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Nature Genetics | Article

日本語要約

A single natural nucleotide mutation alters bacterial pathogen host tropism

Journal name:
Nature Genetics
Volume:
47,
Pages:
361–366
Year published:
DOI:
doi:10.1038/ng.3219
Received
Accepted
Published online

Abstract

The capacity of microbial pathogens to alter their host tropism leading to epidemics in distinct host species populations is a global public and veterinary health concern. To investigate the molecular basis of a bacterial host-switching event in a tractable host species, we traced the evolutionary trajectory of the common rabbit clone of Staphylococcus aureus. We report that it evolved through a likely human-to-rabbit host jump over 40 years ago and that only a single naturally occurring nucleotide mutation was required and sufficient to convert a human-specific S. aureus strain into one that could infect rabbits. Related mutations were identified at the same locus in other rabbit strains of distinct clonal origin, consistent with convergent evolution. This first report of a single mutation that was sufficient to alter the host tropism of a microorganism during its evolution highlights the capacity of some pathogens to readily expand into new host species populations.

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  1. Rabbit but not human ST121 strains can infect rabbits.
    Figure 1: Rabbit but not human ST121 strains can infect rabbits.

    (a) Percentage of rabbits infected with rabbit or human ST121 clones (day 7 after inoculation). Rabbits (n = 24 per strain) were inoculated intradermally with S. aureus ST121 rabbit strain I, J or DL190 (300 CFU) or human strain G, F or A (105 CFU) (Online Methods). Yates' χ2 test was used to compute P values for between-group comparisons: differences that are statistically significant are indicated by an asterisk (*P < 0.0001) and all other comparisons did not show significant differences. (b) Representative rabbit skin lesions (day 7 after inoculation). Images of gross and microscopic histopathologies are presented for representative rabbits as follows. Top, gross skin pathology. Scale bars, 1 cm. Middle, transversal sections of skin lesions. Lesions resulting from infection with rabbit strains were characterized by dermal abscesses (DA) up to 2 cm in diameter, with epidermal ulcers (U) in the most severe cases. Scale bars, 1 cm. Bottom, sections of the skin inoculated with human or rabbit ST121 clone stained with hematoxylin and eosin. Scale bars, 0.5 cm. Microscopically, we observed areas of purulent material surrounded by fibrosis that were infiltrated by inflammatory cells. (c) Experimental skin lesions (day 10 after inoculation) were indistinguishable from naturally occurring lesions. Top, gross skin pathology; middle, transversal sections of skin lesions; bottom, sections of the skin stained with hematoxylin and eosin. Scale bars, 0.5 cm.

  2. Evolutionary history of the ST121 clonal complex suggests a human-to-rabbit host jump leading to the emergence of an epidemic rabbit-specific clone.
    Figure 2: Evolutionary history of the ST121 clonal complex suggests a human-to-rabbit host jump leading to the emergence of an epidemic rabbit-specific clone.

    Bayesian phylogenetic reconstruction of the CC121 lineage based on core genome alignment with branches colored according to host species association (blue, human; red, rabbit). The presence or absence of MGEs in the accessory genome is denoted by filled and empty squares, respectively. The MGEs identified include staphylococcal pathogenicity islands (SaPI), phages (φ) or plasmids (p) containing genes—lukF-lukS, Panton-Valentine leukocidin; eta, exfoliative toxin A; etb, exfoliative toxin B; seb, staphylococcal enterotoxin B; IEC, immune evasion cluster; SCCmec, staphylococcal cassette chromosome mec—and plasmids encoding resistance to bleomycin, kanamycin, quaternary ammonium compounds and trimethoprim (pR1) or teicoplanin (pR2). Branch lengths are scaled according to the timescale indicated on the x axis. Numbers at nodes represent posterior probability values.

  3. A single mutation is sufficient to confer rabbit infectivity to a human S. aureus strain.
    Figure 3: A single mutation is sufficient to confer rabbit infectivity to a human S. aureus strain.

    (a,b) Percentage of rabbits infected with the different rabbit (a) or human (b) ST121 mutants (day 7 after inoculation; n = 20 per strain in a, n = 30 per strain in b). The percentage of rabbits that developed skin lesions from inoculated bacteria is also shown in b. Yates' χ2 test was used to compute P values for between-group comparisons: differences that are statistically significant are indicated by asterisks (*P < 0.05) and all other comparisons did not show significant differences. WT, wild type. (c) Evolution of the skin lesions produced by rabbit strain J and by the human clone carrying the dltB Thr113Lys allele was indistinguishable. Rabbits were inoculated intradermally with 300 CFU of either the wild-type rabbit or mutant human clone (Online Methods). Images are shown for a representative rabbit. The earliest gross changes were observed 24–48 h after infection, consisting in a slight increase in size and erythema but evolving to form skin abscesses up to 2 cm in diameter (7 d after inoculation). Scale bars, 1.5 cm. (d) Transversal sections of skin lesions. Lesions produced by the rabbit and mutant human clones were characterized by dermal abscesses (DA) up to 2 cm in diameter. Bottom, hematoxylin and eosin staining of sections of skin inoculated with the rabbit or mutant human ST121 clone. Scale bars, 0.5 cm. Microscopically, we observed areas of purulent material surrounded by fibrosis that were infiltrated by inflammatory cells.

  4. Rabbit-infective S. aureus clones have evolved on numerous occasions and are associated with nonsynonymous mutations of dltB.
    Figure 4: Rabbit-infective S. aureus clones have evolved on numerous occasions and are associated with nonsynonymous mutations of dltB.

    Bayesian phylogeny of S. aureus species reconstructed using the nucleotide sequence of 5 non-recombinant MLST loci from 108 sequence types representing the breadth of species diversity. Clonal complexes and sequence types with known rabbit host association are indicated with red circles; the scale bar represents five nucleotides.

  5. Alignment of DltB amino acid sequences from S. aureus human, rabbit, ovine and poultry clones, colored according to relative sequence conservation at each position.
    Supplementary Fig. 1: Alignment of DltB amino acid sequences from S. aureus human, rabbit, ovine and poultry clones, colored according to relative sequence conservation at each position.

    Adapted from an alignment generated by PRALINE. The scoring scheme ranges from 0 for the least conserved alignment position up to 10 (indicated by an asterisk) for the most conserved alignment position.

  6. Analysis of D-alanylation of wall teichoic acid or lipoteichoic acid and growth inhibition by the cationic peptide nisin.
    Supplementary Fig. 2: Analysis of D-alanylation of wall teichoic acid or lipoteichoic acid and growth inhibition by the cationic peptide nisin.

    (a,b) The D-Ala content of the human and rabbit S. aureus clones was tested. (c) Shown is the growth in TSB medium of isogenic strains treated with nisin (10 μg/ml). Cell density was monitored (OD600). J, wild-type rabbit strain; J dlt Bh, derivative J strain expressing the DltB protein from the ST121 human clones; J Δdlt B, J dlt B mutant; F, wild-type human strain; F dlt Br, strain F expressing the DltB protein from the rabbit clones. In both cases, the experiments were performed in triplicate. An ANOVA test was carried out using Bonferroni adjustment. All error bars show s.e.m. Differences that are statistically significant are indicated by an asterisk (P < 0.05); all other comparisons were not significant.

  7. Analysis of peptidoglycan structure and composition.
    Supplementary Fig. 3: Analysis of peptidoglycan structure and composition.

    (a) Muropeptide analysis by HPLC. The cell wall from bacteria growing in exponential phase was isolated, digested with a muramidase and analyzed via HPLC. (b) The amino acid composition of the purified peptidoglycan was analyzed by HPLC. In both panels, a representative experiment is shown.

  8. Predicted membrane topology of the DltB protein.
    Supplementary Fig. 4: Predicted membrane topology of the DltB protein.

    DltB topology was predicted using the TMHMM method. Colored in red are amino acid residues that varied in the rabbit ST121 strains, and green indicates amino acid residues that were variant in the other rabbit clones.

