To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.
In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.
To establish endovascular infections, pathogens require a mechanism to adhere to the endothelium, which lines the vessel wall and the inner surface of the heart, and to persist and establish an infection despite being exposed to the shear stress of rapidly flowing blood. The most frequent pathogen causing life-threatening endovascular infections and infective endocarditis is Staphylococcus aureus (S. aureus)1.
Various bacterial surface-bound adhesive molecules mediate adhesion to host tissue by interacting with extracellular matrix components. These MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) recognize molecules such as fibronectin, fibrinogen, collagen and von Willebrand factor (VWF). MSCRAMMs are important virulence factors of S. aureus and are implicated in the colonization and invasion of the host2. Most studies on these virulence factors have been performed in static conditions, and thus may not be representative for human infections where initial adhesion of the bacteria occurs in flowing blood.
In the case of bloodstream infections, bacteria need to overcome the shearing forces of flowing blood in order to attach to the vessel wall. Models that investigate the interaction between bacteria and endothelium or subendothelium under flow conditions are therefore of particular interest.
A recent study showed that the adhesion of S. aureus to blood vessels under shear stress is mediated by VWF3. VWF, a shear stress-operational protein, is released from endothelial cells upon activation. Circulating VWF binds to collagen fibers of the exposed subendothelial matrix. Our group reported that the von Willebrand factor-binding protein (vWbp) of S. aureus is crucial for shear-mediated adhesion to VWF4.
In this article, we present an in vitro flow chamber model where bacterial adhesion to different components of the extracellular matrix or to endothelial cells can be evaluated. To validate the findings from in vitro data, we have developed an in vivo model that visualizes and quantifies the direct interaction of bacteria with the vessel wall and the formation of bacteria-platelet thrombi in the mesenteric circulation of mice, using real-time intravital vascular microscopy.
Animal experiments were approved by the Ethical Committee of the KU Leuven.
1. Preparing Bacteria for In Vitro Perfusions and In Vivo Experiments
2. In Vitro Perfusion Experiments
3. In Vivo Mesenteric Perfusion Model
S. aureus adhesion to VWF, subendothelial matrix and endothelial cells is a shear stress dependent phenomenon
To emphasize the role of shear stress in the interaction between S. aureus and VWF, we performed perfusions over VWF coated coverslips at different shear rates (a schematic overview of the in vitro perfusion model is given in Figure 1. Adhesion of S. aureus to VWF increased with increasing shear rates from 250 sec-1 to 2,000 sec-1 (Figure 2), indicating that high shear forces do not inhibit but reinforce the adhesion of bacteria to VWF.
In order to investigate the contribution of VWF to bacterial adhesion to collagen, the main component of the subendothelial matrix, we perfused fluorescently labeled S. aureus over collagen in the presence or absence of VWF. In the absence of VWF, adhesion of S. aureus to collagen decreased with increasing shear rates. However, when VWF was present in the medium, the adhesion of S. aureus increased with increasing shear rates (Figure 3).
The in vitro flow model also allows us to examine the adhesion of bacteria to endothelial cells under flow. We perfused HUVECs with fluorescently labeled S. aureus at shear rates from 500 to 2,000 sec-1. Where indicated, HUVECs were activated with a Ca2+-ionophore, to cause release of VWF. Endothelial cell activation and the subsequent VWF release, increased adhesion of S. aureus (Figure 4A), which formed typical “string-like” patterns of fluorescently labeled bacterial clusters aligned in the direction of the shear force (Figure 4B), suggesting the binding of bacteria along a linear-stretched VWF molecule.
Initial in vivo bacterial adhesion in splanchnic veins is mediated by VWF
Since S. aureus is able to adhere to VWF, we used wildtype mice (Vwf+/+) and VWF-deficient mice (Vwf−/−) to investigate bacterial adhesion to the activated vessel wall in vivo. Real-time videomicroscopy of splanchnic veins allowed the in vivo visualization of circulating fluorescently labeled S. aureus (Schematic overview of the in vivo perfusion model is represented in Figure 5).
After pharmacological activation of the endothelium by the Ca2+-ionophore, we observed rapid local accumulation of individual bacteria and aggregates of bacteria to the vessel wall of WT mice (supplemental Videos 1 and 2). Almost no adhesion of bacteria was observed on the activated vessel wall of Vwf-deficient mice (supplemental Video 3) compared with adhesion in WT mice (Figure 6). The absence of VWF decreases the ability of S. aureus to adhere to the activated vessel wall.
