Host-pathogen interactions in staphylococcal pyoderma

Superficial bacterial folliculitis of dogs (pyoderma) caused by Staphylococcus pseudintermedius is one of the most common diseases seen in small animal veterinary practice, worldwide. Treatment of canine pyoderma often requires aggressive antibiotic therapy and is subject to common relapse of infection. Worringly, methicillin-resistant S. pseudintermedius has recently emerged as a major problem in veterinary clinics worldwide. Although rare, several episodes of life-threatening infections of humans by S. pseudintermedius have been reported with the typical route of transmission being through dog bite wounds. Gram-positive bacteria cell-wall anchored (CWA) protein play a key role in host colonisation and disease pathogenesis and represent targets for therapeutic intervention. For example, the fibrinogen-binding protein ClfA made by S. aureus has been a major focus of antibody-based therapeutics. Very recently, in a team led by our collaborator Prof. Magnus Hook, we have published the structure for ClfA binding to its major ligand fibrinogen, resulting in the design of peptides which block ClfA function. These peptides are currently under investigation as potential therapeutics for treating staphylococcal disease. We have recently sequenced the whole genome of a clinical isolate of S. pseudintermedius isolated from a case of canine pyoderma (unpublished data). Importantly we have identified genes encoding 18 predicted CWA proteins, each of which is unique to S. pseudintermedius. Through the expression of purified recombinant proteins and the use of a heterologous expression host, Lactococcus lactis, we have discovered 2 S. pseudintermedius CWA proteins which mediate binding to both fibrinogen and fibronectin (SpsD and SpsL) and cytokeratin (SpsD) and which are encoded by all strains examined. These data suggest that they play a critical role in host-pathogen interactions and could represent excellent targets for novel therapeutics. In the current proposal we will characterise the molecular interaction between the CWA proteins SpsD and SpsL with their ligands in order to facilitate the design of novel therapeutic approaches for the control of canine pyoderma. 1) Examination of the allelic variation and expression of SpsD and SpsL among S. pseudintermedius strains. Sequencing of genes encoding SpsD and SpsL from 20 S. pseudintermedius strains representing the breadth of species diversity will allow us to determine how conserved the proteins are among the common pathogenic clones. In addition, surface expression of the proteins by the same panel of strains will be examined by ELISA-type assays with specific IgY antibodies. 2). Identification of the bacterial protein domains and host ligand domains required for binding. This phase of the project will involve cloning and recombinant expression of selected sub-domains of SpsD and SpsL in order to test their ligand-binding activity in ELISA-like assays. Using similar assays, recombinant or protease-cleaved fragments of ECM proteins, fibrinogen, fibronectin and cytokeratin will be employed to determine which region of each host protein is the target of adherence. Recombinant proteins will then be used to block the interaction of L. lactis strains expressing each protein or S. pseudintermedius wild type strains, to ECM proteins. 3) Identification of peptides which block the function of SpsD and SpsL. Structural modeling of SpsD and SpsL will be carried out using the programme PyMol based on the crystal structure of ClfA and other related CWA proteins of S. aureus in order to predict amino acid residues which may be involved in ligand binding. Based on this analysis, we will design short peptides which we predict could interfere with ligand binding. In addition, antibodies specific for the ligand-binding domains will be raised and the ability of each of these molecules to block function and inhibit binding to ECM proteins will be tested.