Interaction of Streptococcus pneumoniae with factor of the coagulation system

Streptococcus pneumoniae besiedelt den oberen Respirationstrakt des Menschen als opportunistisches Pathogen. Die auch als Pneumokokken bezeichneten Bakterien lösen vor allem bei Kindern, älteren Menschen und immunsupprimierten Personen bis zu 50% aller ambulant erworbenen Lungenentzündungen aus. Im Infektionsverlauf migriert der Erreger durch das alveoläre Lungenepithel und kann sich systemisch im Gefäßsystem ausbreiten. Die Folge sind Septikämien und Meningitiden. In dieser Arbeit konnte gezeigt werden, das klinische Pneumokokken Isolate das humane Glykoprotein „von Willebrand Faktor“ (VWF) binden. Hierbei handelt es sich um ein mechanoresponsives Protein der Hämostase. Durch Oberflächenplasmonresonanz-(SPR) und Microscale Thermophorese-Studien konnte die oberflächenassoziierte Enolase der Pneumokokken als bakterielles VWF-Bindungsprotein identifiziert werden und eine Dissoziationskonstante im nanomolaren Bereich ermittelt werden. Die Ergebnisse eines Peptid-Spot Arrays der Enolase gaben erste Hinweise auf ein mögliches VWF-Bindungsmotif, dass eine strukturelle Bindungsfurche in der oktameren Molekülstruktur der Enolase bildet. In Zellkultur-basierten Infektionsstudien konnte der VWF als Adhäsionskofaktor charakterisiert werden, welcher die bakterielle Anheftung an Endothelzellen in Heparin-sensitiver Weise vermittelt. In SPR-Studien konnte zudem eine starke Interaktion zwischen der immobilisierten Enolase und der heterolog exprimierten A1-Domäne des VWF bestätigt und näher charakterisiert werden. Um die VWF-Interaktion unter Scherstress-Bedingungen im Blutgefäßsystem simulieren zu können wurde ein Mikrofluidik System (ibidi®) etabliert. Die mikroskopische Visualisierung lies rot-fluoreszierende Pneumokokken erkennen, die an den VWF-Fäden im konstanten Scherstress von 10 dyn/cm² über einen Zeitraum von bis zu 25 min stabil adhärierten. Darüber hinaus rekrutierten Pneumokokken nach Injektion in 5-Tage alte Zebrafisch-Larven das endogene VWF auf ihrer Oberfläche und adhärierten in vivo an das Gefäßendothel. Nach VWF Präinkubation konnte die Bildung von Bakterien-Aggregaten im Gefäßsystem der Zebrafischlarven nachgewiesen werden. Diese Aggregate sind von hoher pathophysiologischer Relevanz, da sie zu thrombotischen Gefäßverschlüssen führen können. Insgesamt beschreiben die Ergebnisse dieser Arbeit eine neue, medizinisch hochrelevante Erreger-Wirt-Interaktion.Streptococcus pneumoniae can colonize the upper respiratory tract as a human opportunistic pathogen. Causing mild infections like otitis media or sinusitis, as well as community acquired pneumonia (CAP) Pneumococci are the infective agent in 50% of all pneumonia in children, elderly people and immune suppressed patients. During the infection, Pneumococci transmigrate through the alveolar epithelium and spread into the vascular system thereby causing septicaemia and meningitides. Results on this PhD thesis confirm the binding of the human glycoprotein “Von Willebrand factor” (VWF) to clinical Pneumococcus isolates. VWF is known as mechanoresponsive protein of the haemostasis. Surface-plasmon-resonanz- (SPR) and microscale thermophoresis-approaches identified the surface localized Enolase of S. pneumonia as VWF-binding protein and determined a dissociation constant within the nanomolar range. Peptide-spot-array results elucidated a putative VWF- binding motif, forming a structural binding-pocket at adjacent sides of two monomers of the octameric pneumococcal Enolase. In addition to the biochemical characterization, functional studies were performed to analyse pathophysiological consequences of the VWF binding of S. pneumoniae. Results of cell-culture infection analyses identified VWF as adhesion cofactor mediating pneumococcus adhesion to the endothelial cell surface in heparin-sensitive manner. SPR-analyses demonstrated a strong interaction between the immobilized Enolase and the heterologous expressed A1-domain of VWF. A microfluidic system (ibidi) was established to analyse the VWF-interaction in shear stress-condition comparable to the bloodstream. This system enabled the generation of multimerised VWF-strings on histamine-stimulated endothelia cells. Microscopic analyses visualized a stable, 25 min. lasting attachment of red fluorescent Pneumococci to VWF strings at a shear force of 10 dyne/cm². Moreover, the recruitment of zebrafish-derived VWF on the surface of pneumococci and bacterial adherence to the vascular endothelium was detected in in vivo-infection analyses of 5-day old zebrafisch larvae. After VWF preincubation, the formation of large bacterial aggregates within the vascular system of the larvae could be visualized. These aggregates are of high pathophysiological relevance, since they can cause thrombus-like vascular occlusion. In conclusion, the results of this thesis describe a new pathogen-host interaction of highest medical relevance

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells. The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes

Plasma Protein Layer Concealment Protects Streptococcus pyogenes From Innate Immune Attack

Early recognition and elimination of invading pathogens by the innate immune system, is one of the most efficient host defense mechanisms preventing the induction of systemic complications from infection. To this end the host can mobilize endogenous antimicrobials capable of killing the intruder by perforating the microbial cell wall. Here, we show that Streptococcus pyogenes can shield its outer surface with a layer of plasma proteins. This mechanism protects the bacteria from an otherwise lytic attack by LL-37 and extracellular histones, allowing the bacteria to adjust their gene regulation to an otherwise hostile environment

Von Willebrand Factor Mediates Pneumococcal Aggregation and Adhesion in Blood Flow.

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