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A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants

Abstract

The ability of gut bacterial pathogens to escape immunity by antigenic variation—particularly via changes to surface-exposed antigens—is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

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Fig. 1: Vaccine-induced IgA exerts a strong selective pressure on O-antigen variants during murine non-typhoidal salmonellosis.
Fig. 2: O-antigen variants rapidly emerge during S.TmWT infection of vaccinated mice.
Fig. 3: The single-repeat O-antigen confers a selective advantage in the presence of broad-specificity vaccine-induced IgA.
Fig. 4: Single-repeat O-antigen mutants arising during infection of vaccinated mice have attenuated virulence and fitness and diminished resistance to phage predation.

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Data availability

All raw flow cytometry data, ordered by figure, are publicly available via the ETH research collection at https://doi.org/10.3929/ethz-b-000477737. All Illumina sequencing data are publicly available from the NCBI BioProject resource (accession PRJNA720270). Source data are provided with this paper.

Code availability

R code used to generate Extended Data Fig. 5 can be downloaded freely from https://github.com/marnoldini/evotrap.

References

  1. Fierer, J. & Guiney, D. G. Diverse virulence traits underlying different clinical outcomes of Salmonella infection. J. Clin. Invest. 107, 775–780 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kong, Q. et al. Effect of deletion of genes involved in lipopolysaccharide core and O-antigen synthesis on virulence and immunogenicity of Salmonella enterica serovar Typhimurium. Infect. Immun. 79, 4227–4239 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Collins, L. V., Attridge, S. & Hackett, J. Mutations at rfc or pmi attenuate Salmonella Typhimurium virulence for mice. Infect. Immun. 59, 1079–1085 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Broadbent, S. E., Davies, M. R. & van der Woude, M. W. Phase variation controls expression of Salmonella lipopolysaccharide modification genes by a DNA methylation-dependent mechanism. Mol. Microbiol. 77, 337–353 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bogomolnaya, L. M., Santiviago, C. A., Yang, H.-J., Baumler, A. J. & Andrews-Polymenis, H. L. ‘Form variation’ of the O12 antigen is critical for persistence of Salmonella Typhimurium in the murine intestine. Mol. Microbiol. 70, 1105–1119 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Van der Ley, P., de Graaff, P. & Tommassen, J. Shielding of Escherichia coli outer membrane proteins as receptors for bacteriophages and colicins by O-antigenic chains of lipopolysaccharide. J. Bacteriol. 168, 449–451 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bentley, A. T. & Klebba, P. E. Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface. J. Bacteriol. 170, 1063–1068 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Slauch, J. M., Lee, A. A., Mahan, M. J. & Mekalanos, J. J. Molecular characterization of the oafA locus responsible for acetylation of Salmonella Typhimurium O-antigen: OafA is a member of a family of integral membrane trans-acylases. J. Bacteriol. 178, 5904–5909 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Davies, M. R., Broadbent, S. E., Harris, S. R., Thomson, N. R. & van der Woude, M. W. Horizontally acquired glycosyltransferase operons drive salmonellae lipopolysaccharide diversity. PLoS Genet. 9, e1003568 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kim, M. L. & Slauch, J. M. Effect of acetylation (O-factor 5) on the polyclonal antibody response to Salmonella Typhimurium O-antigen. FEMS Immunol. Med. Microbiol. 26, 83–92 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Szabo, I., Grafe, M., Kemper, N., Junker, E. & Malorny, B. Genetic basis for loss of immuno-reactive O-chain in Salmonella enterica serovar Enteritidis veterinary isolates. Vet. Microbiol. 204, 165–173 (2017).

