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.
为了使血管内感染和感染性心内膜炎,细菌必须能够粘附于血管壁,同时被暴露于流动的血液的剪切应力。
要识别有助于微生物血管粘附的细菌和宿主因素,需要的研究生理剪切条件下,这些相互作用合适的模型。在这里,我们描述了一种在体外流动室模型,允许调查细菌粘附到细胞外基质的不同成分或与内皮细胞,并开发了一个活体显微镜模型直接可视化的细菌的初始粘合力,以在体内内脏循环。这些方法可用于鉴定所需细菌下流动的粘附的细菌和宿主因素。我们示出了剪切应力的相关性和血管性血友病因子为Staphy的粘合作用黄色葡萄球菌同时使用体外和体内模型。
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.
剪应力是为早期细菌粘附于血管壁和用于随后产生的血管内或心内膜赘生物和转移性感染4,5的一个关键因素。我们的体外和体内模型中所述互补研究生理剪切应力下的血管内感染的发病机制。这些模型使我们能够确定血管性血友病因子结合蛋白(vWbp)作为主要的S.金黄色葡萄球菌蛋白流下互动与一个受伤的血管壁露出VWF 4。
血管内感染,尤其是感染性心内膜炎,值得关注,不仅是因为败血症所致的器官衰竭而死亡,但由于也本地和遥远('转移')的并发症。引起感染性心内膜炎和转移性感染,细菌有附着到容器壁上,从而抗蚀剂流动的血液的剪切应力。最对细菌的研究毒力因子已经在静态条件下被执行。然而,这些相互作用建立可能无法承受剪切力,并研究流量条件下,可以揭示新的,以前没有认识到的因素在细菌宿主相互作用。
使用微平行流动室,我们和其他人已经表明的VWF对血管粘附的重要性。在剪切应力,VWF逐步展现出来的,从它的休息球状结构,并公开通过其受体GPIb的血小板6交互的A1域。流动腔室已被广泛用来研究血小板功能7。
值得注意的是,还S.下流动葡萄球菌粘附需要的VWF,以及尤其是在剪切露出的A1结构域。我们确定vWbp调解VWF结合。 vWbp是凝固酶,有助于S.金黄色葡萄球菌病理生理通过激活宿主的凝血酶原。 Staphylothrombin,水库ulting细菌凝固酶和凝血酶原复合物,纤维蛋白原转变成不溶性纤维蛋白8,9。我们的研究表明,vWbp不仅激活凝血酶原,但触发细菌血纤维蛋白-血小板聚集,这提高下流动4,10,11的粘附到血管的形成。
体外流室模型允许不同的球员 在细菌粘附研究细胞或基质成分。细菌毒力因子可以通过使用突变体或无害的细菌表达特定表面蛋白进行研究。可替代地,药理抑制剂或阻断抗体可以被添加到在流动室中的介质。的宿主因素如细胞外基质的不同成分的作用可以通过使用盖玻片与不同涂层进行研究。盖玻片也可覆盖有内皮细胞,其中的活化状态可以通过添加特异性刺激进行调制。阿帕室温从血管壁,宿主血细胞和血浆蛋白质的贡献可以通过加入这些因素,流动介质进行研究。因此,增加了复杂性不同的条件可以在层流的标准条件进行研究,以解开,允许细菌附着于血管壁体内的相互作用。
在体外模型中鉴定相互作用随后研究的动物模型,以测试其相关性在一个复杂的有机体。其它体内模型来研究下流动的动态相互作用已被描述,如仓鼠背皮褶腔12和提睾模型13。相比较而言,这里所描述的肠系膜灌注模型提供,因为它易于使用的几个优点,有可能以改变承载小鼠的遗传背景,并评价药物干预。
总之,所描述的模型提供研究表面蛋白不仅S的可能性金黄色葡萄球菌 ,但在不同的主机背景许多其它微生物,以更好地理解血管感染的发病机制。
The authors have nothing to disclose.
这项工作是由该基金VOOR Wetenschappelijk Onderzoek(FWO)VLAANDEREN G0466.10,11I0113N支持; “埃迪墨克斯研究基金”和“Sporta研究经费”为小儿心脏病,UZ比利时鲁汶(JC);该中心分子和血管生物学由Programmafinanciering鲁汶(PF / 10/014)的支持下,由鲁汶大学的“Geconcentreerde Onderzoeksacties”(GOA一十三分之二千零九)和勃林格殷格翰研究经费。
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 |