a rainbow coloring with blue to red from N- to C-terminus. The predicted pore-lining helix (as predicted with MEMSAT-SVM) is given in beige in each case. Bell1, 2 and 3 are from Streptomyces species (Actinobacteria) while the three Bell domains on the left, associated to the BASS4, 5 and 9 motifs are from Cyanobacteria (Supplementary Fig. 1). The lower model is obtained by rotating the upper model by 90° around a horizontal axis as marked.

Extended Data Fig. 8 GFP-BASS11Bab forms foci in vivo when expressed in P. anserina.

Representative examples of micrographs of P. anserina transformants expressing wild-type or mutant GFP-Bab(86-109), Bab(86-109)-RFP or Agp(2-25)-RFP and displaying the diffuse fluorescence state ([b*] state, left panels, imaged after 7 days of culture) and the foci state ([b] state, right panels, imaged after 7 days of culture and confrontation to [b] strains). No images of the foci state are given for D99A and N100P because this state is almost never observed. Scale bar is 5 µm. The pie charts give the fraction of transformants showing diffuse fluorescence (grey sector, [b*] state) or foci (black sector, [b] state), 5, 7, 11 and 30 days after transformation, (n.d., not determined). In each case, the number of transformants that were analyzed is given (n). Transformants were also confronted with strains showing foci ([b] strains) (7 d. with induction) and the fraction of strains showing foci after this confrontation is given. P-values correspond to two-tailed Fischer tests comparing foci formation at 5 and 30 days; comparing foci formation in mutants and wild-type at 30 days and comparing induced and spontaneous foci formation at 7 days.

Extended Data Fig. 9 BASS11 aggregate formation in P. anserina and Bab/BASS11 incompatibility.

a, Western-blot of P. anserina crude extracts expressing GFP-BASS11Bab (GFP-Bab(86-109)) in the [b*] and [b] state or GFP-BASS11Bab N100P fractionated by centrifugation at 17,000g probed with an anti-GFP antibody (T, total extract, S, supernanant, P, pellet). b, Representative example of a micrograph of a P. anserina strain co-expressing GFP-BASS11Bab (GFP-Bab(86-109)) and BASS11Agp-RFP (Agp(2-25)-RFP). Scale bar is 5 µm. c, Representative example of a barrage reaction between a strain expressing Bab-GFP (Bab) and strains expressing GFP-BASS11 in the soluble [b*] and aggregated [b] state assayed on corn meal agar medium. The red arrowhead points to the barrage reaction. On the right an interpretative model of Bab/[b] incompatibility is given. In this model, in [b*] strains BASS11 is soluble, upon cell fusion with a strain expressing Bab, no activation of Bab occurs and the fusion cell is viable. In [b] strains, BASS11 is aggregated into an amyloid state and upon fusion with a strain expressing Bab, Bab is converted and activated and causes death of the fusion cell. d, Cell death in fusion cells of strains expressing Bab (Bab(86-109)-RFP) and BASS11 (GFP-Bab(86-109)). The upper panels correspond to methylene blue staining for dead cells, the lower panel to a GFP/RFP overlay. Arrowheads point to the presumed fusion site. Scale bar is 5 µm. Three representative examples are shown. e, GFP-BASS11 [b] prion propagation assay based on acquisition of the barrage reaction to a tester strain expressing Bab. Two rows of six [b*] state strains expressing GFP-BASS11 were inoculated on the plate (grey bracket) together with a row of strains expressing Bab (center row). The left and right rows differ by the strain inoculated at the bottom of the plate, either [b*] (bottom right) or [b] strain (bottom left). Note the barrage formation in the confrontation zone in the left row, indicating that [b*] strains have been converted to the [b] state at the time of contact with the Bab-tester row. f, Sexual incompatibility associated to the Bab/BASS11 interaction. Counts correspond to the number of fertilized sexual organs (perithecia) formed in the following crosses : ΔhellpΔhet-sΔhellf::Bab x ΔhellpΔhet-sΔhellf (ΔΔΔ::Bab x ΔΔΔ) (shades of grey, two independent crosses used as control), ΔΔΔ::GFP-BASS11 x ΔΔΔ (blue, one cross) and ΔΔΔ::Bab x ΔΔΔ::GFP-BASS11 (red and pink, two independent crosses). Counts are means +/− s.d. of numbers of perithecia per cm in the confrontation zone (8 technical replicates). P-values are calculated from one-way ANOVA and are Tukey’s multiple comparison tests comparing the values to the first control cross. Images of the confrontation zone showing individual perithecia (white arrowheads) are given on the right of the histogram. The upper image is ΔΔΔ::Bab x ΔΔΔ (fertile control) and the lower image, ΔΔΔ::Bab x ΔΔΔ::GFP-BASS11. Scale bar is 2 mm. g, Mutations in the BASS11-motif and the N-terminal region of Bab abolish sexual incompatibility. Strains expressing Bab and Bab mutants as marked were crossed with strains expressing aggregated GFP-BASS11 (ΔΔΔ::Bab x ΔΔΔ::GFP-BASS11). All tested mutations restore fertility and increase formation of fertilized sexual organs (perithecia). h, Mutations in the BASS11-motif and the N-terminal region of Bab abolish vegetative incompatibility. Mutations in Bab abolish the barrage reaction in confrontations with strains expressing aggregated BASS11. The confrontations are shown on synthetic medium (left) and corn meal agar (right). The red arrowhead points to the barrage reaction between the strain expressing Bab and GFP-BASS11.

Extended Data Fig. 10 het-S/R0Agp confers resistance to T5.

a, Spot assay with 10-fold serial dilutions of T5 on lawns of MG1655 cells and containing a plasmid with het-S/R0Agp and mutant versions of the same construct (het-S Q240P and R0Agp H9P). MG1655 cells and MG1655::Bab/Agp cells carrying the empty vector are used as controls. Each dilution is spotted in triplicates, serial dilutions are from top to bottom and left to right as in Fig. 2b. b, Growth curves of strains used in a. and infected with T5 at different MOIs as indicated (NI, non-infected). Curves are given as means +/− s.d. of three biological replicates (the same bacterial cultures has been infected in triplicates), (bars may not appear on the graph if smaller than symbols). c, Representative fields in microscopic examination of strains expressing HET-S/R0Agp, HET-S Q240P/R0Agp and HET-S/R0Agp H9P infected with T5 at MOI 10 and imaged 45 min after infection (NI, non infected). Cell ghosts are present in HET-S R0Agp cultures while most cells are lysed in the Q240P and H9P mutants. Scale bar is 5 µm.

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Ibarlosa, L., Dheur, S., Sanchez, C. et al. Characterization of an amyloid-based antiphage defence system in Escherichia coli. Nat Microbiol 10, 2168–2178 (2025). https://doi.org/10.1038/s41564-025-02074-9

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