    Article  CAS  PubMed  Google Scholar 

  12. Rojas, E. R. et al. The outer membrane is an essential load-bearing element in Gram-negative bacteria. Nature 559, 617–621 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ricci, V., Zhang, D., Teale, C. & Piddock, L. J. V. The O-antigen epitope governs susceptibility to colistin in Salmonella enterica. mBio 11, e02831-19 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Endt, K. et al. The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea. PLoS Pathog. 6, e1001097 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Moor, K. et al. High-avidity IgA protects the intestine by enchaining growing bacteria. Nature 544, 498–502 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Porter, N. T., Canales, P., Peterson, D. A. & Martens, E. C. A subset of polysaccharide capsules in the human symbiont Bacteroides thetaiotaomicron promote increased competitive fitness in the mouse gut. Cell Host Microbe 22, 494–506 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Dolowschiak, T. et al. IFN-γ hinders recovery from mucosal inflammation during antibiotic therapy for Salmonella gut infection. Cell Host Microbe 20, 238–249 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. Felmy, B. et al. NADPH oxidase deficient mice develop colitis and bacteremia upon infection with normally avirulent, TTSS-1- and TTSS-2-deficient Salmonella Typhimurium. PLoS ONE 8, e77204 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Grassl, G. A., Valdez, Y., Bergstrom, K. S. B., Vallance, B. A. & Finlay, B. B. Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis. Gastroenterology 134, 768–780 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Diard, M. et al. Inflammation boosts bacteriophage transfer between Salmonella spp. Science 355, 1211–1215 (2017).

    Article  CAS  PubMed  Google Scholar 

  21. Moor, K. et al. Peracetic acid treatment generates potent inactivated oral vaccines from a broad range of culturable bacterial species. Front. Immunol. 7, 34 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Wotzka, S. Y. et al. Escherichia coli limits Salmonella Typhimurium infections after diet shifts and fat-mediated microbiota perturbation in mice. Nat. Microbiol. 4, 2164–2174 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Barthel, M. et al. Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host. Infect. Immun. 71, 2839–2858 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kaiser, P., Diard, M., Stecher, B. & Hardt, W.-D. The streptomycin mouse model for Salmonella diarrhea: functional analysis of the microbiota, the pathogen’s virulence factors, and the host’s mucosal immune response. Immunol. Rev. 245, 56–83 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Hauser, E., Junker, E., Helmuth, R. & Malorny, B. Different mutations in the oafA gene lead to loss of O5-antigen expression in Salmonella enterica serovar Typhimurium. J. Appl. Microbiol. 110, 248–253 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Cherry, J. L. Selection-driven gene inactivation in Salmonella. Genome Biol. Evol. 12, 18–34 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Murray, G. L., Attridge, S. R. & Morona, R. Altering the length of the lipopolysaccharide O antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with macrophages and complement. J. Bacteriol. 188, 2735–2739 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kim, M. & Ryu, S. Spontaneous and transient defence against bacteriophage by phase-variable glucosylation of O-antigen in Salmonella enterica serovar Typhimurium. Mol. Microbiol. 86, 411–425 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Nobrega, F. L. et al. Targeting mechanisms of tailed bacteriophages. Nat. Rev. Microbiol. 16, 760–773 (2018).

    Article  CAS  PubMed  Google Scholar 

  30. Levin, B. R. Selection and evolution of virulence in bacteria: an ecumenical excursion and modest suggestion. Parasitology 100, S103–S115 (1990).

    Article  PubMed  Google Scholar 

  31. Diard, M. & Hardt, W.-D. Evolution of bacterial virulence. FEMS Microbiol. Rev. 41, 679–697 (2017).

    Article  CAS  PubMed  Google Scholar 

  32. Wildschutte, H., Wolfe, D. M., Tamewitz, A. & Lawrence, J. G. Protozoan predation, diversifying selection, and the evolution of antigenic diversity in Salmonella. Proc. Natl Acad. Sci. USA 101, 10644–10649 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Brussow, H., Canchaya, C. & Hardt, W.-D. Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol. Mol. Biol. Rev. 68, 560–602 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Hsieh, S. et al. Polysaccharide capsules equip the human symbiont Bacteroides thetaiotaomicron to modulate immune responses to a dominant antigen in the intestine. J. Immunol. 204, 1035–1046 (2020).

