Die Bedeutung von Plasmaprotein-Rezeptoren bei Streptokokkeninfektionen Schlussbericht

SIGLEAvailable from TIB Hannover: F95B1247+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman

Lebend-Impfstoffe fuer Schleimhautimmunitaet: Vergleichsstudien mit Shigella, Salmonella und Streptokokken als Traegerstaemme fuer rekombinante Antigene aus Krankheitserregern Schlussbericht

Vaccines represent an effective and unexpensive means of flighting and controlling infectious diseases. Most of the infectious agents cause disease by colonizing or invading the mucosa. It is therefore of interest to protect against infectious agents through mucosal immunity. This would allow to stop the infections process at an early stage. Moreover, mucosal vaccines have less side effects and more acceptance in the population. The aim of this project was to compare the vaccine carrier strains for the development of live vaccines. Three model antigens from Streptococcus and Bordetalla were cloned in Streptococcus gordonii and Salmonella typhimurium aroA as carrier strains. The expression of antigen was tested in Western Blot analysis. All constructs showed substantial expression of the cloned recombinant antigens. The results show that Streptococcus gordonii and Salmonella strains with intracellulary activated promotors are suitable as carriers of live mucosal vaccines. (orig.)SIGLEAvailable from TIB Hannover: F98B166 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Supplementary Material for: The M1 Protein of <b><i>Streptococcus pyogenes</i></b> Triggers an Innate Uptake Mechanism into Polarized Human Endothelial Cells

Serotype M1 <i>Streptococcus pyogenes</i> is a major human pathogen associated with severe invasive diseases causing high morbidity and mortality. In a substantial number of cases, invasive disease develops in previously healthy individuals with no obvious port of entry. This has led to the hypothesis that the source of streptococci in these cases is a transient bacteraemia. This study focuses on the analysis of interaction of tissue-invasive serotype M1 <i>S. pyogenes</i> with human endothelial cells (EC) of the vascular system. We identify the M1 surface protein of <i>S. pyogenes</i> as the EC invasin which is recognised by polarized human blood EC, thereby triggering rapid, phagocytosis-like uptake of streptococci into polarized EC layers. Upon internalization, the M1 <i>S. pyogenes</i> serotype is incorporated into phagosomes which traffic via the endosomal/lysosomal pathway. However, some of the streptococci successfully evade this innate killing process and hereby mediate their escape into the cytoplasm of the host cell. The results of this study demonstrate that blood EC possess an efficient uptake mechanism for serotype M1 <i>S. pyogenes</i>. Despite efficient phagocytosis, streptococcal survival within EC constitutes one potential mechanism which favours intracellular persistence and thus facilitates continuous infection and dissemination from the primary side of infection into deep tissue

Cooperative binding of human fibronectin to SfbI protein triggers streptococcal invasion into respiratory epithelial cells

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii, we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N-terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen-binding fragment of fibronectin. In the case of native surface-expressed SfbI protein, an induced fit mode of bacteria-fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co-operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion

Opacity factor activity and epithelial cell binding by the serum opacity factor protein of Streptococcus pyogenes are functionally discrete

Serum opacity factor (SOF) is a unique multifunctional virulence determinant expressed at the surface of Streptococcus pyogenes and has been shown to elicit protective immunity against GAS infection in a murine challenge model. SOF consists of two distinct domains with different binding capacities: an N-terminal domain that binds apolipoprotein AI and a C-terminal repeat domain that binds fibronectin and fibrinogen. The capacity of SOF to opacify serum by disrupting the structure of high density lipoproteins may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100-fold) or abrogated opacity factor (OF) activity, for use as vaccine antigens. However, alterations introduced into the N-terminal SOF peptide (SOF Delta Fn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic S. pyogenes challenge in a murine model. Mutant forms of purified SOF Delta Fn peptide were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Using latex beads coated with full-length SOF, SOF Delta Fn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOF Delta Fn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOF Delta Fn peptide is independent of OF activity. We demonstrate that while the N terminus of SOF does not directly mediate intracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone