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• W2776278222 abstract "Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15–34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15–34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90–120 and 5–30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15–34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15–34) binds to and disrupts lipid membranes and also observed that Ctn(15–34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15–34), suggesting Ctn(15–34) as a promising lead for development as an antibacterial/antitumor agent. Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15–34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15–34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90–120 and 5–30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15–34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15–34) binds to and disrupts lipid membranes and also observed that Ctn(15–34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15–34), suggesting Ctn(15–34) as a promising lead for development as an antibacterial/antitumor agent. New antimicrobial drugs are urgently needed to address the growing challenge of bacterial resistance to existing antibiotics. Misuse of classical antibiotics has increased the number of superbugs and created a critical situation whereby previously controlled pathogens could in the future cause major morbidity or mortality (1Thabit A.K. Crandon J.L. Nicolau D.P. Antimicrobial resistance: impact on clinical and economic outcomes and the need for new antimicrobials.Expert Opin. Pharmacother. 2015; 16 (25496207): 159-17710.1517/14656566.2015.993381Crossref PubMed Scopus (117) Google Scholar, 2Michael C.A. Dominey-Howes D. Labbate M. The antimicrobial resistance crisis: causes, consequences, and management.Front. Public Health. 2014; 2 (25279369): 145Crossref PubMed Scopus (444) Google Scholar). This alarming growth of multidrug-resistant pathogens has prompted an intensive search for anti-infective drugs with novel mechanisms of action (3Högberg L.D. Heddini A. Cars O. The global need for effective antibiotics: challenges and recent advances.Trends Pharmacol. Sci. 2010; 31 (20843562): 509-51510.1016/j.tips.2010.08.002Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 4Wright G.D. Sutherland A.D. New strategies for combating multidrug-resistant bacteria.Trends Mol. Med. 2007; 13 (17493872): 260-26710.1016/j.molmed.2007.04.004Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In particular, antimicrobial peptides (AMPs) 9The abbreviations used are: AMPantimicrobial peptideACNacetonitrileAFMatomic force microscopyCF5(6)-carboxyfluoresceinCholcholesterolCtncrotalicidinFCAflow cytometry assayLPSlipopolysaccharideLTAlipoteichoic acidLUVlarge unilamellar vesicleMBCminimal bactericidal concentrationMHBIIMueller Hinton broth cation-adjustedMICminimal inhibitory concentrationPCphosphatidylcholinePEphosphatidylethanolaminePGphosphatidylglycerolPOPC1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholinePOPE1-palmitoyl-2-oleoyl-sn-glycero-phospho-l-ethanolaminePOPG1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerolPOPS1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serinePSphosphatidylserineRBCred blood cellsRhBrhodamine BSMsphingomyelinSPRsurface plasmon resonanceSUVsmall unilamellar vesicleLALLimulus amebocyte lysateCbfcathelicidin-BFCATHcathelicidincfucolony-forming units. have emerged as promising alternatives due to their broad-spectrum activity (including superbugs), selectivity, and mechanisms of action that potentially hinder the development of resistance (5Rios A.C. Moutinho C.G. Pinto F.C. Del Fiol F.S. Jozala A. Chaud M.V. Vila M.M. Teixeira J.A. Balcão V.M. Alternatives to overcoming bacterial resistances: state-of-the-art.Microbiol. Res. 2016; 191 (27524653): 51-8010.1016/j.micres.2016.04.008Crossref PubMed Scopus (161) Google Scholar). antimicrobial peptide acetonitrile atomic force microscopy 5(6)-carboxyfluorescein cholesterol crotalicidin flow cytometry assay lipopolysaccharide lipoteichoic acid large unilamellar vesicle minimal bactericidal concentration Mueller Hinton broth cation-adjusted minimal inhibitory concentration phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-phospho-l-ethanolamine 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine phosphatidylserine red blood cells rhodamine B sphingomyelin surface plasmon resonance small unilamellar vesicle Limulus amebocyte lysate cathelicidin-BF cathelicidin colony-forming units. AMPs are ancient weapons of the host defense machinery, present in all life domains (6Zasloff M. Antimicrobial peptides of multicellular organisms.Nature. 2002; 415 (11807545): 389-39510.1038/415389aCrossref PubMed Scopus (6743) Google Scholar). Although they can act in several possible ways to accomplish microbial cell death (e.g. membrane disruption, apoptosis induction, and internal target inhibition) (7Yeaman M.R. Yount N.Y. Mechanisms of antimicrobial peptide action and resistance.Pharmacol. Rev. 2003; 55 (12615953): 27-5510.1124/pr.55.1.2Crossref PubMed Scopus (2333) Google Scholar, 8Hancock R.E. Sahl H.G. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.Nat. Biotechnol. 2006; 24 (17160061): 1551-155710.1038/nbt1267Crossref PubMed Scopus (3095) Google Scholar), an initial common step in the process is their recruitment onto the bacterial cell surface (9Sani M.A. Separovic F. How membrane-active peptides get into lipid membranes.Acc Chem. Res. 2016; 49 (27187572): 1130-113810.1021/acs.accounts.6b00074Crossref PubMed Scopus (264) Google Scholar, 10Li J. Koh J.J. Liu S. Lakshminarayanan R. Verma C.S. Beuerman R.W. Membrane active antimicrobial peptides: translating mechanistic insights to design.Front. Neurosci. 2017; 11 (28261050): 7310.3389/fnins.2017.00073Crossref PubMed Scopus (313) Google Scholar). Accordingly, most AMPs display fairly conserved structural and physicochemical properties, such as positive net charge, high content of hydrophobic amino acid residues, or amphipathic structure, all favoring interaction with and insertion into membranes (11Marín-Medina N. Ramírez D.A. Trier S. Leidy C. Mechanical properties that influence antimicrobial peptide activity in lipid membranes.Appl. Microbiol. Biotechnol. 2016; 100 (27837316): 10251-1026310.1007/s00253-016-7975-9Crossref PubMed Scopus (21) Google Scholar, 12Chen L. Zhang Q. Yuan X. Cao Y. Yuan Y. Yin H. Ding X. Zhu Z. Luo S.Z. How charge distribution influences the function of membrane-active peptides: lytic or cell-penetrating?.Int. J. Biochem. Cell Biol. 2017; 83 (28013149): 71-7510.1016/j.biocel.2016.12.011Crossref PubMed Scopus (13) Google Scholar). Cathelicidins are a large family of AMPs whose unifying feature is the presence of a conserved cathelin (cathepsin L inhibitor) domain at the N terminus. In contrast, their C-terminal domains contain a mature and active AMP and display high inter- and intraspecies diversity (13Tomasinsig L. Zanetti M. The cathelicidins: structure, function and evolution.Curr. Protein Pept. Sci. 2005; 6 (15638766): 23-3410.2174/1389203053027520Crossref PubMed Scopus (197) Google Scholar). Cathelicidins have been shown to be active against a broad range of targets, including bacteria, enveloped viruses, and fungi (14Kościuczuk E.M. Lisowski P. Jarczak J. Strzałkowska N. Jóźwik A. Horbańczuk J. Krzyżewski J. Zwierzchowski L. Bagnicka E. Cathelicidins: family of antimicrobial peptides: a review.Mol. Biol. Rep. 2012; 39 (23065264): 10957-1097010.1007/s11033-012-1997-xCrossref PubMed Scopus (327) Google Scholar). In addition to causing direct pathogen killing, cathelicidins can modulate the immune response by assisting with pathogen clearance (15Wong J.H. Ye X.J. Ng T.B. Cathelicidins: peptides with