    Article  CAS  PubMed  Google Scholar 

  35. Gerlach, D. et al. Methicillin-resistant Staphylococcus aureus alters cell wall glycosylation to evade immunity. Nature 563, 705–709 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Hughes, D. & Andersson, D. I. Evolutionary trajectories to antibiotic resistance. Annu. Rev. Microbiol. 71, 579–596 (2017).

    Article  CAS  PubMed  Google Scholar 

  37. Pires, D. P., Costa, A. R., Pinto, G., Meneses, L. & Azeredo, J. Current challenges and future opportunities of phage therapy. FEMS Microbiol. Rev. 44, 684–700 (2020).

    Article  CAS  PubMed  Google Scholar 

  38. Bikard, D. et al. Exploiting CRISPR–Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Harriman, G. R. et al. Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes. J. Immunol. 162, 2521–2529 (1999).

    CAS  PubMed  Google Scholar 

  40. Gu, H., Zou, Y. R. & Rajewsky, K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73, 1155–1164 (1993).

    Article  CAS  PubMed  Google Scholar 

  41. Mombaerts, P. et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 (1992).

    Article  CAS  PubMed  Google Scholar 

  42. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sternberg, N. L. & Maurer, R. Bacteriophage-mediated generalized transduction in Escherichia coli and Salmonella Typhimurium. Methods Enzymol. 204, 18–43 (1991).

    Article  CAS  PubMed  Google Scholar 

  44. Stecher, B. et al. Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice. Infect. Immun. 72, 4138–4150 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014).

    PubMed  PubMed Central  Google Scholar 

  48. Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6, 80–92 (2012).

    Article  CAS  Google Scholar 

  49. Hoiseth, S. K. & Stocker, B. A. D. Aromatic-dependent Salmonella Typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981).

    Article  CAS  PubMed  Google Scholar 

  50. Moor, K. et al. Analysis of bacterial-surface-specific antibodies in body fluids using bacterial flow cytometry. Nat. Protoc. 11, 1531–1553 (2016).

    Article  PubMed  Google Scholar 

  51. Arnoldini, M. et al. Bistable expression of virulence genes in Salmonella leads to the formation of an antibiotic-tolerant subpopulation. PLoS Biol. 12, e1001928 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Van Vliet, S. et al. Spatially correlated gene expression in bacterial groups: the role of lineage history, spatial gradients, and cell-cell interactions. Cell Syst 6, 496–507 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ilg, K., Zandomeneghi, G., Rugarabamu, G., Meier, B. H. & Aebi, M. HR-MAS NMR reveals a pH-dependent LPS alteration by de-O-acetylation at abequose in the O-antigen of Salmonella enterica serovar Typhimurium. Carbohydr. Res. 382, 58–64 (2013).

    Article  CAS  PubMed  Google Scholar 

  54. Westphal, O. & Jann, K. Bacterial lipopolysaccharides extraction with phenol–water and further applications of the procedure. Methods Carbohydr. Chem. 5, 83–91 (1965).

    CAS  Google Scholar 

  55. Steffens, T. et al. The lipopolysaccharide of the crop pathogen Xanthomonas translucens pv. translucens: chemical characterization and determination of signaling events in plant cells. Glycobiology 27, 264–274 (2017).

    CAS  PubMed  Google Scholar 

  56. Ardissone, S. et al. Cell cycle constraints and environmental control of local DNA hypomethylation in α-proteobacteria. PLoS Genet. 12, e1006499 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Li, H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27, 2987–2993 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Barnett, D. W., Garrison, E. K., Quinlan, A. R., Strömberg, M. P. & Marth, G. T. BamTools: a C++ API and toolkit for analyzing and managing BAM files. Bioinformatics 27, 1691–1692 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kersey, P. J. et al. Ensembl Genomes 2016: more genomes, more complexity. Nucleic Acids Res. 44, D574–D580 (2016).

    Article  CAS  PubMed  Google Scholar 

  61. R Core Development Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2019).

  62. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Yamashita, H. et al. Single-molecule imaging on living bacterial cell surface by high-speed AFM. J. Mol. Biol. 422, 300–309 (2012).

