title: Lipopeptide-Induced Suicidal Erythrocyte Death Correlates with the Degree of Acylation.
abstract: OBJECTIVE
  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+]<Sub>i</Sub>), oxidative stress and cellular accumulation of ceramide. The present study aimed to define the molecular requirements for lipopeptide-induced cell membrane scrambling.
  
  
  METHODS
  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+]<Sub>i</Sub> from Fluo3-fluorescence, ROS formation from DCF dependent fuorescence, and ceramide abundance utilizing specific antibodies.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Abdulla
authors.initials: A
authors.lastname: Al Mamun Bhuyan
authors.forename: Minh-Thu
authors.initials: MT
authors.lastname: Nguyen
authors.forename: Rosi
authors.initials: R
authors.lastname: Bissinger
authors.forename: Friedrich
authors.initials: F
authors.lastname: Götz
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D000072817:Eryptosis / Q000187:drug effects*; D004910:Erythrocyte Membrane / Q000187:drug effects; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D055666:Lipopeptides / Q000737:chemistry / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects; D010718:Phosphatidylserines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism
chemical_list: D002518:Ceramides; D055666:Lipopeptides; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2; D002118:Calcium
keywords: Apoptosis; Eryptosis; Lipopeptide; Pam; Phosphatidylserine