1887

Abstract

We previously reported a series of engineered cationic antibiotic peptides (eCAPs) consisting exclusively of arginine and tryptophan (WR) that display potent activity against diverse multidrug-resistant (MDR) bacterial strains. In this study, we sought to examine the influence of arginine compared to lysine on antibacterial properties by direct comparison of the WR peptides (8–18 residues) with a parallel series of engineered peptides containing only lysine and tryptophan. WR and WK series were compared for antibacterial activity by bacterial killing and growth inhibition assays and for mechanism of peptide–bacteria interactions by surface plasmon resonance and flow cytometry. Mammalian cytotoxicity was also assessed by flow cytometry, haemolytic and tetrazolium-based assays. The shortest arginine-containing peptides (8 and 10 mers) displayed a statistically significant increase in activity compared to the analogous lysine-containing peptides. The WR and WK peptides achieved maximum antibacterial activity at the 12-mer peptide (WK12 or WR12). Further examination of antibacterial mechanisms of the optimally active 12-mer peptides using surface plasmon resonance and flow cytometry demonstrates stronger interactions with , greater membrane permeabilizing activity, and lower inhibitory effects of divalent cations on activity and membrane permeabilization properties of WR12 compared to WK12 ( < 0.05). Importantly, WK12 and WR12 displayed similar negligible haemolytic and cytotoxic effects at peptide concentrations up to ten times the MIC or 20 times the minimum bactericidal concentration. Thus, arginine, compared to lysine, can indeed yield enhanced antibacterial activity to minimize the required length to achieve functional antimicrobial peptides.

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2016-06-01
2024-10-05
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References

  1. Agodi A., Voulgari E., Barchitta M., Quattrocchi A., Bellocchi P., Poulou A., Santangelo C., Castiglione G., Giaquinta L, Santangelo G. 2014; Spread of a carbapenem- and colistin-resistant Acinetobacter baumannii ST2 clonal strain causing outbreaks in two Sicilian hospitals. J Hosp Infect 86:260–266 [View Article][PubMed]
    [Google Scholar]
  2. Boucher H. W., Talbot G. H., Bradley J. S., Edwards J. E., Gilbert D., Rice L. B., Scheld M., Spellberg B., Bartlett J. 2009; Bad bugs, no drugs: No ESKAPE! an update from the infectious diseases society of america. Clin Infect Dis 48:1–12 [View Article][PubMed]
    [Google Scholar]
  3. Bow E. J. 2013; There should be no ESKAPE for febrile neutropenic cancer patients: The dearth of effective antibacterial drugs threatens anticancer efficacy. J Antimicrob Chemother 68:492–495 [View Article][PubMed]
    [Google Scholar]
  4. Brogden K. A. 2005; Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria?. Nat Rev Microbiol 3:238–250 [View Article][PubMed]
    [Google Scholar]
  5. Bucki R., Pastore J. J., Randhawa P., Vegners R., Weiner D. J., Janmey P. A. 2004; Antibacterial activities of rhodamine b-conjugated gelsolin-derived peptides compared to those of the antimicrobial peptides cathelicidin LL37, magainin II, and melittin. Antimicrob Agents Chemother 48:1526–1533 [View Article][PubMed]
    [Google Scholar]
  6. Clifton L. A., Skoda M. W., Le Brun A. P., Ciesielski F., Kuzmenko I., Holt S. A., