&lt;em&gt;In Vitro&lt;/em&gt; and &lt;em&gt;In Vivo&lt;/em&gt; Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

Claes, Jorien; Liesenborghs, Laurens; Lox, Marleen; Verhamme, Peter; Vanassche, Thomas; Peetermans, Marijke

doi:10.3791/52862

Cited by 12 publications

(19 citation statements)

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“…In vitro perfusion experiments were performed as previously described . Glass coverslips (24 × 50 mm, VWR International, Leuven, Belgium) were coated with 50 μg mL −1 VWF (Haemate P, CSL Behring, Mechelen, Belgium), 200 μg mL −1 Horm collagen (Takeda, Linz, Austria), 30 μg mL −1 rvWbp or 50 μg mL −1 VWF A1‐domain in a humidified container at room temperature for 4 h. The coverslips were mounted in a micro‐parallel flow chamber and perfused for 10 min with a high‐accuracy Harvard pump (PHD 2000 Infusion, Harvard Apparatus, Holliston, MA, USA) generating flow rates of 1000 s −1 .…”

Section: Methodsmentioning

confidence: 99%

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Clumping factor A, von Willebrand factor‐binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall

Claes

Liesenborghs

Peetermans

et al. 2017

Journal of Thrombosis and Haemostasis

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Summary. Objective: When establishing endovascular infections, Staphylococcus aureus (S. aureus) overcomes shear forces of flowing blood by binding to von Willebrand factor (VWF). Staphylococcal VWF-binding protein (vWbp) interacts with VWF, but it is unknown how this secreted protein binds to the bacterial cell wall. We hypothesized that vWbp interacts with a staphylococcal surface protein, mediating the adhesion of S. aureus to VWF and vascular endothelium under shear stress. Methods: We studied the binding of S. aureus to vWbp, VWF and endothelial cells in a micro-parallel flow chamber using various mutants deficient in Sortase A (SrtA) and SrtA-dependent surface proteins, and Lactococcus lactis expressing single staphylococcal surface proteins. In vivo adhesion of bacteria was evaluated in the murine mesenteric circulation using real-time intravital vascular microscopy. Results: vWbp bridges the bacterial cell wall and VWF, allowing shear-resistant binding of S. aureus to inflamed or damaged endothelium. Absence of SrtA and Clumping factor A (ClfA) reduced adhesion of S. aureus to vWbp, VWF and activated endothelial cells. ADAMTS-13 and an anti-VWF A1 domain antibody, when combined, reduced S. aureus adhesion to activated endothelial cells by 90%. Selective overexpression of ClfA in the membrane of Lactococcus lactis enabled these bacteria to bind to VWF and activated endothelial cells but only in the presence of vWbp. Absence of ClfA abolished bacterial adhesion to the activated murine vessel wall. Conclusions: vWbp interacts with VWF and with the SrtA-dependent staphylococcal surface protein ClfA. The complex formed by VWF, secreted vWbp and bacterial ClfA anchors S. aureus to vascular endothelium under shear stress.

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Section: Methodsmentioning

confidence: 99%

“…Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords of healthy donors as described before . HUVECs were seeded on 1% gelatin‐coated (Sigma‐Alderich) plastic coverslips (Sarstedt, Nümbrecht, Germany) and grown to confluence.…”

Section: Methodsmentioning

confidence: 99%

Clumping factor A, von Willebrand factor‐binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall

Claes

Liesenborghs

Peetermans

et al. 2017

Journal of Thrombosis and Haemostasis

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“…[14][15][16] In catch bonding, FimH ligands are bound more tightly as a consequence of an allosteric process, in which FimH adopts a conformation of increased mannose affinity. [17,18] Bacterial adhesion under shear stress has been investigated since some time [19][20][21][22] and recently the influence of shear stress on…”

