Recombination-mediated remodelling of host-pathogen interactions during Staphylococcus aureus niche adaptation

Abstract

Large-scale recombination events have led to the emergence of epidemic clones of several major bacterial pathogens. However, the functional impact of the recombination on clonal success is not understood. Here, we identified a novel widespread hybrid clone (ST71) of livestock-associated Staphylococcus aureus that evolved from an ancestor belonging to the major bovine lineage CC97, through multiple large-scale recombination events with other S. aureus lineages occupying the same ruminant niche. The recombination events, affecting a 329 kb region of the chromosome spanning the origin of replication, resulted in allele replacement and loss or gain of an array of genes influencing host-pathogen interactions. Of note, molecular functional analyses revealed that the ST71 hybrid clone has acquired multiple novel pathogenic traits associated with acquired and innate immune evasion and bovine extracellular matrix adherence. These findings provide a paradigm for the impact of large-scale recombination events on the rapid evolution of bacterial pathogens within defined ecological niches.

Keywords: Staphylococcus aureus; host–pathogen interactions; niche adaptation; recombination; remodelling.

Fig. 1.

ST71 is a divergent subtype of CC97 containing a 323 kb genomic region with a distinct evolutionary origin. Maximum-likelihood tree constructed from core genome alignment with (a) and without (b) the SNP-dense region. The outgroup is strain MW2; ST71 strains are highlighted in red. CC97 strains included are those listed in Table S1 in addition to the ST71 strains. Scale is indicated for each tree in substitutions per site. (c) Pairwise strain comparisons of core genome SNP density over 10 000 bp windows between ST71 strain RF103 and ST97 strain RF116 (blue line). (d) Identification of multiple large-scale recombination events in the ST71 genome. The names of each strain are shown on the right with MLST sequence genotype indicated in parentheses. On the left side of the diagram is the proportion of shared ancestry tree as determined by BratNextGen (Marttinen et al., 2012). The coloured region indicates detected recombination events along the length of the core genome alignment. The same colour at overlapping genomic locations in different strains indicates those segments are of the same origin. Faint grey lines indicate alignment gaps.

Fig. 2.

Tracing the evolutionary origin of ST71 recombinant sequences. The circle represents the whole-genome sequence of S. aureus ST71, with the 10 predicted recombinant regions spanning the origin of replication indicated. Inside the circle is a neighbour-joining phylogenetic tree of 178 isolates representing the full breadth of S. aureus species diversity. Filled blue and red lines connect each predicted recombinant region with its likely clonal origin across the S. aureus species, based on sequence identity. Red and blue denote a minimum 99.75 and 99.5 % shared non-variant sites, respectively. Yellow depicts the recombinant region which did not share nucleotide sequence identity of at least 99.5 % with any sequence in the database. The locations of selected acquired genes are indicated. The scale represents substitutions per site.

Fig. 3.

Acquisition of cna by ST71 confers the capacity to bind bovine type I collagen. (a) Cna is expressed on the surface of ST71 strains. Western blot analysis of cell wall-associated proteins of ST71 and ST97 strains with anti-Cna mouse mAbs. S. aureus ATCC 25923 was used as a positive control and S. aureus Newman was used as a negative control. (b) ST71 strains bind to immobilized type I collagen. Plates were coated with doubling dilutions of bovine collagen type I and incubated with S. aureus cultured to exponential phase. Results are expressed as mean ± sd A₅₉₀ values of triplicate results. S. aureus strain ATCC 25923 and strain Newman were used as controls. (c) ST71 Collagen binding is inhibited by anti-Cna antibodies. Exponential phase S. aureus were pre-incubated with anti-Cna antibody before addition to the plate. Results are expressed as mean ± sd A₅₉₀ values of triplicate results. S. aureus strain ATCC 25923 and strain Newman were used as controls (data not shown). (d) Invasion of bovine mammary epithelial cells (MAC-T) by S. aureus. Bacteria were co-cultured with MAC-T cells for 2 h at 37 °C, followed by addition of gentamicin to deplete extracellular bacteria. Percentage invasion was estimated by measuring viable counts after cell lysis compared with initial inoculum. Data represent mean ± sem of at least four independent experiments. The reduction in invasion between S. aureus Phillips and Phillips Δcna was statistically significant using the Mann–Whitney test (P = 0.014).

Fig. 4.

SElZ is a novel superantigen acquired by ST71 which stimulates proliferation of specific bovine T-cell subfamilies. (a) Neighbour-joining tree based on the nucleotide sequences of all known staphylococcal superantigens and the superantigen-like protein SSL7. The branch specific for the novel superantigen SElZ is indicated in red. The scale represents substitutions per site. (b) Proliferation of bovine peripheral blood mononuclear cell cultures in response to recombinant SElZ (red) and SElX (blue) measured by [³H]thymidine incorporation. (c) Relative fold change in Vβ expression (mean ± sem) for bovine T-cells from two donors after stimulation with SElZ in triplicate. *Statistical significance for Vβ3 (P = 0.029) and Vβ11 (P = 0.05).

Fig. 5.

Schematic representation of the evolution and pathogenic diversification of S. aureus ST71.