Antimicrobial activity of synthetic cationic peptides and lipopeptides derived from human lactoferricin against Pseudomonas aeruginosa planktonic cultures and biofilms

BMC Microbiology

Guillermo Martínez de Tejada³  Karl Lohner¹  Betsey Pitts²  Philip S. Stewart²  Raquel Ferrer-Espada³  Susana Sánchez-Gómez⁴ 

[1] Institute of Molecular Biosciences, Biophysics Division, University of Graz, Graz, Austria;Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA;Department of Microbiology, University of Navarra, Irunlarrea 1, Pamplona, E-31008, Spain;Present address: Susana Sánchez-Gómez, Bionanoplus, Noain, 31110, Spain

关键词: Biofilm;    Pseudomonas aeruginosa;    Lactoferricin;    Antimicrobial peptides;   

Others  :  1227626
DOI  :  10.1186/s12866-015-0473-x

 received in 2015-03-18, accepted in 2015-06-26,  发布年份 2015

【 摘 要 】

Background

Infections by Pseudomonas aeruginosa constitute a serious health threat because this pathogen –particularly when it forms biofilms – can acquire resistance to the majority of conventional antibiotics. This study evaluated the antimicrobial activity of synthetic peptides based on LF11, an 11-mer peptide derived from human lactoferricin against P. aeruginosa planktonic and biofilm-forming cells. We included in this analysis selected N-acylated derivatives of the peptides to analyze the effect of acylation in antimicrobial activity. To assess the efficacy of compounds against planktonic bacteria, microdilution assays to determine the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill studies were conducted. The anti-biofilm activity of the agents was assessed on biofilms grown under static (on microplates) and dynamic (in a CDC-reactor) flow regimes.

Results

The antimicrobial activity of lipopeptides differed from that of non-acylated peptides in their killing mechanisms on planktonic and biofilm-forming cells. Thus, acylation enhanced the bactericidal activity of the parental peptides and resulted in lipopeptides that were uniformly bactericidal at their MIC. In contrast, acylation of the most potent anti-biofilm peptides resulted in compounds with lower anti-biofilm activity. Both peptides and lipopeptides displayed very rapid killing kinetics and all of them required less than 21 min to reduce 1,000 times the viability of planktonic cells when tested at 2 times their MBC. The peptides, LF11-215 (FWRIRIRR) and LF11-227 (FWRRFWRR), displayed the most potent anti-biofilm activity causing a 10,000 fold reduction in cell viability after 1 h of treatment at 10 times their MIC. At that concentration, these two compounds exhibited low citotoxicity on human cells. In addition to its bactericidal activity, LF11-227 removed more that 50 % of the biofilm mass in independent assays. Peptide LF11-215 and two of the shortest and least hydrophobic lipopeptides, DI-MB-LF11-322 (2,2-dimethylbutanoyl-PFWRIRIRR) and DI-MB-LF11-215, penetrated deep into the biofilm structure and homogenously killed biofilm-forming bacteria.

Conclusion

We identified peptides derived from human lactoferricin with potent antimicrobial activity against P. aeruginosa growing either in planktonic or in biofilm mode. Although further structure-activity relationship analyses are necessary to optimize the anti-biofilm activity of these compounds, the results indicate that lactoferricin derived peptides are promising anti-biofilm agents.