  9. Survival in rabbit blood.
    Supplementary Fig. 5: Survival in rabbit blood.

    S. aureus strains were grown to mid-exponential growth phase, washed and resuspended in sterile PBS. 4 × 104 CFU of S. aureus in 100 μl of PBS were pipetted slowly into 3 ml of heparinized rabbit blood, mixed gently for 30 s and incubated at 37 °C for 2 h. To determine survival rates, 100 μl of heparinized blood was plated on TSA for S. aureus detection. Data shown represent the means ± s.e.m. of three separate experiments. An ANOVA test was performed, using Bonferroni adjustment. Differences that are statistically significant are indicated by an asterisk (P < 0.05); all other comparisons were not significant.

  10. SDS-PAGE analysis of cell wall-associated protein profiles.
    Supplementary Fig. 6: SDS-PAGE analysis of cell wall–associated protein profiles.

    S. aureus strains were grown to mid-exponential or stationary growth phase, washed and resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 20 mM MgCl2, supplemented with 30% raffinose) with lysostaphin. Protoplasts were sedimented by centrifugation at 6,000g, and the supernatant fraction, which contained the wall-associated proteins, was analyzed by SDS-PAGE.

  11. Alignment of the DltB amino acid sequences from Bacillus amyloliquefaciens and Streptococcus pneumoniae strains, colored according to relative sequence conservation at each position.
    Supplementary Fig. 7: Alignment of the DltB amino acid sequences from Bacillus amyloliquefaciens and Streptococcus pneumoniae strains, colored according to relative sequence conservation at each position.

    Adapted from an alignment generated by PRALINE. The scoring scheme ranges from 0 for the least conserved alignment position up to 10 (indicated by an asterisk) for the most conserved alignment position. (a) B_amyl_FZB42: B. amyloliquefaciens strain FZB42 (plant-associated bacterium). Accession number: YP_001423132. B_amyl_DSM7: B. amyloliquefaciens strain DSM7 (soil adapted). Accession number: YP_003922279. (b) DltB_TIGR4: S. pneumoniae strain TIGR4. Accession number: ZP_01408978. DltB_GA41301: S. pneumoniae strain GA41301. Accession number: ZP_12336902. DltB_GA17227: S. pneumoniae strain GA17227. Accession number: ZP_12797610. DltB_GA47901: S. pneumoniae strain GA47901. Accession number: ZP_12344230.

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Sequence Read Archive

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Author information

  1. These authors contributed equally to this work.

    • David Viana,
    • María Comos &
    • Paul R McAdam

  2. These authors jointly supervised this work.

    • J Ross Fitzgerald &
    • José R Penadés

Affiliations

  1. Biomedical Research Institute, Universidad CEU Cardenal Herrera, Valencia, Spain.

    • David Viana &
    • Laura Selva

  2. Centro de Investigación y Tecnología Animal, Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Segorbe, Spain.

    • María Comos

  3. The Roslin Institute, University of Edinburgh, Easter Bush Campus, Edinburgh, UK.

    • Paul R McAdam,
    • Caitriona M Guinane &
    • J Ross Fitzgerald

  4. Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK.

    • Melissa J Ward

  5. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.

    • Beatriz M González-Muñoz &
    • Simon J Foster

  6. Centre National de Référence des Staphylocoques, Université Lyon, Lyon, France.

    • Anne Tristan

  7. Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (IBV-CSIC), Valencia, Spain.

    • José R Penadés

  8. Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

    • José R Penadés

Contributions

J.R.F. and J.R.P. conceived and designed the study. D.V., M.C. and L.S. generated and characterized the different mutant strains. P.R.M., M.J.W. and C.M.G. performed the genomic studies. B.M.G.-M. and S.J.F. measured D-Ala content. A.T. provided human strains. J.R.P., J.R.F. and S.J.F. supervised the research. J.R.F. and J.R.P. wrote the manuscript and obtained funding.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Alignment of DltB amino acid sequences from S. aureus human, rabbit, ovine and poultry clones, colored according to relative sequence conservation at each position. (1,419 KB)

    Adapted from an alignment generated by PRALINE. The scoring scheme ranges from 0 for the least conserved alignment position up to 10 (indicated by an asterisk) for the most conserved alignment position.