Section: Introductionmentioning

confidence: 99%

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow

Möckl

Fessele

Despras

et al. 2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…Cell culture-based static infection models are widely accepted in cellular microbiology and pathogenicity research. Recently, dynamic cellular and live murine infection models have been applied for analyzing the interaction of bacteria with ECs in vitro or using intravital microscopy, e.g., for B. henselae, Bartonella quintana, Yersinia enterocolitica, Neisseria meningitidis, Borrelia burgdorferi, and Staphylococcus aureus (6,(35)(36)(37)(38). Although these methods are of high importance in the field of pathogenicity research, they suffer from some shortcomings by nature: (i) dynamic in vitro adherence assays are restricted to one particular cell line in an artificial experimental environment (lacking the complexity of natural human tissues; e.g., cell-cell-adhesions, extracellular matrices, mixed cell types, elasticity of human blood vessels) and (ii) cell growth conditions (addition of growth factors, fetal calf serum, etc.)…”

Section: Discussionmentioning

confidence: 99%

Analysis of Endothelial Adherence of Bartonella henselae and Acinetobacter baumannii Using a Dynamic Human Ex Vivo Infection Model

et al. 2016

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f Bacterial adherence determines the virulence of many human-pathogenic bacteria. Experimental approaches elucidating this early infection event in greater detail have been performed using mainly methods of cellular microbiology. However, in vitro infections of cell monolayers reflect the in vivo situation only partially, and animal infection models are not available for many human-pathogenic bacteria. Therefore, ex vivo infection of human organs might represent an attractive method to overcome these limitations. We infected whole human umbilical cords ex vivo with Bartonella henselae or Acinetobacter baumannii under dynamic flow conditions mimicking the in vivo infection situation of human endothelium. For this purpose, methods for quantifying endotheliumadherent wild-type and trimeric autotransporter adhesin (TAA)-deficient bacteria were set up. Data revealed that (i) A. baumannii binds in a TAA-dependent manner to endothelial cells, (ii) this organ infection model led to highly reproducible adherence rates, and furthermore, (iii) this model allowed to dissect the biological function of TAAs in the natural course of human infections. These findings indicate that infection models using ex vivo human tissue samples ("organ microbiology") might be a valuable tool in analyzing bacterial pathogenicity with the capacity to replace animal infection models at least partially.T he analysis of adhesion to host cells is crucial for the elucidation of bacterial infection mechanisms. Animal infection models or cellular microbiology-based approaches are well established, and many important findings have thereby been gathered. By way of example, the protein injection machineries and the autotransporter adhesins (TAA) of Bartonella henselae and Acinetobacter baumannii have been elucidated in that manner (1-3). Although these methods are well established and widely accepted, they still are hampered by several limitations: animal models might reflect the course of human infections only partially (4) or might not even be available for many pathogens (e.g., B. henselae). Cellular infection models allow very detailed analyses of the interaction of bacteria with host cells under standardized conditions. However, they are usually done statically using cell monolayers, and this mimics the complexity of in vivo infection situation only to some extent. Methods to overcome the species barrier in infection models and to analyze host-pathogen interactions close to the dynamic human situation are urgently needed. Ex vivo infection models, using human organ grafts, might represent an attractive alternative or addition to animal or cellular microbiology experiments.B. henselae causes cat scratch disease and endocarditis, whereas immunosuppressed individuals can suffer from vasculoproliferative disorders such as bacillary angiomatosis. B. henselae is believed to be an endotheliotropic pathogen. Bartonella adhesin A (BadA) has been identified as the key factor involved in the adherence to endothelial cells (ECs) and to extracellular matrix (EC...

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<em>In Vitro</em> and <em>In Vivo</em> Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

Cited by 12 publications

References 13 publications

Clumping factor A, von Willebrand factor‐binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall

Clumping factor A, von Willebrand factor‐binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow

Analysis of Endothelial Adherence of Bartonella henselae and Acinetobacter baumannii Using a Dynamic Human Ex Vivo Infection Model

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