Evans Dominique C S, Khamas Amanda B, Payne-Dwyer Alex, Wollman Adam J M, Rasmussen Kristian S, Klitgaard Janne K, Kallipolitis Birgitte, Leake Mark C, Meyer Rikke L
School of Physics, Engineering and Technology, University of York, York, UK.
Interdisciplinary Nanoscience Centre, Aarhus University, Aarhus, Denmark.
Biofilm. 2024 Oct 23;8:100233. doi: 10.1016/j.bioflm.2024.100233. eCollection 2024 Dec.
The major human pathogen forms biofilms comprising of a fibrin network that increases attachment to surfaces and shields bacteria from the immune system. It secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of this fibrin clot. However, it is unclear how Coa and vWbp contribute differently to the localisation and dynamics of clot assembly in growing biofilms. Here, we address this question using high-precision time-resolved confocal microscopy of fluorescent fibrin to establish the spatiotemporal dynamics of fibrin clot formation in functional biofilms. We also use fluorescent fusion proteins to visualise the locations of Coa and vWbp in biofilms using both confocal laser scanning and high resolution highly inclined and laminated optical sheet microscopy. We visualise and quantify the spatiotemporal dynamics of fibrin production during initiation of biofilms in plasma amended with fluorescently labelled fibrinogen. We find that human serum stimulates coagulase production, and that Coa and vWbp loosely associate to the bacterial cell surface. Coa localises to cell surfaces to produce a surface-attached fibrin pseudocapsule but can diffuse from cells to produce matrix-associated fibrin. vWbp produces matrix-associated fibrin in the absence of Coa, and furthermore accelerates pseudocapsule production when Coa is present. Finally, we observe that fibrin production varies across the biofilm. A sub-population of non-dividing cells does not produce any pseudocapsule but remains within the protective extended fibrin network, which could be important for the persistence of biofilm infections as antibiotics are more effective against actively growing cells. Our findings indicate a more cooperative role between Coa and vWbp in building fibrin networks than previously thought, and a bet-hedging cell strategy where some cells produce biofilm matrix while others do not, but instead assume a dormant phenotype that could be associated with antibiotic tolerance.
这种主要的人类病原体形成由纤维蛋白网络组成的生物膜，该网络增加了对表面的附着并保护细菌免受免疫系统的攻击。它分泌两种凝固酶，即凝固酶（Coa）和血管性血友病因子结合蛋白（vWbp），它们劫持宿主凝血级联反应并触发这种纤维蛋白凝块的形成。然而，尚不清楚Coa和vWbp如何对生长中的生物膜中凝块组装的定位和动态产生不同的影响。在这里，我们使用荧光纤维蛋白的高精度时间分辨共聚焦显微镜来解决这个问题，以确定功能性生物膜中纤维蛋白凝块形成的时空动态。我们还使用荧光融合蛋白，通过共聚焦激光扫描和高分辨率高倾斜和层叠光学片显微镜来观察生物膜中Coa和vWbp的位置。我们可视化并量化在用荧光标记的纤维蛋白原改良的血浆中生物膜形成起始阶段纤维蛋白产生的时空动态。我们发现人血清刺激凝固酶的产生，并且Coa和vWbp松散地与细菌细胞表面结合。Coa定位于细胞表面以产生表面附着的纤维蛋白假包膜，但可以从细胞扩散以产生与基质相关的纤维蛋白。vWbp在没有Coa的情况下产生与基质相关的纤维蛋白，并且当存在Coa时还会加速假包膜的产生。最后，我们观察到生物膜中纤维蛋白的产生各不相同。一部分不分裂的细胞不产生任何假包膜，但仍留在保护性的扩展纤维蛋白网络内，这对于生物膜感染的持续存在可能很重要，因为抗生素对活跃生长的细胞更有效。我们的研究结果表明，Coa和vWbp在构建纤维蛋白网络中发挥的协同作用比以前认为的更大，并且存在一种“风险对冲”细胞策略，即一些细胞产生生物膜基质，而另一些细胞则不产生，而是呈现出可能与抗生素耐受性相关的休眠表型。