【 授权许可】

   
2015 Sánchez-Gómez et al.

【 图 表 】

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【 参考文献 】

• [1]Lodise TP, Patel N, Kwa A, Graves J, Furuno JP, Graffunder E, et al. Predictors of 30-day mortality among patients with Pseudomonas aeruginosa bloodstream infections: impact of delayed appropriate antibiotic selection. Antimicrob Agents Chemother. 2007;51:3510–5.
• [2]Tam VH, Rogers CA, Chang KT, Weston JS, Caeiro JP, Garey KW. Impact of multidrug-resistant Pseudomonas aeruginosa bacteremia on patient outcomes. Antimicrob Agents Chemother. 2010; 54:3717-3722.
• [3]Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001; 45:999-1007.
• [4]Davey ME, O’Toole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev. 2000; 64:847-867.
• [5]Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev. 2002; 15:167-193.
• [6]Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet. 2001; 358:135-138.
• [7]Jenssen H, Hamill P, Hancock REW. Peptide antimicrobial agents. Clin Microbiol Rev. 2006; 19:491-511.
• [8]Lohner K. New strategies for novel antibiotics: peptides targeting bacterial cell membranes. Gen Physiol Biophys. 2009; 28:105-116.
• [9]Navon-Venezia S, Feder R, Gaidukov L, Carmeli Y, Mor A. Antibacterial properties of dermaseptin S4 derivatives with in vivo activity. Antimicrob Agents Chemother. 2002; 46:689-694.
• [10]Zhang L, Parente J, Harris SM, Woods DE, Hancock REW, Falla TJ. Antimicrobial peptide therapeutics for cystic fibrosis. Antimicrob Agents Chemother. 2005; 49:2921-2927.
• [11]Batoni G, Maisetta G, Brancatisano FL, Esin S, Campa M. Use of antimicrobial peptides against microbial biofilms: advantages and limits. Curr Med Chem. 2011; 18:256-279.
• [12]Gifford JL, Hunter HN, Vogel HJ. Lactoferricin: a lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological properties. Cell Mol Life Sci. 2005; 62:2588-2598.
• [13]Orsi N. The antimicrobial activity of lactoferrin: current status and perspectives. Biometals. 2004; 17:189-196.
• [14]Sanchez-Gomez S, Lamata M, Leiva J, Blondelle SE, Jerala R, Andra J, et al. Comparative analysis of selected methods for the assessment of antimicrobial and membrane-permeabilizing activity: a case study for lactoferricin derived peptides. BMC Microbiol. 2008;8:196.
• [15]Sanchez-Gomez S, Japelj B, Jerala R, Moriyon I, Fernández Alonso M, Leiva J, et al. Structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against Pseudomonas aeruginosa in vitro and in vivo. Antimicrob Agents Chemother. 2011;55:218–28.
• [16]Zweytick D, Deutsch G, Andra J, Blondelle SE, Vollmer E, Jerala R, et al. Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides. J Biol Chem. 2011;286:21266–76.
• [17]Martinez de Tejada G, Sanchez-Gomez S, Razquin-Olazaran I, Kowalski I, Kaconis Y, Heinbockel L, et al. Bacterial cell wall compounds as promising targets of antimicrobial agents I. Antimicrobial peptides and lipopolyamines. Curr Drug Targets. 2012;13:1121–30.
• [18]Zorko M, Japelj B, Hafner-Bratkovic I, Jerala R. Expression, purification and structural studies of a short antimicrobial peptide. Biochim Biophys Acta. 2009; 1788:314-323.
• [19]Andra J, Lohner K, Blondelle SE, Jerala R, Moriyon I, Koch MHJ, et al. Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide. Biochem J. 2005;385:135–43.
• [20]Zweytick D, Pabst G, Abuja PM, Jilek A, Blondelle SE, Andra J, et al. Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity. Biochim Biophys Acta. 2006;1758:1426–35.
• [21]Japelj B, Zorko M, Majerle A, Pristovsek P, Sanchez-Gomez S, Martinez de Tejada G, et al. The acyl group as the central element of the structural organization of antimicrobial lipopeptide. J Am Chem Soc. 2007;129:1022–3.
