Physiological Concentrations of Calcium Interact with Alginate and Extracellular DNA in the Matrices of Pseudomonas aeruginosa Biofilms to Impede Phagocytosis by Neutrophils
Physiological Concentrations of Calcium Interact with Alginate and Extracellular DNA in the Matrices of Pseudomonas aeruginosa Biofilms to Impede Phagocytosis by Neutrophils
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DOI：
10.1021/acs.langmuir.3c01637
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发表时间：
2023-11-16
期刊：
影响因子：
3.9
通讯作者：
Gordon，Vernita D.
中科院分区：
文献类型：
--
作者：
Wells，Marilyn J.;Currie，Hailey;Gordon，Vernita D.
生物膜是嵌入聚合物、蛋白质和其他材料基质中的相互作用的微生物群落。生物膜形成取决于其主要基质组分的独特机械特性。铜绿假单胞菌（P. aeruginosa）是一种人类病原体，能形成强大的生物膜，广泛耐受抗生素，有效地逃避免疫系统的清除。两个重要的细菌产生的聚合物的基质中的P。在一些实施方案中，厌氧生物膜是藻酸盐和细胞外DNA（eDNA），两者都是阴离子的，因此具有与阳离子静电相互作用的潜力。许多感染的生理部位含有显著浓度的钙离子（Ca2+）。在这项研究中，我们研究了Ca2+补充对体外生长的藻酸盐为主的生物膜的结构和机械影响，并评估了靶向酶处理对免疫细胞清除的影响。我们使用多粒子跟踪微流变学来评估由藻酸盐裂解酶或DNA酶I处理引起的生物膜粘弹性的变化。对于生长无Ca2+的生物膜，我们将相对弹性的降低与吞噬成功率的增加相关联。然而，我们发现，与Ca2+补充生长破坏这种相关性，除了在两种酶都适用的情况下。这表明钙阳离子可能以非平凡的方式影响生物膜的微观结构。事实上，共聚焦激光扫描荧光显微镜和扫描电子显微镜揭示了独特的Ca2+依赖的eDNA和藻酸盐微结构。我们的研究结果表明，Ca2+的存在下，驱动形成的结构和成分离散的微区内的生物膜通过静电相互作用与阴离子基质成分eDNA和藻酸盐。此外，我们观察到，这些结构起到保护作用，因为这两种组分的溶解是使生物膜细菌易于被中性粒细胞吞噬所必需的。
Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infectionsin vivo, incorporated from the host environment.Pseudomonas aeruginosa(P. aeruginosa) is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices ofP. aeruginosabiofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study, we investigate the structural and mechanical impacts of Ca2+supplementation in alginate-dominated biofilms grownin vitro, and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple-particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase or DNase I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in nontrivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils.