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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Fierer, J. & Guiney, D. G. Diverse virulence traits underlying different clinical outcomes of Salmonella infection. J. Clin. Invest. 107, 775–780 (2001).
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).
Collins, L. V., Attridge, S. & Hackett, J. Mutations at rfc or pmi attenuate Salmonella Typhimurium virulence for mice. Infect. Immun. 59, 1079–1085 (1991).
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).
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).
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).
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).
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).
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).
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).
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).
Rojas, E. R. et al. The outer membrane is an essential load-bearing element in Gram-negative bacteria. Nature 559, 617–621 (2018).
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).
Endt, K. et al. The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal Salmonella diarrhea. PLoS Pathog. 6, e1001097 (2010).
Moor, K. et al. High-avidity IgA protects the intestine by enchaining growing bacteria. Nature 544, 498–502 (2017).
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).
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).
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).
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).
Diard, M. et al. Inflammation boosts bacteriophage transfer between Salmonella spp. Science 355, 1211–1215 (2017).
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).
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).
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).
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).
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).
Cherry, J. L. Selection-driven gene inactivation in Salmonella. Genome Biol. Evol. 12, 18–34 (2020).
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).
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).
Nobrega, F. L. et al. Targeting mechanisms of tailed bacteriophages. Nat. Rev. Microbiol. 16, 760–773 (2018).
Levin, B. R. Selection and evolution of virulence in bacteria: an ecumenical excursion and modest suggestion. Parasitology 100, S103–S115 (1990).
Diard, M. & Hardt, W.-D. Evolution of bacterial virulence. FEMS Microbiol. Rev. 41, 679–697 (2017).
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).
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).
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).
Gerlach, D. et al. Methicillin-resistant Staphylococcus aureus alters cell wall glycosylation to evade immunity. Nature 563, 705–709 (2018).
Hughes, D. & Andersson, D. I. Evolutionary trajectories to antibiotic resistance. Annu. Rev. Microbiol. 71, 579–596 (2017).
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).
Bikard, D. et al. Exploiting CRISPR–Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 (2014).
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).
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).
Mombaerts, P. et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 (1992).
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).
Sternberg, N. L. & Maurer, R. Bacteriophage-mediated generalized transduction in Escherichia coli and Salmonella Typhimurium. Methods Enzymol. 204, 18–43 (1991).
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).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014).
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).
Hoiseth, S. K. & Stocker, B. A. D. Aromatic-dependent Salmonella Typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981).
Moor, K. et al. Analysis of bacterial-surface-specific antibodies in body fluids using bacterial flow cytometry. Nat. Protoc. 11, 1531–1553 (2016).
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).
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).
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).
Westphal, O. & Jann, K. Bacterial lipopolysaccharides extraction with phenol–water and further applications of the procedure. Methods Carbohydr. Chem. 5, 83–91 (1965).
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).
Ardissone, S. et al. Cell cycle constraints and environmental control of local DNA hypomethylation in α-proteobacteria. PLoS Genet. 12, e1006499 (2016).
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).
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).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Kersey, P. J. et al. Ensembl Genomes 2016: more genomes, more complexity. Nucleic Acids Res. 44, D574–D580 (2016).
R Core Development Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2019).
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).
Yamashita, H. et al. Single-molecule imaging on living bacterial cell surface by high-speed AFM. J. Mol. Biol. 422, 300–309 (2012).
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).
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).
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).
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).
Maier, L. et al. Microbiota-derived hydrogen fuels Salmonella Typhimurium invasion of the gut ecosystem. Cell Host Microbe 14, 641–651 (2013).
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).
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).
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).
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.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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.
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.
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. 1–4). 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 (S.Tmwt, O:4, 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.
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.
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.
Text descriptions for Supplementary Tables 1–4 and Supplementary Videos 1 and 2, and Supplementary Figs. 1–10.
Supplementary Tables 1–4.
Visualization of O:12 phase variation using live-cell immunofluorescence (loss of antibody binding).
Visualization of O:12 phase variation using live-cell immunofluorescence (gain of antibody binding).
Numerical source data for Supplementary Figs. 1–10.
All numerical source data for Fig. 1.
All numerical source data for Fig. 2.
All numerical source data for Fig. 3.
Uncropped gel image for Fig. 3g.
All numerical source data for Fig. 4.
Uncropped phage plaque images for Fig. 4g (ancestral strain and evolved clone).
All numerical source data for Extended Data Fig. 3.
All numerical source data for Extended Data Fig. 4.
All numerical source data for Extended Data Fig. 7.
All numerical source data for Extended Data Fig. 9.
All numerical source data for Extended Data Fig. 10.
Uncropped phage plaque images for Extended Data Fig 10a (long and short O-antigens).
About this article
Cite this article
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 (2021). https://doi.org/10.1038/s41564-021-00911-1