Evolutionary genomics of Staphylococcus aureus reveals insights into the origin and molecular basis of ruminant host adaptation

Abstract

Phenotypic biotyping has traditionally been used to differentiate bacteria occupying distinct ecological niches such as host species. For example, the capacity of Staphylococcus aureus from sheep to coagulate ruminant plasma, reported over 60 years ago, led to the description of small ruminant and bovine S. aureus ecovars. The great majority of small ruminant isolates are represented by a single, widespread clonal complex (CC133) of S. aureus, but its evolutionary origin and the molecular basis for its host tropism remain unknown. Here, we provide evidence that the CC133 clone evolved as the result of a human to ruminant host jump followed by adaptive genome diversification. Comparative whole-genome sequencing revealed molecular evidence for host adaptation including gene decay and diversification of proteins involved in host-pathogen interactions. Importantly, several novel mobile genetic elements encoding virulence proteins with attenuated or enhanced activity in ruminants were widely distributed in CC133 isolates, suggesting a key role in its host-specific interactions. To investigate this further, we examined the activity of a novel staphylococcal pathogenicity island (SaPIov2) found in the great majority of CC133 isolates which encodes a variant of the chromosomally encoded von Willebrand-binding protein (vWbp(Sov2)), previously demonstrated to have coagulase activity for human plasma. Remarkably, we discovered that SaPIov2 confers the ability to coagulate ruminant plasma suggesting an important role in ruminant disease pathogenesis and revealing the origin of a defining phenotype of the classical S. aureus biotyping scheme. Taken together, these data provide broad new insights into the origin and molecular basis of S. aureus ruminant host specificity.

FIG. 1.—

The majority of Staphylococcus aureus isolates from ruminants belong to three clonal lineages. Neighbor-Joining consensus tree inferred from 500 bootstrap replicates was constructed using concatenated sequences of 33 S. aureus STs from ruminants and 97 selected representative STs from humans and other animals. STs from large (cows) and small (sheep or goats) ruminants are depicted by blue and red branches, and the major lineages associated with large and small ruminants are indicated by blue and red shading, respectively. Branches depicting STs of human, avian, or unknown host origin are indicated in black. ST945 represents the human-associated ST, which is most closely related to CC133, used to calculate the date range for the predicted host jump.

FIG. 2.—

Ovine Staphylococcus aureus strain ED133 contains novel MGE including staphylococcal pathogenicity islands (SaPIs) (A) and prophages (B) encoding putative virulence factors. Regions of nucleotide identity (>94%) between MGE are indicated by gray shading, genes in different MGE with the same predicted function have the same color, genes in gray encode hypothetical proteins of unknown function, and green squares denote SaPI attachment sites.

FIG. 3.—

Several novel MGE identified in strain ED133 are widely distributed among CC133 isolates but not among human isolates. Strains of ruminant origin associated with the common lineages CC133, CC151, and CC97 and the less common genotypes ST126, ST130, ST30, and ST39, in addition to representative human strains were examined for the presence of MGE identified in strain ED133, determined by a combination of comparative genome hybridization, PCR and/or comparative genome sequence analysis. A filled square indicates the presence of an MGE (confirmed by at least 2 out of 2 positive distinct MGE-specific PCR reactions), an empty space its absence, and a hatched square indicates the presence of a distinct but genetically related element (determined by at least 1 out of 2 positive distinct MGE-specific PCR reaction). Red, blue, and green boxes denote strains of ruminant, human, or avian origin, respectively. A Neighbor-Joining consensus tree inferred from 500 bootstrap replicates was constructed based on the concatenated sequences of STs to indicate the genetic relatedness of the strains examined.

FIG. 4.—

SaPIov2 confers the ability to coagulate ruminant plasma. Ruminant plasma coagulase assays represented by inverted test tubes with or without plasma clots indicating positive or negative results, respectively. (A) Wild-type clinical isolates of ruminant, human, or avian origin with (i) or without (ii) the novel MGE SaPIov2. (B) (i) Ruminant plasma coagulase activity of genetically modified isolates including clinical isolates with SaPIov2 replaced by SaPIN1 from human strain N315, and lab strain RN4220 which has acquired SaPIov2. (ii) Rabbit plasma coagulase activity of selected strains (all strains examined were positive for rabbit plasma coagulase activity, data not shown).