The pneumococcal eukaryotic-type serine/threonine protein kinase StkP co-localizes with the cell division apparatus and interacts with FtsZ in vitro.

The importance of serine/threonine phosphorylation in signalling and regulation of gene expression in prokaryotes has been widely recognized. Driven by our interest in StkP (the pneumococcal serine/threonine kinase homologue) for vaccine development, we studied its cellular localization. We found that the C-terminally located PASTA (penicillin-binding protein and serine/threonine kinase associated) domains, but not the N-terminal kinase domain of StkP, were located on the surface of live pneumococcal cells grown in vitro and were also accessible to antibodies during pneumococcal infection in mice and man. Most importantly, we discovered, by immunofluorescence microscopy, that StkP co-localized with the cell division apparatus. StkP and FtsZ, the prokaryotic tubulin homologue, co-localized at mid-cell in most cells. Formation and constriction of the ring-like structure of StkP followed the dynamic changes of FtsZ in dividing cells. This pattern resembles that of the 'late' divisome protein penicillin-binding protein 2X. The lack of StkP in gene deletion mutants did not disturb FtsZ ring formation, further suggesting that StkP joins the divisome after the FtsZ ring is assembled. We also present evidence that StkP binds and phosphorylates recombinant FtsZ in vitro; however, we could not detect changes in the phosphorylation of FtsZ in a stkP deletion strain relative to wild-type cells. Based on its cell-division-dependent localization and interaction with FtsZ, we propose that StkP plays a currently undefined role in cell division of pneumococcus.

The antigenome: from protein subunit vaccines to antibody treatments of bacterial infections?

New strategies are needed to master infectious diseases. The so-called "passive vaccination", i.e., prevention and treatment with specific antibodies, has a proven record and potential in the management of infections and entered the medical arena more than 100 years ago. Progress in the identification of specific antigens has become the hallmark in the development of novel subunit vaccines that often contain only a single immunogen, frequently proteins, derived from the microbe in order to induce protective immunity. On the other hand, the monoclonal antibody technology has enabled biotechnology to produce antibody species in unlimited quantities and at reasonable costs that are more or less identical to their human counterparts and bind with high affinity to only one specific site of a given antigen. Although, this technology has provided a robust platform for launching novel and successful treatments against a variety of devastating diseases, it is up till now only exceptionally employed in therapy of infectious diseases. Monoclonal antibodies engaged in the treatment of specific cancers seem to work by a dual mode; they mark the cancerous cells for decontamination by the immune system, but also block a function that intervenes with cell growth. The availability of the entire genome sequence of pathogens has strongly facilitated the identification of highly specific protein antigens that are suitable targets for neutralizing antibodies, but also often seem to play an important role in the microbe's life cycle. Thus, the growing repertoire of well-characterized protein antigens will open the perspective to develop monoclonal antibodies against bacterial infections, at least as last resort treatment, when vaccination and antibiotics are no options for prevention or therapy. In the following chapter we describe and compare various technologies regarding the identification of suitable target antigens and the foundation of cognate monoclonal antibodies and discuss their possible applications in the treatment of bacterial infections together with an overview of current efforts.

Anti-infective antibodies: a novel tool to prevent and treat nosocomial diseases.

The emergence of multidrug-resistant bacteria is a growing challenge for healthcare in the treatment of infectious diseases. In particular, nosocomial infections are getting out of control and reduce the likelihood to recover without, sometimes lethal, complications and long-term damage. Current antibiotics are unable to keep nosocomial infections in check and novel ones move only reluctantly forward and are expected to only delay the problem of multidrug resistance. Progress made in the identification of suitable pathogen targets, a better understanding of host-parasite interactions and the recent inclusion of monoclonal antibodies into the arsenal of novel therapies has provoked the interest to revitalize a historical concept of medicine to treat and prevent bacterial infections with antibodies.

Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies.

Pneumococcus is one of the most important human pathogens that causes life-threatening invasive diseases, especially at the extremities of age. Capsular polysaccharides (CPSs) are known to induce protective antibodies; however, it is not feasible to develop CPS-based vaccines that cover all of the 90 disease-causing serotypes. We applied a genomic approach and described the antibody repertoire for pneumococcal proteins using display libraries expressing 15-150 amino acid fragments of the pathogen's proteome. Serum antibodies of exposed, but not infected, individuals and convalescing patients identified the ANTIGENome of pneumococcus consisting of approximately 140 antigens, many of them surface exposed. Based on several in vitro assays, 18 novel candidates were preselected for animal studies, and 4 of them showed significant protection against lethal sepsis. Two lead vaccine candidates, protein required for cell wall separation of group B streptococcus (PcsB) and serine/threonine protein kinase (StkP), were found to be exceptionally conserved among clinical isolates (>99.5% identity) and cross-protective against four different serotypes in lethal sepsis and pneumonia models, and have important nonredundant functions in bacterial multiplication based on gene deletion studies. We describe for the first time opsonophagocytic killing activity for pneumococcal protein antigens. A vaccine containing PcsB and StkP is intended for the prevention of infections caused by all serotypes of pneumococcus in the elderly and in children.

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.