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Von Willebrand factor mediates pneumococcal aggregation and adhesion in blood flow

Jagau, Hilger LU ; Behrens, Ina Kristin ; Lahme, Karen ; Lorz, Georgina ; Köster, Reinhard W. ; Schneppenheim, Reinhard ; Obser, Tobias ; Brehm, Maria A. ; König, Gesa and Kohler, Thomas P. , et al. (2019) In Frontiers in Microbiology 10.
Abstract

Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture... (More)

Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture infection analyses with primary endothelial cells characterized VWF as bridging molecule that mediates bacterial adherence to endothelial cells in a heparin-sensitive manner. Due to the mechanoresponsive changes of the VWF protein conformation and multimerization status, which occur in the blood stream, we used a microfluidic pump system to generate shear flow-induced multimeric VWF strings on endothelial cell surfaces and analyzed attachment of RFP-expressing pneumococci in flow. By applying immunofluorescence visualization and additional electron microscopy, we detected a frequent and enduring bacterial attachment to the VWF strings. Bacterial attachment to the endothelium was confirmed in vivo using a zebrafish infection model, which is described in many reports and acknowledged as suitable model to study hemostasis mechanisms and protein interactions of coagulation factors. Notably, we visualized the recruitment of zebrafish-derived VWF to the surface of pneumococci circulating in the blood stream and detected a VWF-dependent formation of bacterial aggregates within the vasculature of infected zebrafish larvae. Furthermore, we identified the surface-exposed bacterial enolase as pneumococcal VWF binding protein, which interacts with the VWF domain A1 and determined the binding kinetics by surface plasmon resonance. Subsequent epitope mapping using an enolase peptide array indicates that the peptide 181YGAEIFHALKKILKS195 might serve as a possible core sequence of the VWF interaction site. In conclusion, we describe a VWF-mediated mechanism for pneumococcal anchoring within the bloodstream via surface-displayed enolase, which promotes intravascular bacterial aggregation.

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Contribution to journal
publication status
published
subject
keywords
Endothelium, Enolase, Streptococcus pneumoniae, Von Willebrand factor, Zebrafish
in
Frontiers in Microbiology
volume
10
article number
511
publisher
Frontiers
external identifiers
  • pmid:30972039
  • scopus:85066434042
ISSN
1664-302X
DOI
10.3389/fmicb.2019.00511
language
English
LU publication?
no
id
c9dc3096-6c75-4ade-94c3-23f554dea8a5
date added to LUP
2020-09-23 15:37:14
date last changed
2020-10-20 02:22:35
@article{c9dc3096-6c75-4ade-94c3-23f554dea8a5,
  abstract     = {<p>Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture infection analyses with primary endothelial cells characterized VWF as bridging molecule that mediates bacterial adherence to endothelial cells in a heparin-sensitive manner. Due to the mechanoresponsive changes of the VWF protein conformation and multimerization status, which occur in the blood stream, we used a microfluidic pump system to generate shear flow-induced multimeric VWF strings on endothelial cell surfaces and analyzed attachment of RFP-expressing pneumococci in flow. By applying immunofluorescence visualization and additional electron microscopy, we detected a frequent and enduring bacterial attachment to the VWF strings. Bacterial attachment to the endothelium was confirmed in vivo using a zebrafish infection model, which is described in many reports and acknowledged as suitable model to study hemostasis mechanisms and protein interactions of coagulation factors. Notably, we visualized the recruitment of zebrafish-derived VWF to the surface of pneumococci circulating in the blood stream and detected a VWF-dependent formation of bacterial aggregates within the vasculature of infected zebrafish larvae. Furthermore, we identified the surface-exposed bacterial enolase as pneumococcal VWF binding protein, which interacts with the VWF domain A1 and determined the binding kinetics by surface plasmon resonance. Subsequent epitope mapping using an enolase peptide array indicates that the peptide <sup>181</sup>YGAEIFHALKKILKS<sup>195</sup> might serve as a possible core sequence of the VWF interaction site. In conclusion, we describe a VWF-mediated mechanism for pneumococcal anchoring within the bloodstream via surface-displayed enolase, which promotes intravascular bacterial aggregation.</p>},
  author       = {Jagau, Hilger and Behrens, Ina Kristin and Lahme, Karen and Lorz, Georgina and Köster, Reinhard W. and Schneppenheim, Reinhard and Obser, Tobias and Brehm, Maria A. and König, Gesa and Kohler, Thomas P. and Rohde, Manfred and Frank, Ronald and Tegge, Werner and Fulde, Marcus and Hammerschmidt, Sven and Steinert, Michael and Bergmann, Simone},
  issn         = {1664-302X},
  language     = {eng},
  publisher    = {Frontiers},
  series       = {Frontiers in Microbiology},
  title        = {Von Willebrand factor mediates pneumococcal aggregation and adhesion in blood flow},
  url          = {http://dx.doi.org/10.3389/fmicb.2019.00511},
  doi          = {10.3389/fmicb.2019.00511},
  volume       = {10},
  year         = {2019},
}