antimicrobial, immunomodulatory, anti-inflammatory, angiogenic, anticancer and procancer activities.Curr. Protein Pept. Sci. 2013; 14 (23968350): 504-51410.2174/13892037113149990067Crossref PubMed Scopus (51) Google Scholar). Cathelicidins have been isolated from a wide range of organisms, including mammals (16Dürr U.H. Sudheendra U.S. Ramamoorthy A. LL-37, the only human member of the cathelicidin family of antimicrobial peptides.Biochim. Biophys. Acta. 2006; 1758 (16716248): 1408-142510.1016/j.bbamem.2006.03.030Crossref PubMed Scopus (744) Google Scholar, 17Termén S. Tollin M. Olsson B. Svenberg T. Agerberth B. Gudmundsson G.H. Phylogeny, processing and expression of the rat cathelicidin rCRAMP: a model for innate antimicrobial peptides.Cell Mol. Life Sci. 2003; 60 (12737313): 536-54910.1007/s000180300045Crossref PubMed Scopus (49) Google Scholar18Wang J. Wong E.S. Whitley J.C. Li J. Stringer J.M. Short K.R. Renfree M.B. Belov K. Cocks B.G. Ancient antimicrobial peptides kill antibiotic-resistant pathogens: Australian mammals provide new options.PLoS One. 2011; 6 (21912615): e2403010.1371/journal.pone.0024030Crossref PubMed Scopus (68) Google Scholar), birds (19Xiao Y. Cai Y. Bommineni Y.R. Fernando S.C. Prakash O. Gilliland S.E. Zhang G. Identification and functional characterization of three chicken cathelicidins with potent antimicrobial activity.J. Biol. Chem. 2006; 281 (16326712): 2858-286710.1074/jbc.M507180200Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), fish (20Uzzell T. Stolzenberg E.D. Shinnar A.E. Zasloff M. Hagfish intestinal antimicrobial peptides are ancient cathelicidins.Peptides. 2003; 24 (15019197): 1655-166710.1016/j.peptides.2003.08.024Crossref PubMed Scopus (139) Google Scholar), frogs (21Mu L. Zhou L. Yang J. Zhuang L. Tang J. Liu T. Wu J. Yang H. The first identified cathelicidin from tree frogs possesses anti-inflammatory and partial LPS neutralization activities.Amino Acids. 2017; 49 (28593346): 1571-158510.1007/s00726-017-2449-7Crossref PubMed Scopus (22) Google Scholar), and marine (22Wei L. Gao J. Zhang S. Wu S. Xie Z. Ling G. Kuang Y.Q. Yang Y. Yu H. Wang Y. Identification and characterization of the first cathelicidin from sea snakes with potent antimicrobial and anti-inflammatory activity and special mechanism.J. Biol. Chem. 2015; 290 (26013823): 16633-1665210.1074/jbc.M115.642645Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and terrestrial snakes (23Zhao H. Gan T.X. Liu X.D. Jin Y. Lee W.H. Shen J.H. Zhang Y. Identification and characterization of novel reptile cathelicidins from elapid snakes.Peptides. 2008; 29 (18620012): 1685-169110.1016/j.peptides.2008.06.008Crossref PubMed Scopus (86) Google Scholar, 24Zhang Y. Zhao H. Yu G.Y. Liu X.D. Shen J.H. Lee W.H. Zhang Y. Structure-function relationship of king cobra cathelicidin.Peptides. 2010; 31 (20576537): 1488-149310.1016/j.peptides.2010.05.005Crossref PubMed Scopus (36) Google Scholar25Blower R.J. Barksdale S.M. van Hoek M.L. Snake cathelicidin NA-CATH and smaller helical antimicrobial peptides are effective against Burkholderia thailandensis.PLoS Negl. Trop. Dis. 2015; 9 (26196513): e000386210.1371/journal.pntd.0003862Crossref PubMed Scopus (35) Google Scholar). We recently identified a new family of cathelicidin-like peptides named vipericidins in the venom glands of various South American pit viper snakes and experimentally validated them as AMPs (26Falcao C.B. de La Torre B.G. Pérez-Peinado C. Barron A.E. Andreu D. Rádis-Baptista G. Vipericidins: a novel family of cathelicidin-related peptides from the venom gland of South American pit vipers.Amino Acids. 2014; 46 (25100358): 2561-257110.1007/s00726-014-1801-4Crossref PubMed Scopus (48) Google Scholar). Crotalicidin (Ctn), the most active vipericidin, is a 34-residue helical peptide found in Crotalus durissus terrificus and was selected for further study. Ctn displays powerful antimicrobial, as well as antitumor and antifungal, activity. However, it is moderately hemolytic and unstable in serum (27Falcao C.B. Pérez-Peinado C. de la Torre B.G. Mayol X. Zamora-Carreras H. Jiménez M.Á. Rádis-Baptista G. Andreu D. Structural dissection of crotalicidin, a rattlesnake venom cathelicidin, retrieves a fragment with antimicrobial and antitumor activity.J. Med. Chem. 2015; 58 (26465972): 8553-856310.1021/acs.jmedchem.5b01142Crossref PubMed Scopus (52) Google Scholar, 28Cavalcante C.S. Falcão C.B. Fontenelle R.O. Andreu D. Rádis-Baptista G. Anti-fungal activity of Ctn[15–34], the C-terminal peptide fragment of crotalicidin, a rattlesnake venom gland cathelicidin.J. Antibiot. 2017; 70 (27876749): 231-237Crossref PubMed Scopus (22) Google Scholar). To overcome these limitations, a rational dissection of the Ctn sequence was undertaken, aimed at identifying active motifs with enhanced properties. The search produced as main lead Ctn(15–34), a structurally disordered 20-mer that spans the C terminus and replicates the antibacterial and antitumor activities of the parental peptide but is less toxic toward healthy cells and significantly more stable in human serum (27Falcao C.B. Pérez-Peinado C. de la Torre B.G. Mayol X. Zamora-Carreras H. Jiménez M.Á. Rádis-Baptista G. Andreu D. Structural dissection of crotalicidin, a rattlesnake venom cathelicidin, retrieves a fragment with antimicrobial and antitumor activity.J. Med. Chem. 2015; 58 (26465972): 8553-856310.1021/acs.jmedchem.5b01142Crossref PubMed Scopus (52) Google Scholar). Investigating the mechanism of action on bacteria of both Ctn and Ctn(15–34) is of considerable interest, particularly given the potential therapeutic applications of the latter peptide. In this study, the mode of action of Ctn and Ctn(15–34) toward two Gram-negative bacterial species was investigated in detail. We determined bactericidal concentrations and quantitative kinetics of cell death and measured peptide accumulation on bacterial cell surfaces. Combining flow cytometry and colony count procedures, we established that bacterial death is accomplished by membrane disruption. We also monitored peptide uptake and membrane permeabilization in real time to ascertain whether both effects are achieved simultaneously or peptide pre-accumulation precedes loss of viability. To visualize peptide localization and structural damage on bacteria surface, we carried out confocal and atomic force microscopy experiments. Furthermore, to gain insights into the cell selectivity and the ability to disrupt lipid bilayers of the fragment Ctn(15–34), we performed experiments with model vesicles mimicking healthy human and bacterial cell membranes. Altogether, our results provide strong evidence that Ctn and Ctn(15–34) act by inducing disruption of the bacterial cell; they also exemplify how a set of optimized methodologies can be combined to evaluate the action of AMPs at the membrane level. The amino acid sequences in Table 1 were prepared in C-terminal amide form by Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase synthesis and purified to >95% purity (see Fig. S1 for HPLC and MS data). For the N-terminal rhodamine B (RhB)-labeled peptides, the two peaks observed in the chromatograms of purified compounds (Fig. S1a) are due to RhB atropisomerism. The overall hydrophobicity of unlabeled and RhB-labeled peptides can be compared by the percentage of acetonitrile (ACN) at which they elute in HPLC (Table 1). Values for unlabeled Ctn and Ctn(15–34) were 30.9 and 30.3% ACN, respectively, whereas RhB-labeled versions eluted at 34.7 and 34.2% ACN, respectively, underlining the increase in hydrophobicity brought about by RhB labeling.Table 1Peptides used in this studyPeptideAmino acid sequenceTheoretical massaTheoretical molecular mass was calculated using GPMAW version 8.10.Experimental massbExperimental molecular mass was determined from the MS spectra shown in Fig. S1b.PuritycPeptide purity was estimated by peak integration of the analytical HPLC chromatograms shown in Fig. S1a.ACN gradientdACN gradient over 15 min used to run the analytical HPLC. Range indicates the initial and final ACN concentrations.Retention timeACNePercentage of ACN corresponding to the eluted peptide, calculated from the gradient used and the retention time.DaDa%min%CtnfProtein Data Bank accession code of Ctn: 2MWT.KRFKKFFKKVKKSVKKRLKKIFKKPMVIGVTIPF-amide4151.394152.459815–409.55630.9RhB-CtnRhB-KRFKKFFKKVKKSVKKRLKKIFKKPMVIGVTIPF-amide4576.944575.979920–4010.99734.7Ctn(15–34)KKRLKKIFKKPMVIGVTIPF-amide2371.112371.239910–4010.13130.3RhB-Ctn(15–34)RhB-KKRLKKIFKKPMVIGVTIPF-amide2796.662795.909820–4010.67034.2a Theoretical molecular mass was calculated using GPMAW version 8.10.b Experimental molecular mass was determined from the MS spectra shown in Fig. S1b.c Peptide purity was estimated by peak integration of the analytical HPLC chromatograms shown in Fig. S1a.d ACN gradient over 15 min used to run the analytical HPLC. Range indicates the initial and final ACN concentrations.e Percentage of ACN corresponding to the eluted peptide, calculated from the gradient used and the retention time.f Protein Data Bank accession code of Ctn: 2MWT. Open table in a new tab We evaluated the antimicrobial activity of Ctn and Ctn(15–34) against two Gram-negative bacterial strains: Escherichia coli ATCC 25922 (E. coli) and Pseudomonas aeruginosa ATCC 27853 (P. aeruginosa). As shown in Table 2, Ctn has the lowest minimal inhibitory concentration (MIC) for E. coli as well as for P. aeruginosa (0.78 and 1.56 μm, respectively). Ctn(15–34) also has a low MIC for E. coli (3.13 μm), but the concentration required to prevent P. aeruginosa visible growth is higher (12.5 μm). To determine whether the peptides were bactericidal or bacteriostatic, the minimal bactericidal concentrations (MBCs) were also determined (Table 2). The data show both Ctn and Ctn(15–34) to be bactericidal, as MBCs exceed MICs by <2-fold (29Levison M.E. Levison J.H. Pharmacokinetics and pharmacodynamics of antibacterial agents.Infect. Dis. Clin. North Am. 2009; 23 (19909885, vii): 791-81510.1016/j.idc.2009.06.008Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). The MIC/MBC determinations reveal that more peptide is required for bacterial death when higher bacterial inocula are used. This difference is significant for E. coli, for which up to 3 times more peptide is needed. These results support previous observations suggesting that the therapeutic effect of AMPs depends on the peptide/cell ratio rather than directly on peptide concentration (30Roversi D. Luca V. Aureli S. Park Y. Mangoni M.L. Stella L. How many antimicrobial peptide molecules kill a bacterium? The case of PMAP-23.ACS Chem. Biol. 2014; 9 (25058470): 2003-200710.1021/cb500426rCrossref PubMed Scopus (88) Google Scholar, 31Hällbrink M. Oehlke J. Papsdorf G. Bienert M. Uptake of cell-penetrating peptides is dependent on peptide-to-cell ratio rather than on peptide concentration.Biochim. Biophys. Acta. 2004; 1667 (15581859): 222-22810.1016/j.bbamem.2004.10.009Crossref PubMed Scopus (82) Google Scholar). In tune with this suggestion, and to avoid the results being influenced by the use of different peptide/cell ratios, peptide concentrations for mechanism-of-action studies were adjusted to the bacterial inoculum used for each specific experiment.Table 2MIC and MBC of Ctn and Ctn(15–34) against two standard Gram-negative bacterial strainsPeptideE. coli ATCC 25922P. aeruginosa ATCC 278535 × 105 cfu/ml107 cfu/ml5 × 105 cfu/ml107 cfu/mlMIC (μm)Ctn0.786.251.563.13Ctn(15–34)3.135012.525MBC (μm)Ctn1.566.253.133.13Ctn(15–34)6.255025–5025 Open table in a new tab The killing kinetics of E. coli and P. aeruginosa by Ctn and Ctn(15–34) at their respective MBCs was evaluated using a time-kill assay. The results in Fig. 1a show that for both peptides, a bactericidal effect (99.9% reduction of bacterial viability) against E. coli is observed within 4 h. At 90 min, Ctn kills >90% of bacteria, whereas Ctn(15–34) needs 120 min for a similar effect. Ctn and Ctn(15–34) are faster at killing P. aeruginosa, requiring only 5 or 30 min for 90% cell death, respectively, with bactericidal effect observed at 15 and 90 min, respectively. This relatively fast effect against E. coli, even faster against P. aeruginosa, suggests that both peptides act by direct damage to the bacterial membrane rather than by inhibition of an internal target. To investigate whether surface charge can be neutralized by the accumulation of the peptides at the membrane of E. coli or P. aeruginosa, ζ potential measurements were carried out. The results show that, as expected (32Alves C.S. Melo M.N. Franquelim H.G. Ferre R. Planas M. Feliu L. Bardají E. Kowalczyk W. Andreu D. Santos N.C. Fernandes M.X. Castanho M.A. Escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides BP100 and pepR.J. Biol. Chem. 2010; 285 (20566635): 27536-2754410.1074/jbc.M110.130955Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 33Shephard J. McQuillan A.J. Bremer P.J. Mechanisms of Cation Exchange by Pseudomonas aeruginosa PAO1 and PAO1 wbpL, a Strain with a truncated lipopolysaccharide.Appl. Environ. Microbiol. 2008; 74 (18820073): 6980-698610.1128/AEM.01117-08Crossref PubMed Scopus (33) Google Scholar), in the absence of peptide, both bacteria possess a negative potential (Fig. 1b, ∼−24 mV for E. coli and ∼−21 mV for P. aeruginosa). In the presence of increasing peptide concentrations, a shift to positive ζ potential values is observed. In E. coli, complete neutralization of the surface charge occurs at 0.78–3.13 μm Ctn and 1.56–3.13 μm Ctn(15–34), whereas for P. aeruginosa, total surface charge neutralization is reached at 0.78–3.13 μm Ctn and 3.13–6.25 μm Ctn(15–34). For both bacteria, membrane neutralization occurs at concentrations below the MIC/MBC of either peptide. To clarify whether E. coli and P. aeruginosa death induced by Ctn and Ctn(15–34) is due to direct membrane disruption, correlation between membrane permeabilization and bacterial viability was studied. To this end, a flow cytometry assay (FCA) with SYTOX® Green to examine membrane permeabilization and a plate colony count to quantify viable cells were performed. As a cell-impermeable nucleic acid stain that only labels bacteria with compromised plasma membrane, SYTOX® Green is a useful tool in bacterial viability studies. As shown in Fig. 2, an increased percentage of bacterial cells with SYTOX® Green-permeable membrane is accompanied by a decrease in cell viability by the colony count method. FCA histograms on the SYTOX® Green channel (Fig. 2a) shift to higher fluorescence intensities indicative of E. coli and P. aeruginosa membrane disruption with increasing peptide concentration. Negative and positive controls (live and dead bacteria; black and gray histograms, respectively) were used to define gates and calculate permeabilization percentages, shown in Fig. 2b along with the viability percentages obtained in the colony count assay. A direct correlation between both sets of data (Fig. 2b) confirms that antibacterial activity of Ctn and Ctn(15–34) is mediated by membrane permeabilization. To correlate the membrane permeabilization effect with peptide uptake (including both membrane-bound and intracellularly internalized), a parallel FCA experiment was performed with RhB-labeled versions of both peptides. From the SYTOX® Green and RhB channel readings (Fig. S2, a and b), the percentages of permeabilized and of peptide-internalizing cells, respectively, were determined (Fig. S2c). For both Ctn and Ctn(15–34), an increase in E. coli permeabilization coincides with increased peptide uptake, suggesting a direct correlation between the membrane damage observed and peptide