RESUMO
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 haptoglobin receptor 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.
Assuntos
Proteínas de Bactérias/metabolismo , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Staphylococcus aureus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Divisão Celular/fisiologia , Parede Celular/metabolismo , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Staphylococcus aureus/citologia , Staphylococcus aureus/genéticaRESUMO
For the design of potent subunit vaccines, it is of paramount importance to identify all antigens immunologically recognized by a patient population infected with a pathogen. We have developed a rapid and efficient procedure to identify such commonly recognized antigens, and here we provide a comprehensive in vivo antigenic profile of Staphylococcus aureus, an important human pathogen. S. aureus peptides were displayed on the surface of Escherichia coli via fusion to one of two outer membrane proteins (LamB and FhuA) and probed with sera selected for high Ab titer and opsonic activity. A total of 60 antigenic proteins were identified, most of which are located or predicted to be located on the surface of the bacterium or secreted. The identification of these antigens and their reactivity with individual sera from patients and healthy individuals greatly facilitate the selection of promising vaccine candidates for further evaluation. This approach, which makes use of whole genome sequence information, has the potential to greatly accelerate and facilitate the formulation of novel vaccines and is applicable to any pathogen that induces Abs in humans and/or experimental animals.