• [22]Brandenburg K, Howe J, Sanchez-Gomez S, Garidel P, Roessle M, Andra J, et al. Effective antimicrobial and anti-endotoxin activity of cationic peptides based on lactoferricin: a biophysical and microbiological study. Antiinfect Agents Med Chem. 2010;9:9–22.
• [23]Radzishevsky IS, Rotem S, Zaknoon F, Gaidukov L, Dagan A, Mor A. Effects of acyl versus aminoacyl conjugation on the properties of antimicrobial peptides. Antimicrob Agents Chemother. 2005; 49:2412-2420.
• [24]Jiang Z, Vasil AI, Gera L, Vasil ML, Hodges RS. Rational design of α-helical antimicrobial peptides to target gram-negative pathogens, acinetobacter baumannii and pseudomonas aeruginosa: utilization of charge, “Specificity Determinants”, total hydrophobicity, hydrophobe type and location as design parameters to improve the therapeutic ratio. Chem Biol Drug Des. 2011; 77:225-240.
• [25]Rathinakumar R, Walkenhorst WF, Wimley WC. Broad-spectrum antimicrobial peptides by rational combinatorial design and high-throughput screening: the importance of interfacial activity. J Am Chem Soc. 2009; 131:7609-7617.
• [26]Rathinakumar R, Wimley WC. Biomolecular engineering by combinatorial design and high-throughput screening: small, soluble peptides that permeabilize membranes. J Am Chem Soc. 2008; 130:9849-9858.
• [27]Yeaman MR. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev. 2003; 55:27-55.
• [28]Chen Y, Guarnieri MT, Vasil AI, Vasil ML, Mant CT, Hodges RS. Role of peptide hydrophobicity in the mechanism of action of -helical antimicrobial peptides. Antimicrob Agents Chemother. 2007; 51:1398-1406.
• [29]Kustanovich I. Structural requirements for potent versus selective cytotoxicity for antimicrobial dermaseptin S4 derivatives. J Biol Chem. 2002; 277:16941-16951.
• [30]Zelezetsky I, Pag U, Sahl H-G, Tossi A. Tuning the biological properties of amphipathic α-helical antimicrobial peptides: rational use of minimal amino acid substitutions. Peptides. 2005; 26:2368-2376.
• [31]Jiang Z, Vasil AI, Hale JD, Hancock REW, Vasil ML, Hodges RS. Effects of net charge and the number of positively charged residues on the biological activity of amphipathic α-helical cationic antimicrobial peptides. Biopolymers. 2008; 90:369-383.
• [32]Gopal R, Kim YG, Lee JH, Lee SK, Chae JD, Son BK, et al. Synergistic effects and antibiofilm properties of chimeric peptides against multidrug-resistant Acinetobacter baumannii strains. Antimicrob Agents Chemother. 2014;58:1622–9.
• [33]Zairi A, Ferrieres L, Latour-Lambert P, Beloin C, Tangy F, Ghigo J-M, et al. In vitro activities of dermaseptins K4S4 and K4K20S4 against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa planktonic growth and biofilm formation. Antimicrob Agents Chemother. 2014;58:2221–8.
• [34]Beckloff N, Laube D, Castro T, Furgang D, Park S, Perlin D, et al. Activity of an antimicrobial peptide mimetic against planktonic and biofilm cultures of oral pathogens. Antimicrob Agents Chemother. 2007;51:4125–32.
• [35]Roveta S, Marchese A, Schito GC. Activity of daptomycin on biofilms produced on a plastic support by Staphylococcus spp. Int J Antimicrob Agents. 2008; 31:321-328.
• [36]Wei GX. Effect of MUC7 peptides on the growth of bacteria and on Streptococcus mutans biofilm. J Antimicrob Chemother. 2006; 57:1100-1109.
• [37]Singh PK, Parsek MR, Greenberg EP, Welsh MJ. A component of innate immunity prevents bacterial biofilm development. Nature. 2002; 417:552-555.
• [38]Singh PK. Iron sequestration by human lactoferrin stimulates P. aeruginosa surface motility and blocks biofilm formation. Biometals. 2004; 17:267-270.
• [39]Kamiya H, Ehara T, Matsumoto T. Inhibitory effects of lactoferrin on biofilm formation in clinical isolates of Pseudomonas aeruginosa. J Infect Chemother. 2012; 18:47-52.
• [40]Arslan SY, Leung KP, Wu CD. The effect of lactoferrin on oral bacterial attachment. Oral Microbiol Immunol. 2009; 24:411-416.