    Article  CAS  PubMed  Google Scholar 

  64. Hoffmann, M. et al. Complete genome sequence of a multidrug-resistant Salmonella enterica serovar Typhimurium var. 5− strain isolated from chicken breast. Genome Announc. 1, e01068–13 (2013).

    PubMed  PubMed Central  Google Scholar 

  65. Silva, C., Calva, E., Puente, J. L., Zaidi, M. B. & Vinuesa, P. Complete genome sequence of Salmonella enterica serovar Typhimurium strain SO2 (sequence type 302) isolated from an asymptomatic child in Mexico.Genome Announc. 4, e00285–16 (2016).

    PubMed  PubMed Central  Google Scholar 

  66. Hong, Y., Liu, M. A. & Reeves, P. R. Progress in our understanding of Wzx flippase for translocation of bacterial membrane lipid-linked oligosaccharide. J. Bacteriol. 200, e00154–17 (2018).

    Article  CAS  PubMed  Google Scholar 

  67. Hapfelmeier, S. et al. The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar Typhimurium to trigger colitis via myd88-dependent and myd88-independent mechanisms. J. Immunol. 174, 1675–1685 (2005).

    Article  CAS  PubMed  Google Scholar 

  68. Maier, L. et al. Microbiota-derived hydrogen fuels Salmonella Typhimurium invasion of the gut ecosystem. Cell Host Microbe 14, 641–651 (2013).

    Article  CAS  PubMed  Google Scholar 

  69. Suar, M. et al. Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model. Infect. Immun. 74, 632–644 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Fransen, F. et al. BALB/c and C57BL/6 mice differ in polyreactive IgA abundance, which impacts the generation of antigen-specific IgA and microbiota diversity. Immunity 43, 527–540 (2015).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

O.H. acknowledges H. Käßner for recording NMR spectra, R. Engel for gas/liquid chromatography–mass spectrometry and K. Jakob and S. Düpow for technical support. We thank M. Schneider, C. Kiessling, E. Schultheiss, R.-M. Vesco and C. Straub for the DNA extraction, library preparations and sequencing of the bacterial isolates. M.D. acknowledges D. Cornillet and the group of D. Bumann for serum resistance measurements. E.S., W.-D.H. and M.D. acknowledge M. Aebi, M. Loessner, J. Cherry and the group of A. Harms for helpful discussion and insight, as well as members of the W.-D.H., E.S. and M.D. groups for comments. E.S., W.-D.H. and M.D. further acknowledge the staff of the ETH Phenomics Center and Rodent Centre HCI for support with animal experimentation. M.D. is supported by a Swiss National Science Foundation professorship (PP00PP_176954) and Gebert Rüf Microbials (PhagoVax GRS-093/20). E.S. acknowledges support from the Swiss National Science Foundation (40B2-0_180953 and 310030_185128; NCCR Microbiomes), a European Research Council Consolidator Grant and Gebert Rüf Microbials (GR073_17). M.D. and E.S. acknowledge the Botnar Research Centre for Child Health Multi-Investigator Project 2020. B.M.S. acknowledges support from R01 AI041239/AI/NIAID NIH HHS/United States. W.-D.H. acknowledges support from the Swiss National Science Foundation (310030B-173338 and 310030_192567; NCCR Microbiomes), ProMedica Foundation, Gebert Rüf Foundation (with A.E.) and Helmut Horten Foundation. E.B. is supported by a Boehringer Ingelheim Fonds PhD fellowship. B.H.M. acknowledges support from the Swiss National Science Foundation (200020_159707).