  2. Supplementary Figure 2: Analysis of D-alanylation of wall teichoic acid or lipoteichoic acid and growth inhibition by the cationic peptide nisin. (138 KB)

    (a,b) The D-Ala content of the human and rabbit S. aureus clones was tested. (c) Shown is the growth in TSB medium of isogenic strains treated with nisin (10 μg/ml). Cell density was monitored (OD600). J, wild-type rabbit strain; J dlt Bh, derivative J strain expressing the DltB protein from the ST121 human clones; J Δdlt B, J dlt B mutant; F, wild-type human strain; F dlt Br, strain F expressing the DltB protein from the rabbit clones. In both cases, the experiments were performed in triplicate. An ANOVA test was carried out using Bonferroni adjustment. All error bars show s.e.m. Differences that are statistically significant are indicated by an asterisk (P < 0.05); all other comparisons were not significant.

  3. Supplementary Figure 3: Analysis of peptidoglycan structure and composition. (177 KB)

    (a) Muropeptide analysis by HPLC. The cell wall from bacteria growing in exponential phase was isolated, digested with a muramidase and analyzed via HPLC. (b) The amino acid composition of the purified peptidoglycan was analyzed by HPLC. In both panels, a representative experiment is shown.

  4. Supplementary Figure 4: Predicted membrane topology of the DltB protein. (129 KB)

    DltB topology was predicted using the TMHMM method. Colored in red are amino acid residues that varied in the rabbit ST121 strains, and green indicates amino acid residues that were variant in the other rabbit clones.

  5. Supplementary Figure 5: Survival in rabbit blood. (42 KB)

    S. aureus strains were grown to mid-exponential growth phase, washed and resuspended in sterile PBS. 4 × 104 CFU of S. aureus in 100 μl of PBS were pipetted slowly into 3 ml of heparinized rabbit blood, mixed gently for 30 s and incubated at 37 °C for 2 h. To determine survival rates, 100 μl of heparinized blood was plated on TSA for S. aureus detection. Data shown represent the means ± s.e.m. of three separate experiments. An ANOVA test was performed, using Bonferroni adjustment. Differences that are statistically significant are indicated by an asterisk (P < 0.05); all other comparisons were not significant.

  6. Supplementary Figure 6: SDS-PAGE analysis of cell wall–associated protein profiles. (362 KB)

    S. aureus strains were grown to mid-exponential or stationary growth phase, washed and resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 20 mM MgCl2, supplemented with 30% raffinose) with lysostaphin. Protoplasts were sedimented by centrifugation at 6,000g, and the supernatant fraction, which contained the wall-associated proteins, was analyzed by SDS-PAGE.

  7. Supplementary Figure 7: Alignment of the DltB amino acid sequences from Bacillus amyloliquefaciens and Streptococcus pneumoniae strains, colored according to relative sequence conservation at each position. (742 KB)

    Adapted from an alignment generated by PRALINE. The scoring scheme ranges from 0 for the least conserved alignment position up to 10 (indicated by an asterisk) for the most conserved alignment position. (a) B_amyl_FZB42: B. amyloliquefaciens strain FZB42 (plant-associated bacterium). Accession number: YP_001423132. B_amyl_DSM7: B. amyloliquefaciens strain DSM7 (soil adapted). Accession number: YP_003922279. (b) DltB_TIGR4: S. pneumoniae strain TIGR4. Accession number: ZP_01408978. DltB_GA41301: S. pneumoniae strain GA41301. Accession number: ZP_12336902. DltB_GA17227: S. pneumoniae strain GA17227. Accession number: ZP_12797610. DltB_GA47901: S. pneumoniae strain GA47901. Accession number: ZP_12344230.

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