• [41]Berlutti F, Ajello M, Bosso P, Morea C, Petrucca A, Antonini G, et al. Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans. Biometals. 2004;17:271–8.
• [42]Caraher EM, Gumulapurapu K, Taggart CC, Murphy P, McClean S, Callaghan M. The effect of recombinant human lactoferrin on growth and the antibiotic susceptibility of the cystic fibrosis pathogen Burkholderia cepacia complex when cultured planktonically or as biofilms. J Antimicrob Chemother. 2007; 60:546-554.
• [43]O’May CY, Sanderson K, Roddam LF, Kirov SM, Reid DW. Iron-binding 3compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions. J Med Microbiol. 2009;58:765–73.
• [44]Wakabayashi H, Yamauchi K, Kobayashi T, Yaeshima T, Iwatsuki K, Yoshie H. Inhibitory effects of lactoferrin on growth and biofilm formation of Porphyromonas gingivalis and Prevotella intermedia. Antimicrob Agents Chemother. 2009; 53:3308-3316.
• [45]Xu G, Xiong W, Hu Q, Zuo P, Shao B, Lan F, et al. Lactoferrin-derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa. J Appl Microbiol. 2010;109:1311–8.
• [46]la Fuente-Núñez de C, Reffuveille F, Haney EF, Straus SK, Hancock REW. Broad-spectrum anti-biofilm peptide that targets a cellular stress response. PLoS Pathog. 2014; 10:e1004152.
• [47]la Fuente-Núñez de C, Korolik V, Bains M, Nguyen U, Breidenstein EBM, Horsman S, et al. Inhibition of bacterial biofilm formation and swarming motility by a small synthetic cationic peptide. Antimicrob Agents Chemother. 2012;56:2696–704.
• [48]Nagant C, Seil M, Nachtergael A, Dulanto S, Dehaye JP. Contribution of the production of quormones to some phenotypic characteristics of Pseudomonas aeruginosa clinical strains. J Med Microbiol. 2013; 62:951-958.
• [49]Hill D, Rose B, Pajkos A, Robinson M, Bye P, Bell S, et al. Antibiotic susceptabilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions. J Clin Microbiol. 2005;43:5085–90.
• [50]Pamp SJ, Gjermansen M, Johansen HK, Tolker-Nielsen T. Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes. Mol Microbiol. 2008; 68:223-240.
• [51]Herrmann G, Yang L, Wu H, Song Z, Wang H, Høiby N, et al. Colistin-Tobramycin combinations are superior to monotherapy concerning the killing of biofilm Pseudomonas aeruginosa. J Infect Dis. 2010;202:1585–92.
• [52]Eckert R, Brady KM, Greenberg EP, Qi F, Yarbrough DK, He J, et al. Enhancement of antimicrobial activity against Pseudomonas aeruginosa by coadministration of G10KHc and tobramycin. Antimicrob Agents Chemother. 2006;50:3833–8.
• [53]Banin E, Brady KM, Greenberg EP. Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm. Appl Environ Microbiol. 2006; 72:2064-2069.
• [54]Nivens DE, Ohman DE, Williams J, Franklin MJ. Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J Bacteriol. 2001; 183:1047-1057.
• [55]Pitts B, Hamilton MA, Zelver N, Stewart PS. A microtiter-plate screening method for biofilm disinfection and removal. J Microbiol Methods. 2003; 54:269-276.
• [56]Andra J, Monreal D, Martinez de Tejada G, Olak C, Brezesinski G, Gomez SS, et al. Rationale for the design of shortened derivatives of the NK-lysin-derived antimicrobial peptide NK-2 with improved activity against Gram-negative pathogens. J Biol Chem. 2007;282:14719–28.
• [57]O’Toole GA, Kolter R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol. 1998; 30:295-304.
• [58]Goeres DM, Loetterle LR, Hamilton MA, Murga R, Kirby DW, Donlan RM. Statistical assessment of a laboratory method for growing biofilms. Microbiology (Reading, Engl). 2005; 151:757-762.
• [59]Wimley WC, Creamer TP, White SH. Solvation energies of amino acid side chains and backbone in a family of host-guest pentapeptides. Biochemistry. 1996; 35:5109-5124.
• [60]Snider C, Jayasinghe S, Hristova K, White SH. MPEx: a tool for exploring membrane proteins. Protein Sci. 2009; 18:2624-2628.