Identification of a novel iron regulated staphylococcal surface protein with haptoglobin‐haemoglobin binding activity

Summary Staphylococcus aureus is an extremely adaptable pathogen causing a wide variety of infections. Staphylococcal surface proteins that directly interact with host extracellular proteins greatly contribute to virulence and are involved in adhesion, immune escape and nutrient acquisition. In our extensive search for highly immunogenic, in vivo ‐expressed, staphylococcal proteins, previously, we identified a novel member of the family of Gram‐positive anchor motif proteins with a predicted 895 amino acid long sequence. In order to determine the ligand for this novel LPXTG cell wall protein, we employed affinity purification of human plasma using the recombinant form of the protein. Two‐dimensional electrophoresis of eluted plasma proteins identified haptoglobin as a specific binding partner. Importantly, we also observed this specific ligand binding when living S. aureus cells were exposed to biotin‐labelled haptoglobin (Hp) in a FACS‐based assay. Targeted deletion of the gene eliminated Hp‐binding, a function that has not been attributed to S. aureus before. Based on these data we specified the protein as the staphylococcal ha ptoglobin r eceptor A (HarA). Similarly to other haptoglobin receptors identified in Gram‐negative pathogens, HarA binds not only Hp, but also haptoglobin‐haemoglobin complexes with an even higher affinity, as demonstrated in in vitro binding assays. Employing specific deletion mutants, ligand binding was localized to two homologous regions with about 145 amino acid residues located within the N‐terminal part of the protein. In addition, we demonstrated that expression of HarA was strictly controlled by iron through the iron‐dependent transcriptional regulator Fur. Based on these data we propose that HarA can be added to the list of staphylococcal virulence factors with a most likely function related to iron acquisition.