Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts
Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts
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DOI：
10.1128/iai.63.2.585-590.1995
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发表时间：
1995-02
影响因子：
3.1
通讯作者：
P. Vaudaux;Patrice François;R. A. Proctor;Damien McDevitt;Timothy J. Foster;Ralph M. Albrecht;D. Lew;Hugh Wabers;Stuart L. Cooper
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文献类型：
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作者：
P. Vaudaux;Patrice François;R. A. Proctor;Damien McDevitt;Timothy J. Foster;Ralph M. Albrecht;D. Lew;Hugh Wabers;Stuart L. Cooper
我们使用了离体犬动静脉分流模型，该模型先前开发用于研究聚合生物材料上的血浆蛋白吸附和血栓形成，以确定宿主蛋白在促进金黄色葡萄球菌粘附中的作用。将聚乙烯或聚氯乙烯管以 300 ml/min 的流速暴露于犬血液中 5、15 或 60 分钟，然后在磷酸盐缓冲盐水 (PBS) 中冲洗，切成 1.5 厘米的片段，并保存在 -70°C 下。解冻后，将每个片段在 PBS 中的 0.5% 白蛋白中预孵育，以防止非特异性葡萄球菌附着在未暴露的表面上到血。然后在体外粘附测定中，将每个节段与每毫升 4 x 10(6) CFU 的[3H]胸苷标记的金黄色葡萄球菌在 37 摄氏度下孵育 60 分钟。测试了金黄色葡萄球菌的两种位点特异性突变体：一种在与表面结合的纤连蛋白（FnAd-def）的粘附方面存在特异性缺陷，另一种在与纤维蛋白原（FgAD-def）的粘附方面存在缺陷[已校正]。与其各自的亲本菌株相比，金黄色葡萄球菌的 FgAd-def（而非 FnAd-def）突变体显示出与离体管道的附着强烈（> 80%）减少。每种金黄色葡萄球菌菌株在聚乙烯上的粘附始终比在暴露于流动血液 5 或 15 分钟的聚氯乙烯片段上的粘附高出两倍以上，但在暴露 60 分钟后，粘附变得与在聚氯乙烯上的粘附相似。总之，金黄色葡萄球菌的特异性粘附缺陷突变体表明纤维蛋白原是短期血液-物质相互作用中最活跃的粘附促进蛋白。本研究中描述的实验方法对于筛选被认为对蛋白质介导的葡萄球菌粘附和定植具有抵抗力的材料应该是有用的。
We used an ex vivo canine arteriovenous shunt model, previously developed to study plasma protein adsorption and thrombogenesis on polymeric biomaterials, to define the role of host proteins in promoting adhesion of Staphylococcus aureus. Either polyethylene or polyvinyl chloride tubings were exposed to canine blood for 5, 15, or 60 min at a flow rate of 300 ml/min and then were flushed in phosphate-buffered saline (PBS), cut into 1.5-cm segments, and stored at -70 degrees C. After thawing, each segment was preincubated in 0.5% albumin in PBS to prevent nonspecific staphylococcal attachment to surfaces that were not exposed to blood. Each segment was then incubated with 4 x 10(6) CFU of [3H]thymidine-labelled S. aureus per ml for 60 min at 37 degrees C in an in vitro adhesion assay. Two site-specific mutants of S. aureus were tested: one specifically defective in adhesion to surface-bound fibronectin (FnAd-def) and the other defective in adhesion to fibrinogen (FgAD-def) [corrected]. Compared with their respective parental strains, the FgAd-def, but not the FnAd-def, mutant of S. aureus showed a strong (> 80%) decrease in attachment to ex vivo tubings. The adhesion of each strain of S. aureus onto polyethylene was consistently more than twofold higher than the adhesion onto polyvinyl chloride segments exposed to flowing blood for 5 or 15 min, but adhesion became similar to that on polyvinyl chloride after 60 min of exposure. In conclusion, the specific adhesion-defective mutants of S. aureus suggested that fibrinogen was the most active adhesion-promoting protein in a short-term blood-material interaction. The experimental approach described in this study should prove useful for screening materials thought to be resistant to protein-mediated staphylococcal adhesion and colonization.