Abstract
Gramicidin S (GS) is a cyclo-decapeptide antibiotic with wide Gram+ and Gram− antimicrobial spectrum. However, its therapeutic application is very limited due to hemolytic activity of GS. The presence of cholesterol defines one of the most significant differences between eukaryotic plasma membranes and bacterial inner membranes. To find out the cholesterol effect on the GS hemolytic efficiency we compared GS-induced hemolysis of erythrocytes extracted from the blood of healthy donors against donors with atherosclerosis, “naturally” enriched with cholesterol. Our results show that increased cholesterol levels significantly attenuates yet does not abolishes the GS hemolytic activity. High levels of cholesterol content in erythrocyte membranes results in a decrease in the membrane fluidity and deformability leading to a decrease in the rate of GS interaction with membranes. The results obtained confirm that hydrophobic as well as electrostatic interactions must be involved in the binding of GS to cell membranes. Lipid peroxidation occurring within atherosclerotic erythrocytes leads to considerable decrease in the degree of GS-induced erythrocyte hemolysis in vitro. These results can be applied to the rational design of GS analogs with increased antibacterial efficiency but reduced hemolytic activity.
References
Abraham T, Lewis RN, Hodges RS, McElhaney RN (2005) Isothermal titration calorimetry studies of the binding of a rationally designed analogue of the antimicrobial peptide gramicidin s to phospholipid bilayer membranes. Biochemistry 44(6):2103–2112
Abraham T, Marwaha S, Kobewka DM, Lewis RN, Prenner EJ, Hodges RS, McElhaney RN (2007) The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S. Biochim Biophys Acta 1768(9):2089–2098
Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1994) Molecular biology of the cell, 3rd edn. Garland Publishing, Inc., New York
Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC (1974) Enzymatic determination of total serum cholesterol. Clin Chem 20:470–521
Ashrafuzzaman Md, Andersen OS, McElhaney RN (2008) The antimicrobial peptide gramicidin S permeabilizes phospholipid bilayer membranes without forming discrete ion channels. Biochim Biophys Acta 1778(12):2814–2822
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Cazzola R, Rondanelli M, Russo-Volpe S, Ferrari E, Cestaro B (2004) Decreased membrane fluidity and altered susceptibility to peroxidation and lipid composition in overweight and obese female erythrocytes. J Lipid Res 45:1846–1851
Chance B, Sies H, Boveris A (1979) Hydroperoxide metabolism in mammalian organs. Physiol Rev 59:527–605
Dincer Yi, Akcay T, Konukoglu D, Hatemi H (1999) Erythrocyte susceptibility to lipid peroxidation in patients with coronary atherosclerosis. Acta Med Okayama 53(6):259–264
Folch J, Lees M, Sloan Stanley GH (1957) A single method for the isolation and purification of total lipid from animal tissue. J Biol Chem 226:497–509
Gilbert HS, Stump DD, Roth EF Jr (1984) A method to correct for errors caused by generation of interfering compounds during erythrocyte lipid peroxidation. Anal Biochem 137:282–286
Giulivi C, Hochstein P, Davies KJ (1994) Hydrogen peroxide production by red blood cells. Free Radic Biol Med 16(1):123–129
Halliwell B, Clement MV, Long LH (2000) Hydrogen peroxide in the human body. FEBS Lett 486:10–13
Hodis HN, Crawford DW, Sevanian A (1991) Cholesterol feeding increases plasma and aortic tissue cholesterol oxide levels in parallel: further evidence for the role of cholesterol oxidation in atherosclerosis. Atherosclerosis 89:117–126
Horwitt MK, Harvey CC, Duncan CD, Wilson WC (1956) Effects of limited tocopherol intake in man with relationships to erythrocyte hemolysis and lipid oxidations. Am J Clin Nutr 4:408–419
Ivanov IT (1999) Low pH-induced hemolysis of erythrocytes is related to the entry of the acid into cytosole and oxidative stress on cellular membranes. Biochim Biophys Acta 1415(2):349–360
Jelokhani-Niaraki M, Kondejewski LH, Wheaton LC, Hodges RS (2009) Effect of ring size on conformation and biological activity of cyclic cationic antimicrobial peptides. J Med Chem 52(7):2090–2097
Kamysz E, Mickiewicz B, Kamysz W, Bielińska S, Rodziewicz-Motowidło S, Ciarkowski J (2011) Synthesis, biological activity and solution structure of new analogues of the antimicrobial Gramicidin S. J Pept Sci 17(3):211–217
Katsu T, Ninomiya C, Kuroko M, Kobayashi H, Hirota T, Fujita Y (1988) Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane. Biochim Biophys Acta 939(1):57–63
Knijnenburg AD, Kapoerchan VV, Grotenbreg GM, Spalburg E, de Neeling AJ, Mars-Groenendijk RH, Noort D, Otero JM, Llamas-Saiz AL, van Raaij MJ, Ravensbergen B, Nibbering PH, van der Marel GA, Overkleeft HS, Overhand M (2011) Synthesis and evaluation of strand and turn modified ring-extended gramicidin S derivatives. Bioorg Med Chem 19(11):3402–3409
Kondejewski LH, Farmer SW, Wishart DS, Hancock RE, Hodges RS (1996) Gramicidin S is active against both gram-positive and gram-negative bacteria. Int J Pept Protein Res 47(6):460–466
Koter M, Franiak I, Broncel M, Chojnowska-Jezierska J (2003) Effects of simvastatin and pravastatin on peroxidation of erythrocyte plasma membrane lipids in patients with type 2 hypercholesterolemia. Can J Physiol Pharmacol 81:485–492
Lee DL, Powers JP, Pflegerl K, Vasil ML, Hancock RE, Hodges RS (2004) Effects of single d-amino acid substitutions on disruption of beta-sheet structure and hydrophobicity in cyclic 14-residue antimicrobial peptide analogs related to gramicidin S. J Pept Res 63(2):69–84
Lewis RN, Kiricsi M, Prenner EJ, Hodges RS, McElhaney RN (2003) Fourier transform infrared spectroscopic study of the interactions of a strongly antimicrobial but weakly hemolytic analogue of gramicidin S with lipid micelles and lipid bilayer membranes. Biochemistry 42(2):440–449
Lurie KG, Chin JH, Hoffman BB (1985) Decreased membrane fluidity and beta-adrenergic responsiveness in atherosclerotic quail. Am J Physiol 249:380–385
Martinez M, Vaya A, Marti R et al (1996) Erythrocyte membrane cholesterol/phospholipid changes and hemorheological modifications in familial hypercholesterolemia treated with lovastatin. Thromb Res 83:375–388
McMullen TP, Lewis RN, McElhaney RN (1999) Calorimetric and spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylethanolamine bilayers. Biochim Biophys Acta 1416(1–2):119–134
Michalak J, Kadziolka A, Pruszkowska R, Ledwozyw A, Madejczyk A (1988) Compensatory mechanisms in erythrocyte lipids in patients with atherosclerosis. Lipids 23:476–480
Mueller S, Riedel H-D, Stremmel W (1997) Direct evidence for catalase as the predominant H2O2-removing enzyme in human erythrocytes. Blood 90(12):4973–4978
Peng SK, Hu B, Morin RJ (1991) Angiotoxicity and atherogenicity of cholesterol oxides. J Clin Lab Anal 5:144–152
Prenner EJ, Lewis RN, McElhaney RN (1999) The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes. Biochim Biophys Acta 1462(1–2):201–221
Prenner EJ, Lewis RN, Jelokhani-Niaraki M, Hodges RS, McElhaney RN (2001) Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes. Biochim Biophys Acta 1510(1–2):83–92
Rose HG, Oklander M (1965) Improved procedure for the extraction of lipids from human erythrocytes. J Lipid Res 6:428–431
Semrau S, Monster MW, van der Knaap M, Florea BI, Schmidt T, Overhand M (2010) Membrane lysis by gramicidin S visualized in red blood cells and giant vesicles. Biochim Biophys Acta 1798(11):2033–2039
Sevanian A, McLeod LL (1987) Cholesterol autoxidation in phospholipid membrane bilayers. Lipids 22(9):627–636
Snyder LM, Fortier NL, Trainor J, Jacobs J, Leb L, Lubin B, Chiu D, Shohet S, Mohandas N (1985) Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin–hemoglobin crosslinking. J Clin Invest 76:1971–1977
Tamaki M, Sasaki I, Nakao Y, Shindo M, Kimura M, Uchida Y (2009) Gramicidin S analogs having six basic amino acid residues. J Antibiot (Tokyo) 62(10):597–599
Tavazzi B, Donato DP, Amorini AM et al (2000) Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress. Eur J Biochem 267:684–689
Tziakas DN, Kaski JC, Chalikias GK et al (2007) Total cholesterol content of erythrocyte membranes is increased in patients with acute coronary syndrome: a new marker of clinical instability? J Am Coll Cardiol 49:2081–2089
Tziakas DN, Chalikias GK, Tentes IK, Stakos D, Chatzikyriakou SV, Mitrousi K, Kortsaris AX, Kaski JC, Boudoulas H (2008) Interleukin-8 is increased in the membrane of circulating erythrocytes in patients with acute coronary syndrome. Eur Heart J 29(22):2713–2722
van den Berg JJM, Op den Kamp JAF, Lubin BH, Roelofsen B, Kuypers FA (1992) Kinetics and site specificity of hydroperoxide-induced oxidative damage in red blood cells. FreeRad Biol Med 12:487–498
van der Knaap M, Engels E, Busscher HJ, Otero JM, Llamas-Saiz AL, van Raaij MJ, Mars-Groenendijk RH, Noort D, van der Marel GA, Overkleeft HS, Overhand M (2009) Synthesis and biological evaluation of asymmetric gramicidin S analogues containing modified d-phenylalanine residues. Bioorg Med Chem 17(17):6318–6328
van der Knaap M, Lageveen LT, Busscher HJ, Mars-Groenendijk R, Noort D, Otero JM, Llamas-Saiz AL, van Raaij MJ, van der Marel GA, Overkleeft HS, Overhand M (2011) Evaluation of readily accessible azoles as mimics of the aromatic ring of d-phenylalanine in the turn region of gramicidin S. Chem Med Chem 6(5):840–847
Zavodnik IB, Piletskaia TP (1997) Acid-induced hemolysis of human erythrocytes. Biofizika 42(5):1106–1112
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hackl, E.V., Berest, V.P. & Gatash, S.V. Effect of Cholesterol Content on Gramicidin S-Induced Hemolysis of Erythrocytes. Int J Pept Res Ther 18, 163–170 (2012). https://doi.org/10.1007/s10989-012-9289-9
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10989-012-9289-9