Biosensorsystem
Final Report Abstract
Streptococcus pneumoniae (pneumococci) and Staphylococcus aureus are Gram-positive asymptomatic colonizer of the nasopharynx in humans. However, pneumococci and staphylococci are also versatile human pathogenic bacteria that cause severe and life-threatening diseases. Pneumococci are the most cause of community-acquired pneumonia progressing to bacteremia and they are able to breach the blood-brain barrier. S. aureus causes infections ranging from mild wound infections to severe nosocomial infections such as pneumonia. The aim of the various studies in our laboratory is to decipher the mechanisms of pneumococcal- and staphylococcalhost interactions. Therefore adhesion mechanisms of pneumococci to human epithelial cells and the analysis of adhesion-induced signal transduction cascades in human host cells are investigated in cell-culture based infection assays and experimental mice infection models. A prerequisite for these studies is the identification of surface-exposed virulence factors contributing to adherence to and invasion into host cells or immune evasion via their interaction with soluble host proteins or cellular receptors. We have identified the pneumococcal adherence and virulence factor B (PavB) as a novel bacterial adhesin, whose repetitive sequences, designated Streptococcal Surface REpeats (SSURE), interact with different human proteins derived from the extracellular matrix (ECM) or serum. Surface plasmon resonance (SPR) was used to investigate the interaction of different His6- tagged SSURE peptides with fibronectin and plasminogen, respectively. The SPR analysis shows the direct interaction of the SSURE peptides in a dose-dependent manner, suggesting, in combination with the results of other experiments, the function of PavB as adhesin contributing to the adherence of pneumococci to human host epithelial cells. One of the most important adhesins of S. pneumoniae is the pneumococcal surface protein C (PspC, also referred to as CbpA or SpsA), which belongs to the family of surface-exposed pneumococcal choline binding proteins. Eleven different PspC subtypes are classified into two subgroups, depending on their anchorage to the bacterial cell envelope. Pneumococcal adhesion and internalization into respiratory epithelial cells are primarily accomplished by the unique human-specific binding of PspC to the secretory component (SC) of the polymeric Ig receptor (pIgR). In addition, PspC has also been shown to recruit the complement regulator Factor H. In our recent studies we identified the multifunctional PspC of S. pneumoniae further as a human vitronectin-binding protein. Using SPR experiments we demonstrated that vitronectin preferentially binds to PspC subtypes consisting of two SC-binding regions, termed R domains, located in the mature N-terminal part of PspC. By employing ELISA and SPR the major binding site of PspC was narrowed down to the C-terminal heparin-binding domain of human vitronectin. Staphylococcus aureus (S. aureus) has evolved sophisticated mechanisms to attach to and invade into epithelial and endothelial cells. These multifaceted mechanisms are one of the major reasons for hospital acquired infections. Understanding the mechanisms of colonization and invasion is therefore of greatest interest to prevent and treat patients. S. aureus exhibits a wide variety of adhesins interacting with human ECM and serum proteins. The principle goal of our studies in this project is to identify and characterize yet unknown binding partners (bacterial surface proteins) for matricellular proteins such as thrombospondin-1 and ECM proteins like vitronectin involved in adhesion and invasion. Surface plasmon resonance is used for the screening of enriched surface protein preparations from S. aureus. Single proteins (identified by 2D-gelelectrophoresis, ligand overlay blots and mass spectrometry) are used in protein-proteininteraction studies such as SPR, ELISA, and flow cytometry, respectively, to characterize the interaction between bacterial and host cell proteins. Functional domains of thrombospondin-1 and vitronectin were cloned and heterologously expressed in E. coli to identify and characterize binding sites within the host molecules. SPR allows us to work with smallest amounts of bacterial or human proteins which are often difficult to purify in large amounts or are expensive when purchased from suppliers.
Publications
- PavB is a surface-exposed adhesin of Streptococcus pneumoniae contributing to nasopharyngeal colonization and airways infections. Mol Microbiol 2010, 77(1):22-43
Jensch I, Gámez G, Rothe M, Ebert S, Fulde M, Somplatzki D, Bergmann S, Petruschka L, Rohde M, Nau R, Hammerschmidt S
- Recognition of drug degradation products by target proteins: isotetracycline binding to Tet repressor. J Med Chem. 2011, 54(14):5108-15
Volkers G, Petruschka L, Hinrichs W
- Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis. Proc Natl Acad Sci U S A. 2012, 109(19):7451-6
Elsholz AK, Turgay K, Michalik S, Hessling B, Gronau K, Oertel D, Mäder U, Bernhardt J, Becher D, Hecker M, Gerth U
- The Choline-binding Protein PspC of Streptococcus pneumoniae Interacts with the C-terminal Heparin-binding Domain of Vitronectin. J Biol Chem. 2013, 288(22):15614-27
Voss S, Hallström T, Saleh M, Burchhardt G, Pribyl T, Singh B, Riesbeck K, Zipfel PF, Hammerschmidt S