entry/binding. The dose–response curve for RhB-Ctn (Fig. S2c, continuous red line) is similar to that for unlabeled Ctn (Fig. 2b, continuous red line), suggesting that RhB does not interfere with the peptide effect. In contrast, the profiles for RhB-labeled (Fig. S2c, continuous blue line) and unlabeled Ctn(15–34) (Fig. 2b, continuous blue line) are different. Whereas the latter induces 50% permeabilization in the 6.25–12.5 μm range, the percentage rises to 80% for the RhB-labeled analogue at similar concentrations. Moreover, at higher (25–100 μm) concentrations, a decrease in membrane permeabilization and uptake of RhB-Ctn(15–34) occurs, an effect not observed for unlabeled Ctn(15–34). This probably reflects a loss in integrity of the bacterial cell, no longer capable of retaining the labeled peptide within, and with its DNA (SYTOX® Green target) either damaged or leaking out. The observation of N-terminal RhB label interfering with the activity of Ctn(15–34) calls for caution in extrapolating results from labeled to unlabeled versions of this peptide. Changes in membrane permeabilization and peptide uptake were monitored by a time-resolved flow cytometry assay, which allows a more accurate study of the kinetics of peptide effect, compared with kinetic studies based on end-point sampling. We studied the membrane permeabilization of E. coli using SYTOX® Green dye right after Ctn or Ctn(15–34) addition, monitoring the changes from negative to positive gates for 90 min. Fig. 3 (a and b) shows time-course results and negative and positive controls (for the whole acquisition, see Movies S1a and S1b for Ctn and Ctn(15–34), respectively). As detailed under “Experimental procedures,” data from the FCA histograms were used to generate the kinetic curves on Fig. 3c. Data were fitted using the two-state kinetic model described previously for other AMPs (34Freire J.M. Gaspar D. de la Torre B.G. Veiga A.S. Andreu D. Castanho M.A. Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry.Biochim. Biophys. Acta. 2015; 1848 (25445678): 554-56010.1016/j.bbamem.2014.11.001Crossref PubMed Scopus (38) Google Scholar), assuming that peptide-bacteria interaction consists of an initial binding step, followed by permeabilization of the membrane. Although both peptides induce permeabilization of ∼90% of bacteria after 90 min (Fig. 3c), the process differs between peptides at the early stages; based on the tendency of the experimental data, Ctn(15–34) seems to start killing bacteria right after its addition, and the permeabilization takes place in a single process. In contrast, Ctn shows an initial lag phase. Fitting of the kinetic data to a two-state model results in statistically poor residuals for some time intervals (Fig. S3), suggesting that for both peptides, permeabilization is more complex than as assumed by the model. An additional slow event leading to permeabilization is suggested because the data deviate from the fit at longer times. In contrast, the fit is quite acceptable at shorter times. k0, the membrane attachment rate constant, is higher for Ctn(15–34) than for Ctn (2 × 10−2 s−1 versus 1 × 10−3 s−1) as well as the permeabilization rate constant (1 × 10−3 s−1 versus 9 × 10−4 s−1). Cooperativity is not observed in either case (f ∼ 0), contrary to other AMPs (34Freire J.M. Gaspar D. de la Torre B.G. Veiga A.S. Andreu D. Castanho M.A. Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry.Biochim. Biophys. Acta. 2015; 1848 (25445678): 554-56010.1016/j.bbamem.2014.11.001Crossref PubMed Scopus (38) Google Scholar). To determine whether permeabilization is concomitant with internalization, kinetic studies with RhB-labe" @default.
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• W2776278222 title "Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15–34), antimicrobial peptides from rattlesnake venom" @default.
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