Function of the Interaction between Enolase and von Willebrand Factor in Pneumococcus-induced Thrombogenesis
Parasitology and Biology of Tropical Infectious Disease Pathogens
Final Report Abstract
Streptococcus pneumoniae is a human pathobiont causing a variety of infections followed by cardiovascular complications. In our initial studies, we confirmed binding of different pneumococcus strains to the coagulation glycoprotein Von Willebrand factor (VWF) and confirmed the pneumococcal surface- exposed enolase as VWF-binding protein. The main scientific question of this project reads as follows: “Is VWF binding to pneumococci via an interaction with the enolase causing a shift of the hemostasis-fibrinolysis balance”? In the first part of the project, we biochemically characterized the enolase-VWF interaction. A VWF-binding site was identified by enolase peptide spot array in combination with computational modelling based on the known enolase structure. Moreover, surface plasmon resonance studies revealed a strong and dose-dependent enolase-VWF interaction and confirmed the A1-domain of VWF as strong interaction partner with a kD of 2.08 x 10^-10 M. In line with this, physiological cleavage of VWF A2-domain by the protease ADAMTS13 was marginally delayed but not prevented by enolase-binding to VWF. The second project part was focused on functional aspects. Microscopic immunofluorescence analyses of cell culture infections demonstrated that globular VWF significantly increased pneumococcus adhesion to primary endothelial cells in dose-dependent and heparin-sensitive manner, whereas bacterial internalization remained unaffected. In complementary analyses, the interaction of the enolase with extracellular nucleic acids again promoted pneumococcus attachment and internalization in endothelial and epithelial cells. Likewise, the generation of multimerized VWF strings on the surface of primary endothelial cells was monitored using a microfluidic cell culture system, which simulated the vascular shear stress in the human host. Immunofluorescence microscopy analyses and live cell microscopy identified the VWF strings as stable attachment sites for pneumococci. To assess pathophysiological phenotypes of the VWF-pneumococcus interaction in an in vivo system, a zebrafish infection model was established. Microscopic monitoring detected a profound VWF-mediated bacterial aggregation in zebrafish larvae. Such aggregate formation might trigger the development of vascular complications post pneumococcus pneumoniae. Additional functional analyses demonstrated that plasmin activity and fibrin degradation were significantly reduced in presence of enolase-coupled particles incubated with VWF. These results provide strong evidence that VWF-binding to enolase inhibits further interaction of enolase with plasminogen. We conclude that the strong Enolase-VWF interaction enhances vascular attachment of pneumococci, promotes bacterial aggregation in vivo, and weakens the interaction with plasminogen, which supports the assumption that pneumococcus-VWF interaction shifts the hemostatic balance towards an enhanced thrombotic coagulation.
Publications
- (2019) Von Willebrand Factor Mediates Pneumococcal Aggregation and Adhesion in Blood Flow. Frontiers in microbiology 10 511
Jagau, Hilger; Behrens, Ina-Kristin; Lahme, Karen; Lorz, Georgina; Köster, Reinhard W.; Schneppenheim, Reinhard; Obser, Tobias; Brehm, Maria A.; König, Gesa; Kohler, Thomas P.; Rohde, Manfred; Frank, Ronald; Tegge, Werner; Fulde, Marcus; Hammerschmidt, Sv
(See online at https://doi.org/10.3389/fmicb.2019.00511) - From Single Cells to Engineered and Explanted Tissues: New Perspectives in Bacterial Infection Biology. International Review of Cell & Molecular Biology (IRCMB), 319: 1-44
Bergmann, S. and Steinert, M.
(See online at https://doi.org/10.1016/bs.ircmb.2015.06.003) - Host-derived extracellular RNA promotes adhesion of Streptococcus pneumoniae to endothelial and epithelial cells. Scientific Reports, 28(6): 37758
Zakrzewicza, D., Bergmann, S.a, Didiasova, M., Giaimo, B. D., Borggrefe, T., Mieth, M., Hocke, A. C., Lochnit, G., Preissner, K. T., Wygrecka M.
(See online at https://doi.org/10.1038/srep37758)
