Evolutionary and Functional Analysis of Coagulase Positivity among the Staphylococci

Abstract

The bacterial genus Staphylococcus comprises a large group of pathogenic and nonpathogenic species associated with an array of host species. Staphylococci are differentiated into coagulase-positive or coagulase-negative groups based on the capacity to promote clotting of plasma, a phenotype historically associated with the ability to cause disease. However, the genetic basis of this important diagnostic and pathogenic trait across the genus has not been examined to date. Here, we selected 54 representative staphylococcal species and subspecies to examine coagulation of plasma derived from six representative host species. In total, 13 staphylococcal species mediated coagulation of plasma from at least one host species including one previously identified as coagulase negative (Staphylococcus condimenti). Comparative genomic analysis revealed that coagulase activity correlated with the presence of a gene (vwb) encoding the von Willebrand binding protein (vWbp) whereas only the Staphylococcus aureus complex contained a gene encoding staphylocoagulase (Coa), the classical mediator of coagulation. Importantly, S. aureus retained vwb-dependent coagulase activity in an S. aureus strain deleted for coa whereas deletion of vwb in Staphylococcus pseudintermedius resulted in loss of coagulase activity. Whole-genome-based phylogenetic reconstruction of the Staphylococcus genus revealed that the vwb gene has been acquired on at least four different occasions during the evolution of the Staphylococcus genus followed by allelic diversification via mutation and recombination. Allelic variants of vWbp from selected coagulase-positive staphylococci mediated coagulation in a host-dependent manner indicative of host-adaptive evolution. Taken together, we have determined the genetic and evolutionary basis of staphylococcal coagulation, revealing vWbp to be its archetypal determinant. IMPORTANCE The ability of some species of staphylococci to promote coagulation of plasma is a key pathogenic and diagnostic trait. Here, we provide a comprehensive analysis of the coagulase positivity of the staphylococci and its evolutionary genetic basis. We demonstrate that the von Willebrand binding protein rather than staphylocoagulase is the archetypal coagulation factor of the staphylococci and that the vwb gene has been acquired several times independently during the evolution of the staphylococci. Subsequently, vwb has undergone adaptive diversification to facilitate host-specific functionality. Our findings provide important insights into the evolution of pathogenicity among the staphylococci and the genetic basis for a defining diagnostic phenotype.

Keywords: Staphylococcus; coagulase; coagulase-positive staphylococci; coagulation; diagnostics; evolution; host specificity; phylogeny; von Willebrand binding protein.

FIG 1

Schematic protein domain structure of Coa and vWbp of S. aureus Newman. Both Coa and vWbp contain N-terminal signal peptides (S) and prothrombin-binding D1 and D2 domains. The C-terminal sequence of Coa contains a linker (L) followed by fibrinogen-binding repeats (R). The C-terminal sequence of vWbp contains a von Willebrand factor binding region between two domains. In S. aureus Newman, Coa and vWbp share 26.3% amino acid identity.

FIG 2

All CoPS species contain the vwb gene. Maximum likelihood phylogenetic tree of 50 staphylococcal species and 5 additional subspecies based on a core genome alignment of 231 concatenated genes. A list of the genome sequences used is included in Table S2. Coagulase-positive species are highlighted in bold, and coagulase-variable species are highlighted with an asterisk. Species containing the vwb gene are represented with orange branches, with orange circles indicating the branch on which this gene was acquired. Red circles indicate the nodes in which additional vwb copies were acquired. The single acquisition event of the coa gene by an ancestor of the S. aureus complex is indicated by a green circle. Genome locations of the vwb and coa genes in each CoPS lineage are represented by colored bars on a blue ring depicting the chromosome. Support values are shown on each branch and were calculated using the Shimodaira-Hasegawa test. The Mammaliicoccus cluster is the root of the tree.

FIG 3

Staphylococcal vWbp has an evolutionary history distinct from that of coagulase. (A) Unrooted maximum-likelihood tree constructed using FastTree v2 of the Coa and vWbp protein sequences. Each of the main clusters is highlighted and labeled. Coagulase positivity has evolved on multiple occasions through acquisition of vwb followed by evolution according to species. (B) Phylogenetic reconstruction of the non-aureus CoPS based on vWbp protein sequences.

FIG 4

The chromosomal vwb has undergone extensive recombination among the S. aureus complex species. Midpoint-rooted maximum-likelihood tree constructed using FastTree v2 of the S. aureus complex vwb nucleotide sequences. Colored circles denote each Staphylococcus species. Plot of the FastGEAR analysis of the vwb nucleotide alignment applied to 100-bp-long windows. For each window (i.e., column in the plot), each color represents different phylogenetic clusters based on the BAPS algorithm. The axis at the top shows, for reference, the location of the coagulase (D1+D2) domain using the amino acid coordinates of the RF122 S. aureus isolate vWbp.

FIG 5

Recombinant vWbp proteins exhibit host-specific coagulation of plasma. Coagulation of plasma from human (A), canine (B), equine (C), porcine (D), avian (E), and rabbit (F) origin by 50 μg ml⁻¹ recombinant vWbp encoded by S. pseudintermedius (pink), S. intermedius (orange), S. delphini (dark green), S. hyicus (light green), and S. aureus (blue), in addition to S. aureus Coa (purple). Coagulation was scored as 0.5 for trace levels of coagulation, 1.0 to 2.5 for partially coagulating plasma, and 3.0 for a complete clot. Data are shown as the mean from 6 replicates with error bars representing standard deviation.

FIG 6

vwb is required for the coagulase phenotype of S. pseudintermedius ED99. (A) Coagulation assay of wild-type and mutant strains of S. pseudintermedius ED99 and S. aureus Newman in human and canine plasma. Average data are provided from three biological replicates. (B) Antibody (Ab)-mediated inhibition of coagulation in canine plasma using ED99 wild type in the presence of increasing concentrations of anti-vWbp^{pseudintermedius} antibody. The mean values are given from three experiments with error bars representing standard deviation (n = 3).