Author information

Authors and Affiliations

Authors

Contributions

M.D., W.D.H. and E.S. designed the project and wrote the paper. M.D. and E.S. designed and carried out the experiments relating to the vaccination and infection of mice, re-isolation of S.Tm clones, phenotyping of S.Tm clones by flow cytometry and gel electrophoresis, characterization of human monoclonal antibodies, analysis of antibody titres and analysis of the fitness of O-antigen variants of S.Tm in vitro and in vivo. M.W.v.d.W., B.H.M., C.L. and R.M. contributed to experimental design and data interpretation. G.Z. carried out the HR-MAS NMR analysis. O.H. carried out the proton NMR analysis. M.A. generated the mathematical model for O:12 switching. N.D. supplied unpublished Salmonella strains. J.A. carried out and analysed all of the AFM imaging. A.R. and N.A.B. carried out phage sensitivity assays. A.E., F.B. and D.W. carried out the Illumina whole-genome resequencing of re-isolated S.Tm isolates. E.B., V.L., D.H., F.B., K.S.-M. and S.A. carried out S.Tm challenge infections in vaccinated mice and analysed the re-isolated clones. A.H. carried out the microfluidic video microscopy of O:12 switching. P.H.V. and L.F. carried out the methylome analysis of re-isolated S.Tm clones. L.P., A.L. and B.M.S. generated the novel antibody reagents. All authors critically reviewed the manuscript.

Corresponding authors

Correspondence to Médéric Diard, Wolf-Dietrich Hardt or Emma Slack.

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Competing interests

Evolutionary trap vaccines are covered by European patent application number EP19177251, submitted by M.D. W.-D.H. and E.S. All other authors declare no competing interests.

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Peer review information Nature Microbiology thanks Reiko Shinkura and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Surface phenotype of S.Tm mutants.

Atomic force microscopy phase images of S.Tmwt, S.TmΔwzyB (single-repeat O-antigen), and S.TmΔwbaP (rough mutant - no O-antigen) at low magnification (A, uncropped image, scale bar = 1µm) and high magnification (B and C, scale bar main image = 150nm, scale bar inset = 15nm). Invaginations in the surface of S.TmΔwbaP (dark colour, B) show a geometry and size consistent with outer membrane pores63. These are already less clearly visible on the surface of S.TmΔwzyB with a single-repeat O-antigen, and become very difficult to discern in S.Tmwt. One representative image of 3 for each genotype is shown. While arrows point to features with consistent size and abundance to be exposed outer membrane porins. C. Fast-Fourier transform of images shown in ‘B’ demonstrating clear regularity on the surface of S.TmΔwbaP, which is progressively lost when short and long O-antigen is present.

Extended Data Fig. 2 Mutations detected in the oafA gene sequence among several strains of S.Tm.

A. Aligned fractions of the oafA ORF from a natural isolate (from chicken) presenting the same 7 bp deletion detected in mutants of S.Tm SL1344 emerging in vaccinated mice. S.Tm SL1344 was used a reference64. B. Aligned oafA promoter sequences from three natural isolates of human origin (stool or cerebrospinal fluid65) showing variations in the number of 9 bp direct repeats.

Extended Data Fig. 3 Loss of the O:12-0 epitope is a reversible phenotype.

A. Wild type and evolved S.Tm clones were picked from LB plates, cultured overnight, phenotypically characterized by O:12-0 (left panel) and O:5 staining (right panel), plated and re-picked. This process was repeated over 3 cycles with lines showing the descendants of each clone. B and C. Wild type 129S1/SvImJ mice were mock-vaccinated or were vaccinated with PA-S.TmΔoafA ΔgtrC as in Fig. 1. On d28, all mice were pre-treated with streptomycin, and infected with the indicated strain. B. Feces recovered at day 10 post-infection, was enriched overnight by culture in streptomycin, and stained for O:12-0 (human monoclonal STA5). Fraction O:12-0-low S.Tm was determined by flow cytometry. Percentage of S.Tm that are O:12-0-negative was quantified over 10 days and is plotted in panel C. Vaccination selects for S.Tm that have lost the O:12-0 epitope, only if the gtrC gene is intact.

Source data

Extended Data Fig. 4 Loss of the O:12-0 epitope can be driven by adoptive transfer of O:12-0-specific IgA.

