Al Mamun Bhuyan Abdulla, Nguyen Minh-Thu, Bissinger Rosi, Götz Friedrich, Lang Florian
Cell Physiol Biochem. 2017;41(1):296-309. doi: 10.1159/000456147. Epub 2017 Jan 24.
BACKGROUND/AIMS: Consequences of bacterial infection include anemia, which could result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Bacterial components known to stimulate eryptosis include lipopeptides. Signaling mediating the triggering of eryptosis include increased cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and cellular accumulation of ceramide. The present study aimed to define the molecular requirements for lipopeptide-induced cell membrane scrambling.
Human erythrocytes were incubated for 48 hours in the absence and presence of 1 or 5 µg/ml of the synthetic lipopeptides Pam1 (lipopeptide with one fatty acid), Pam2 (lipopeptide with two fatty acids), or Pam3 (lipopeptide with three fatty acids). In the following phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fuorescence, and ceramide abundance utilizing specific antibodies.
Pam1 (5 µg/ml), Pam2 (5 µg/ml) and Pam3 (1 and 5 µg/ml) significantly increased the percentage of annexin-V-binding to erythrocytes in a dose dependent manner, which was largely independent of Ca2+. Pam1-3 increased the percentage of both, swollen and shrunken erythrocytes without significantly modifying the average forward scatter. They also increased reactive oxygen species (ROS) and ceramide abundance. In all assays the effect on eryptosis increased with increasing number of fatty acids, with Pam3 showing always the strongest effect. In contrast, a comparison of the effect of Pam1-3 on TLR2 dependent immune stimulation showed that not Pam3 but Pam2 displayed the strongest activity, and that immune stimulation was triggered at much lower concentrations than eryptosis.
Lipopeptides are not only important activators of the immune system; at higher concentrations they also drive host cells into apoptosis thus aggravating a bacterial infection.
背景/目的：细菌感染的后果包括贫血，这可能是由于自杀性红细胞死亡或红细胞凋亡受到刺激所致，其特征为细胞萎缩以及细胞膜磷脂酰丝氨酸易位导致的细胞膜紊乱。已知能刺激红细胞凋亡的细菌成分包括脂肽。介导红细胞凋亡触发的信号传导包括胞质Ca2+活性增加（[Ca2+]i）、氧化应激和神经酰胺的细胞内积累。本研究旨在确定脂肽诱导细胞膜紊乱的分子需求。
将人红细胞在不存在和存在1或5μg/ml合成脂肽Pam1（含一个脂肪酸的脂肽）、Pam2（含两个脂肪酸的脂肽）或Pam3（含三个脂肪酸的脂肽）的情况下孵育48小时。随后，通过膜联蛋白-V结合评估细胞表面磷脂酰丝氨酸的暴露情况，通过前向散射评估细胞体积，通过Fluo3荧光评估[Ca2+]i，通过DCF依赖性荧光评估活性氧生成，并使用特异性抗体评估神经酰胺丰度。
Pam1（5μg/ml）、Pam2（5μg/ml）和Pam3（1和5μg/ml）以剂量依赖性方式显著增加了膜联蛋白-V与红细胞结合的百分比，这在很大程度上与Ca2+无关。Pam1 - 3增加了肿胀和萎缩红细胞的百分比，而未显著改变平均前向散射。它们还增加了活性氧（ROS）和神经酰胺丰度。在所有实验中，对红细胞凋亡的影响随着脂肪酸数量的增加而增强，Pam3始终显示出最强的效果。相比之下，Pam1 - 3对TLR2依赖性免疫刺激作用的比较表明，显示最强活性的不是Pam3而是Pam2，并且免疫刺激在比红细胞凋亡低得多的浓度下触发。
脂肽不仅是免疫系统的重要激活剂；在较高浓度下，它们还会促使宿主细胞凋亡，从而加重细菌感染。
