Musrati Ahmed Ali, Fteita Dareen, Paranko Jorma, Könönen Eija, Gürsoy Ulvi Kahraman
Department of Periodontology, Institute of Dentistry, University of Turku, Turku, Finland.
Department of Periodontology, Institute of Dentistry, University of Turku, Turku, Finland.
Anaerobe. 2016 Jun;39:31-8. doi: 10.1016/j.anaerobe.2016.02.008. Epub 2016 Feb 23.
We recently demonstrated that Fusobacterium nucleatum can resist to human neutrophil peptide (HNP)-1 by decreasing its membrane permeability and increasing its proliferation and biofilm formation. In this continuation study, we aimed to further evaluate and explain these resistance properties by determining the morphological and functional adaptations of F. nucleatum, using transmission electron microscopy (TEM).
Cultures of the type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. polymorphum AHN 9910 and ssp. nucleatum AHN 9508) were incubated without (0 μg/ml) or with four different test concentrations of recombinant HNP-1 (1, 5, 10 and 20 μg/ml). Membrane morphology and thickness, and cell (visualized by TEM), planktonic growth (measured in colony forming units), and biofilm formation (measured as total mass) were analyzed. Scrambled HNP-1 was used in planktonic growth and biofilm formation studies as a negative control.
TEM analyses revealed a decrease in the outer membrane surface corrugations and roughness of the strain AHN 9508 with increasing HNP-1 concentrations. In higher concentrations of HNP-1, the strain AHN 9910 showed thicker outer membranes with a number of associated rough vesicles attached to the outer surface. Intracellular granules became increasingly visible in the strain ATCC 25586 with increasing peptide concentrations. With increased concentrations of HNP-1, planktonic growth of the two clinical strains was significantly enhanced (P < 0.001) and of the type strain significantly suppressed (P < 0.01). HNP-1 decreased the biofilm formation of the two clinical strains, AHN 9910 (P < 0.01) and 9508 (P < 0.001) significantly. Scrambled HNP-1 showed no effect on planktonic growth or biofilm formation of the tested strains.
F. nucleatum has the ability to withstand the lethal effects of HNP-1, and the ultrastructural changes on bacterial membrane and cytoplasm may play role in this adaptive process.
我们最近证明具核梭杆菌可通过降低膜通透性、增加增殖及生物膜形成来抵抗人中性粒细胞肽（HNP）-1。在这项后续研究中，我们旨在通过透射电子显微镜（TEM）确定具核梭杆菌的形态和功能适应性，以进一步评估和解释这些抗性特性。
将具核梭杆菌（核亚种ATCC 25586）的模式菌株培养物以及两株临床菌株（多形亚种AHN 9910和核亚种AHN 9508）在无（0 μg/ml）或含有四种不同测试浓度的重组HNP-1（1、5、10和20 μg/ml）的条件下培养。分析膜的形态和厚度以及细胞（通过TEM观察）、浮游生长（以菌落形成单位测量）和生物膜形成（以总质量测量）。在浮游生长和生物膜形成研究中使用乱序HNP-1作为阴性对照。
TEM分析显示，随着HNP-1浓度增加，AHN 9508菌株外膜表面的波纹和粗糙度降低。在较高浓度的HNP-1作用下，AHN 9910菌株显示外膜更厚，外表面附着有许多相关的粗糙囊泡。随着肽浓度增加，ATCC 25586菌株中的细胞内颗粒变得越来越明显。随着HNP-1浓度增加，两株临床菌株的浮游生长显著增强（P < 0.001），而模式菌株的浮游生长显著受到抑制（P < 0.01）。HNP-1显著降低了两株临床菌株AHN 9910（P < 0.01）和9508（P < 0.001）的生物膜形成。乱序HNP-1对受试菌株的浮游生长或生物膜形成无影响。
具核梭杆菌具有抵抗HNP-1致死作用的能力，细菌膜和细胞质的超微结构变化可能在这一适应性过程中起作用。