C57BL/6 SPF mice received oral streptomycin to deplete the microbiota 23.5h before an intravenous injection with saline only, or with 1mg of recombinant dimeric murine IgA specific for the O:12-0 epitope (STA121). 0.5 h later all mice were orally inoculated with S.TmΔoafA pM965 or S.TmΔoafAΔG4 pM965 (lacking 4 different glucosyl transferases, including gtrC) both carrying pM965 to drive constitutive GFP production. The adoptive transfer was repeated 12h later and all animals were euthanized at 24h post-infection. A. O:12-0 expression on S.Tm enriched from cecum content by overnight culture on 1:1000 dilution LB with selective antibiotics, determined by staining with the monoclonal antibody STA5. Flow cytometry plots shown have been gated on scatter only – see Supplementary Fig. 1 for example. B. Quantification of the O:12-0-high fraction of S.Tm from A. C. Individual clones of S.Tm of the indicated genotype were recovered from the cecal content of mice from A that had received an adoptive transfer of mSTA121 and individual clones, cultured overnight in LB were analysed as in A and B for fraction of O:12-0-high cells.

Source data

Extended Data Fig. 5 Phase-variation and selection, without a shift in switching rate, underly recovery of O:12-2 producing clones from vaccinated mice.

A. Comparison of fractions of O:12-0-positive and O:12-0-negative bacteria (in fact O:12-2) determined by flow cytometry (gating – see Fig.S1) staining with typing sera and by blue-white colony counts using a gtrABC-lacZ reporter strain and overnight cultures from individual clonal colonies. B-D: Results of a mathematical model simulating bacterial growth and antigen switching (see supplementary methods). B. Switching rates from O:12-0 to O:12-2 and from O:12-2 to O:12-0 were varied computationally, and the fraction of O:12-2 was plotted after 16 h of growth. Left-hand plot depicts the results of the deterministic model when starting with 100% O:12-2, right-hand plot depicts the results when starting with 100% O:12-0. C. depicts only the switching rates that comply with the experimentally observed antigen ratios after overnight growth (90% O:12-0 when starting with O:12-0, and 50% O:12-0 when starting with O:12-2). Right-hand plot is a zoomed version showing values for switching rates between 0 – 0.2 h−1 (marked by a grey rectangle). Dashed lines are linear regressions on the values in this range, and their intersection marks the switching rates used for the stochastic simulation in (D). D. Simulation results of bacterial population growth, when starting with only O:12-2 (left-hand plot) or only O:12-0 (right-hand plot). µ = 2.05h−1 was kept constant in all simulations; switching rates were kept constant at s->12-0 = 0.144h−1 and s->12-2 = 0.0365h−1; the starting populations were always individuals of the indicated phenotype; carrying capacity was always K = 109 cells. Time resolution for the simulations is 0.2h.

Extended Data Fig. 6 NMR of purified LPS and HR-MAS 1H-NMR confirms O-antigen structures in evolved clones.

A. Schematic diagram of expected NMR peaks for each molecular species B. HR-MAS 1H-NMR spectra. Spectra show predicted peak positions and observed spectra for C1 protons of the O-antigen sugars. C. 1H NMR of purified LPS from the indicated strains. Note that non-acetylated abequose can be observed in wild type strains due to spontaneous deacetylation at low pH in late stationary phase cultures53. A gtrA mutant strain is used here to over-represent the O:12-2 O-antigen variant due to loss of regulation5.

Extended Data Fig. 7 S.Tm O-antigen variants arise during chronic S.Tm infections, dependent on a specific IgA response.

IgA−/− (A) and Rag1−/− (B) and heterozygote littermate controls (C57BL/6-background) were pre-treated with streptomycin and infected with S.TmΔsseD orally. Fecal S.Tm were enriched overnight by culturing a 1:2500 dilution of feces in LB plus kanamycin. These enrichment cultures were then stained for O:5 and O:12-0 and analysed by flow cytometry (gating as in Supplementary Fig. 14). The fraction of the population that lost O:5 and O:12-0 antisera staining is shown over time, as well as the total CFU/g in feces. Both immunocompetent mouse strains show increased O:5-negative S.Tm in the fecal enrichments from day 14 post-infection: approximately when we expect to see a robust secretory IgA response developing. These changes are not observed in Rag1-deficient or IgA-deficient mice. The kinetics of O:5-loss are likely influenced by development or broader IgA responses as the chronic infection proceeds. Note that lines joining the points are to permit tracking of individual animals through the data set, and may not be representative of what occurs between the measured time-points. C. Titres of intestinal lavage IgA specific for O:4[5] (S.Tmwt, O:4[5], 12-0) and O:4(S.TmΔoafA, O:4,12-0), presented as the dilution of intestinal lavage required to give an IgA-staining MFI=1000 by bacterial flow cytometry, and the ratios of these titres. Samples: d28 post-vaccination with PA-STmwt (n = 12) or d35 post-colonization with live-attenuated S.Tm (n = 8 S.TmΔaroA + n = 8 S.TmΔsseD), This revealed a weaker, but less biased IgA response in mice infected with the live-vaccine strain, when compared to that induced by the inactivated oral vaccine. Results of 2-tailed Mann-Whitney U tests shown.

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Extended Data Fig. 8 Enzymes of the O-antigen synthesis pathway.

Schematic of S.Tm O-antigen synthesis (with reference to ref. 66).

Extended Data Fig. 9 Synthetic and natural deletions of wzyB reduce the fitness of S.Tm in presence of Tris-EDTA, Cholate, SDS and serum complement.

The deletion of wzyB does not affect the growth of S.Tm or S.TmΔoafA ΔgtrC in LB (No stress) (A) but impairs growth in presence of Tris-EDTA (B), 2% cholate (C) and 0.05% SDS (D). Dashed lines represent the range of variations between the n = 4 pooled experiments. (E). Relative fitness of the long versus short O-antigen in the presence of membrane stress as quantified by competitive growth of S.TmGFP against S.TmΔoafA ΔgtrC, S.Tm ΔoafA ΔgtrC ΔwzyB or an evolved S.TmΔwzyB, in LB with or without Tris-EDTA. 2-tailed Mann-Whitney U test. ** p = 0.0013 (F) Loss of complement resistance in evolved and synthetic wzyB mutants revealed by relative CFU recovery after treatment with heat-inactivated and fresh human serum. Mann-Whitney U 2-tailed tests * p = 0.0167.

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Extended Data Fig. 10 Analysis of bacteriophages preferentially infecting short O-antigen S. Tm mutants.

A. Lysis plaques observed on lawns of S.Tm ΔgtrC ΔoafA and S.Tm ΔgtrC ΔoafA ΔwzyB isogenic mutants exposed to wastewater samples. Scale = 1cm. This phenocopies the observation with naturally arising wzyB mutants B. Growth curves of S.Tm ΔgtrC ΔoafA ΔwzyB exposed to purified bacteriophages from Fig. 4D. The re-growing S.Tm clones were isolated for sequencing. The mutations identified and their effects are listed in the table below (C), confirming btuB as the most likely exposed outer-membrane receptor for these phages.

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Supplementary Information

Text descriptions for Supplementary Tables 1–4 and Supplementary Videos 1 and 2, and Supplementary Figs. 1–10.

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Supplementary Tables

Supplementary Tables 1–4.

Supplementary Video 1

Visualization of O:12 phase variation using live-cell immunofluorescence (loss of antibody binding).

Supplementary Video 2

Visualization of O:12 phase variation using live-cell immunofluorescence (gain of antibody binding).

Supplementary Data 1

Numerical source data for Supplementary Figs. 1–10.

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Source Data Fig. 1

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Source Data Fig. 3

Uncropped gel image for Fig. 3g.

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All numerical source data for Fig. 4.

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Uncropped phage plaque images for Fig. 4g (ancestral strain and evolved clone).

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All numerical source data for Extended Data Fig. 10.

Source Data Extended Data Fig. 10

Uncropped phage plaque images for Extended Data Fig 10a (long and short O-antigens).

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Diard, M., Bakkeren, E., Lentsch, V. et al. A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants. Nat Microbiol 6, 830–841 (2021). https://doi.org/10.1038/s41564-021-00911-1

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