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A single natural nucleotide mutation alters bacterial pathogen host tropism

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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Figure 1: Rabbit but not human ST121 strains can infect rabbits.
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.
Figure 3: A single mutation is sufficient to confer rabbit infectivity to a human S. aureus strain.
Figure 4: Rabbit-infective S. aureus clones have evolved on numerous occasions and are associated with nonsynonymous mutations of dltB.

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Acknowledgements

We thank J. Etienne for helpful advice, O. Schneewind, M. Woolhouse and Í. Lasa for comments on the manuscript, C. Cervera and E. Blas for their support with the in vivo experiments, and R. Cartwright for excellent technical assistance. We are grateful to Edinburgh Genomics (Roslin Institute) for sequencing services. This work was supported by grants BIO2011-30503-C02-01, Eranet-pathogenomics PIM2010EPA-00606 and Consolider-Ingenio CSD2009-00006 from Ministerio de Ciencia e Innovación (Spain) and strategic grant funding from the University of Glasgow to J.R.P.; by a project grant (BB/I013873/1) and institute strategic grant funding from the Biotechnology and Biological Sciences Research Council (UK) to J.R.F., in addition to a doctoral training grant from the Medical Research Council (UK) to J.R.F.; and by grants AGL2011-30170-CO2-02 (Ministerio de Ciencia e Innovación, Spain) and GV2013-077 (Conselleria d'Educació, Cultura i Esport, Generalitat Valenciana) to D.V.

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Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to J Ross Fitzgerald or José R Penadés.

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The authors declare no competing financial interests.

Integrated supplementary information

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.

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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

Supplementary Figure 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.

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.

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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Viana, D., Comos, M., McAdam, P. et al. A single natural nucleotide mutation alters bacterial pathogen host tropism. Nat Genet 47, 361–366 (2015). https://doi.org/10.1038/ng.3219

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