High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded beta-barrel fold.

Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for "near transporter") present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (K(D)) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel beta-barrel with the strand order (-beta1 -beta2 -beta3 -beta6 -beta5 -beta4 -beta7 -beta8), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.

Comparison of antibody repertoires against Staphylococcus aureus in healthy individuals and in acutely infected patients.

The management of staphylococcal diseases is increasingly difficult with present medical approaches. Preventive and therapeutic vaccination is considered to be a promising alternative; however, little is known about immune correlates of protection and disease susceptibility. To better understand the immune recognition of Staphylococcus aureus by the human host, we studied the antistaphylococcal humoral responses in healthy people in comparison to those of patients with invasive diseases. In a series of enzyme-linked immunosorbent assay analyses performed using 19 recombinant staphylococcal cell surface and secreted proteins, we measured a wide range of antibody levels, finding a pronounced heterogeneity among individuals in both donor groups. The analysis revealed marked differences in the antibody repertoires of healthy individuals with or without S. aureus carriage, as well as in those of patients in the acute phase of infection. Most importantly, we identified antigenic proteins for which specific antibodies were missing or underrepresented in infected patients. In contrast to the well-described transient nature of disease-induced antistaphylococcal immune response, it was demonstrated that high-titer antistaphylococcal antibodies are stable for years in healthy individuals. In addition, we provide evidence obtained on the basis of opsonophagocytic and neutralizing activity in vitro assays that circulating antistaphylococcal serum antibodies in healthy donors are functional. In light of these data we suggest that proper serological analysis comparing the preexisting antibody repertoires of hospitalized patients with different outcomes for nosocomial staphylococcal infections could be extremely useful for the evaluation of candidate vaccine antigens in addition to protection data generated with animal models.

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

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.

[Utilization of hybridization in situ and PCR in situ methods to detect latent infection by the wild strain of HSV-1 and by a mutant with reduced reactivation]. / Wykorzystanie metod hybrydyzacji in situ i PCR in situ do wykrywania utajonego zakazenia wywolanego dzikim szczepem HSV-1 oraz mutantem o oslabionej reaktywacji.

The aim of the study was to detect the number of latently infected cells with wild type virus and with mutant with reduced reactivation. Using PCR in situ method we established, that the number of cells containing genome of these viruses do not differ especially between them. The number of cells with LAT expression is significantly reduced in the ganglia infected with mutant with impaired reactivation as we showed using in situ hybridization. Based on the observations of other authors, that high expression of LATs takes place in cells with high copy number of viral DNA our results showed, that after infection of mice with mutant with reduced reactivation less cells contain high copy number of viral DNA than after infection with wild type KOS. We suggest that impaired reactivation of an ICP22 mutant occurs as a result of reduced number of cells with high copy number of viral DNA.

Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus.

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.