Figure 1. A schematic representation of the in vitro flow model. The in vitro flow model is a multifunctional model, which allows the study of different shear dependent mechanisms such as bacterial adhesion to the subendothelial matrix but also thrombus formation. The micro-parallel flow chamber is placed on a coverslip (plastic or glass) with different coatings of proteins and endothelial cells. The adhesion of different bacteria (orange and grey dots) can be analyzed, and the impact of the presence of plasma proteins, platelets and whole blood can be evaluated. Fluorescent markers for platelets (blue ovals) or fibrinogen (blue strings) can be used in combination with different inhibitors (black ovals) to distinguish bacterial and host factors. Representative images of bacterial adhesion of S. aureus to collagen coating in the presence (bottom) or absence (top) of VWF are shown (scale bar is 100 µm). Please click here to view a larger version of this figure.
Figure 2. Adhesion of S. aureus to VWF increases with increasing shear rates. Micro-parallel flow chamber perfusion over coated VWF (50 µg/ml) with fluorescently labeled S. aureus Newman at shear rates of 250 to 2,000 sec-1 (sec-1) in medium (n >5). All results are expressed as mean ± SEM. *p <0.05, **p <0.01.
Figure 3. Adhesion of S. aureus to subendothelium is shear and VWF dependent. Micro-parallel flow chamber perfusion over coated collagen (160 µg/ml) with fluorescently labeled S. aureus Newman at shear rates of 250 to 2,000 sec-1 in medium (n >5). VWF (60 µg/ml) was present in the medium where indicated. All results are expressed as mean ± SEM. **p <0.01.
Figure 4. Adhesion of S. aureus to activated endothelial cells is shear dependent. Micro-parallel flow chamber perfusion over endothelial cells. (A) Human umbilical vein endothelial cells were activated with the Ca2+-ionophore A23187 (0.1 mM) followed by a 10 min perfusion of fluorescently labeled S. aureus Newman at shear rates of 500 to 2,000 sec-1 in medium (n >5). All results are expressed as mean ± SEM. *p <0.05. (B) Image of micro-parallel flow chamber perfusion over activated HUVECs with S. aureus at a shear rate of 1,000 sec-2. S. aureus forms strings of ± 200 microns length, suggesting adhesion to VWF multimers (scale bar is 100 µm). Please click here to view a larger version of this figure.
Figure 5. A schematic overview of the in vivo mesenteric perfusion model. A right jugular vein catheter (yellow line) is inserted for the administration of fluorescently labeled bacteria (orange dots), additional anesthetics or other components such as pharmaceutical inhibitors and antibodies. The peritoneal cavity is opened and the mesenterium is spread to visualize the blood vessels (venous and arterial) under a fluorescence microscope. After pharmacological activation of the endothelium by a Ca2+-ionophore, which induces the release of VWF, bacteria can be injected through the jugular vein catheter. Real-time intravascular video microscopy allows the in vivo visualization of circulating fluorescently labeled bacteria and the resulting formation of bacteria-platelet thrombi. Please click here to view a larger version of this figure.
Figure 6. The initial adhesion of S. aureus to activated endothelium in vivo is mediated by VWF. In vivo venous mesenteric perfusion model with C57Bl/6-Vwf+/+ and C57Bl/6-Vwf-/- mice. Adhesion of fluorescently labeled S. aureus to the locally activated vessel wall is significantly lower in Vwf-/- mice. All results are expressed as mean ± SEM. ***p <0.001, n >7.
Video 1: Real-time adhesion of S. aureus to activated vessel wall in Vwf+/+ mice. Please click here to view this video.
Video 2: Real-time aggregate formation and embolization of S. aureus in Vwf+/+ mice. Please click here to view this video.
Video 3: Real-time adhesion of S. aureus to activated vessel wall in Vwf-/- mice. In vivo mesenteric perfusion model with Vwf+/+ and Vwf-/- mice. Five µl of a Ca2+-ionophore (10 mM) was applied to the region of the visualized vascular bed. A suspension of carboxy-fluorescein-labeled S. aureus was injected through the jugular catheter. The mesenteric circulation was visualized under an inverted microscope. Please click here to view this video.
Shear stress is a crucial factor for the early bacterial adhesion to the vessel wall and for the subsequent generation of endovascular or endocardial vegetations and metastatic infections4,5. We described complementary in vitro and in vivo models to study the pathogenesis of endovascular infections under physiological shear stress. These models have allowed us to identify von Willebrand factor-binding protein (vWbp) as the major S. aureus protein to interact under flow with an injured vascular wall exposing VWF4.
Endovascular infections, and infective endocarditis in particular, are of concern not only because of sepsis-induced organ failure and death, but also because of local and distant (‘metastatic’) complications. To cause infective endocarditis and metastatic infections, bacteria have to adhere to the vessel wall and thus resist the shear stress of flowing blood. Most studies on bacteria virulence factors have been performed in static conditions. However, these established interactions might not withstand shear forces and studies under flow conditions can reveal new, previously unrecognized factors in bacteria-host interplay.
