Genomic and surface proteomic analysis of the canine pathogen Staphylococcus pseudintermedius reveals proteins that mediate adherence to the extracellular matrix

Abstract

Cell wall-associated (CWA) proteins made by Gram-positive pathogens play a fundamental role in pathogenesis. Staphylococcus pseudintermedius is a major animal pathogen responsible for the canine skin disease bacterial pyoderma. Here, we describe the bioinformatic analysis of the family of 18 predicted CWA proteins encoded in the genome of S. pseudintermedius strain ED99 and determine their distribution among a phylogenetically diverse panel of S. pseudintermedius clinical isolates and closely related species of the Staphylococcus intermedius group. In parallel, we employed a proteomic approach to identify proteins presented on the surface of strain ED99 in vitro, revealing a total of 60 surface-localized proteins in one or more phases of growth, including 6 of the 18 genome-predicted CWA proteins. Based on these analyses, we selected two CWA proteins (SpsD and SpsL) encoded by all strains examined and investigated their capacity to mediate adherence to extracellular matrix proteins. We discovered that SpsD and SpsL mediated binding of a heterologous host, Lactococcus lactis, to fibrinogen and fibronectin and that SpsD mediated binding to cytokeratin 10, a major constituent of mammalian skin. Of note, the interaction with fibrinogen was host-species dependent, suggestive of a role for SpsD and SpsL in the host tropism of S. pseudintermedius. Finally, we identified IgG specific for SpsD and SpsL in sera from dogs with bacterial pyoderma, implying that both proteins are expressed during infection. The combined genomic and proteomic approach employed in the current study has revealed novel host-pathogen interactions which represent candidate therapeutic targets for the control of bacterial pyoderma.

Fig. 1.

Adherence of S. pseudintermedius strain ED99 to immobilized ligands. Plates were coated with doubling dilutions of fibrinogen (A), fibronectin (B), and cytokeratin 10 (C) and incubated with S. pseudintermedius ED99 grown to exponential (▪) or stationary (•) phase of growth. Absorbance was measured at 590 nm, and results are expressed as mean values of triplicate samples; error bars indicate standard deviations. S. aureus strain SH1000, strain Newman, and an isogenic derivative of strain Newman deficient in ClfA production were used as controls (data not shown).

Fig. 2.

Genome location and distribution of genes encoding CWA proteins. (A) Genome map of S. pseudintermedius ED99 indicating the genomic location of each gene. (B) Distribution of the genes encoding putative CWA proteins among 20 S. pseudintermedius strains, representatives of the closely related S. delphini and S. intermedius, and other staphylococcal species associated with animal skin disease. The diversity of strains is indicated by a phylogenetic tree constructed using the neighbor-joining method, with bootstrap values over 60 indicated; a blue square denotes presence of a gene, and an empty square indicates its absence, based on Southern blot analysis (for spsA to spsO) or PCR amplification (for spsP, spsQ, and spsR).

Fig. 3.

Schematic representation of predicted CWA proteins encoded in the S. pseudintermedius ED99 genome. The predicted length in amino acids of each protein is indicated; typical MSCRAMM features are shown as appropriate with signal sequence (red), the A domain with IgG-like folds (labeled), repeat regions (blue), and a cell wall-anchoring LPX(TSA)/(GANS) motif (black). For SpsP and SpsQ (S. aureus SpA orthologues), the repeated IgG-binding domains (blue) and X repeat regions (Xr; gray) are also indicated. All organisms belong to Staphylococcus species unless otherwise noted. ORF, open reading frame.

Fig. 4.

Adherence of L. lactis expressing specified CWA proteins to immobilized ligands. (A and B) Plates were coated with doubling dilutions of fibrinogen (Fg) of human, bovine, canine, equine, or avian origin or with bovine serum albumin (BSA), as indicated in the legend of panel B, and incubated with L. lactis expressing SpsD or SpsL. L. lactis with empty expression vector pOri23 incubated with immobilized canine fibrinogen was included as a negative control. (C and D) For panel C plates were coated with doubling dilutions of bovine fibronectin and incubated with L. lactis expressing SpsD or SpsL, as indicated on panel D. L. lactis with empty expression vector pOri23 incubated with immobilized bovine fibronectin was included as a negative control. For panel D, plates were coated with doubling dilutions of recombinant mouse cytokeratin 10 and incubated with L. lactis expressing SpsD or SpsL. L. lactis with empty expression vector pOri23 incubated with immobilized recombinant mouse cytokeratin 10 was included as a negative control. Absorbance was measured at 590 nm, and results are expressed as mean values of triplicate samples. Error bars indicate standard deviation. S. aureus strain SH1000, strain Newman, and an isogenic derivative of strain Newman deficient in ClfA production were used as controls (data not shown).

Fig. 5.

SpsD and SpsL are expressed during canine infection. SDS-PAGE (A) and Western immunoblot analysis (B) of recombinant A domains of SpsD and SpsL with sera obtained from dogs with S. pseudintermedius pyoderma. Recombinant SEI encoded by the human pathogen S. aureus was included as a negative control.