Using the micro-parallel flow chamber, we and others have shown the importance of VWF for vascular adhesion. Under shear stress, VWF progressively unfolds from its resting globular structure, and exposes the A1 domain that interacts with platelets via its GPIb receptor6. Flow chambers have been extensively used to study platelet function7.
Remarkably, also S. aureus adhesion under flow requires VWF, and in particular the A1 domain that is exposed upon shear. We identified vWbp to mediate VWF binding. vWbp is a coagulase that contributes to S. aureus pathophysiology by activating the host’s prothrombin. Staphylothrombin, the resulting complex of a bacterial coagulase and prothrombin, converts fibrinogen into insoluble fibrin8,9. Our studies have shown that vWbp does not only activate prothrombin, but triggers the formation of bacteria-fibrin-platelet aggregates, which enhance the adhesion to blood vessels under flow4,10,11.
The in vitro flow chamber model allows to study the different players in bacterial adhesion to cellular or matrix components. Bacterial virulence factors can be studied by using mutants or innocuous bacteria expressing specific surface proteins. Alternatively, pharmacologic inhibitors or blocking antibodies can be added to the medium in the flow chamber. The role of host factors such as different constituents of extracellular matrix can be studied by using coverslips with different coatings. The coverslips can also be covered with endothelial cells, of which the activation status can be modulated by adding specific stimulators. Apart from the vascular wall, the contribution of host blood cells and plasma proteins can be studied by adding these factors to the flowing medium. Thus, different conditions of increasing complexity can be studied under standardized conditions of laminar flow to unravel the interactions that allow bacteria to adhere to the vessel wall in vivo.
Interactions identified in the in vitro model are subsequently studied in an animal model to test their relevance in a complex organism. Other in vivo models to study dynamic interactions under flow have been described, such as the hamster dorsal skinfold chamber12 and the cremaster model13. In comparison, the mesenteric perfusion model described here offers several advantages because of its ease of use, the possibility to vary host genetic background of the mice and to evaluate pharmacological interventions.
In conclusion, the described models offer the possibility to study surface proteins not only of S. aureus, but of many other microorganisms in different host backgrounds, to better understand the pathogenesis of vascular infections.
The authors have nothing to disclose.
This work was supported by the Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen G0466.10, 11I0113N; “Eddy Merckx Research Grant” and the “Sporta research Grant” for Pediatric Cardiology, UZ Leuven, Belgium (J.C.); the Center for Molecular and Vascular Biology is supported by the Programmafinanciering KU Leuven (PF/10/014), by the “Geconcentreerde Onderzoeksacties” (GOA 2009/13) from the University of Leuven and a research grant from Boehringer-Ingelheim.
Brain Heart Infusion (BHI) | BD Plastipak | 237500 | |
Tryptic Soy Broth (TSB) | Oxoid | CM0129 | |
Phosphate Buffered Saline (PBS) | Invitrogen | 14190-169 | D-PBS |
5(6)-carboxy-fluorescein N-hydroxysuccinimidyl ester | Sigma-Aldrich | 21878-25MG-F | fluorescent labeling |
Bovine Serum Albumin Fraction V (BSA) | Roch | 10 735 086 001 | |
Haemate-P | CSL Behring | PL 15036/0010 | VWF |
Horm collagen | Takeda | 10500 | collagen |
1-well PCA cell culture chambers | Sarstedt | ######## | plastic slips |
Temgesic | Reckitt Benckiser | 283716 | bruprenorphine |
Anesketin (Ketamin hydrochloride 115 mg/ml (100 mg/ml ketaminum)) | Eurovet | BE-V136516 | ketamin |
XYL-M 2% (xylazine hydrochloride 23.32 mg/ml (20 mg/ml xylazine)) | VMD Arendonk | BE-V170581 | xylazine |
2 french intravenous catheter green | Portex | 200/300/010 | |
0,9% Sodium chloride (NaCl) | Baxter Healthcare | W7124 | |
cotton swabs | International Medical Product | 300230 | |
Ca2+-ionophore solution A23187 | Sigma-Aldrich | C7522-10 MG | |
26 gauge 1 ml syringe | BD Plastipak | 300013 | |
26 gauge 1 ml syringe with needle | BD Plastipak | 300015 | intra-peritoneal injection |
Centrifuge 5810-R | Eppendorf | 5811 000.320 | |
Glass cover slips (24×50) | VWR | BB02405A11 | Thickness No, 1 |
PHD 2000 Infusion | Harvard Apparatus | 702100 | High-accuracy Harvard infusion pump |
Axio-observer DI | Carl-Zeiss | Inverted fluorescence microscope | |
ImageJ | National Institute of Health | Analysis software | |
Graphpad Prism 5,0 | Graphpad Software | Analysis software | |
AxioCam MRm | Carl-Zeiss | Black and white camera |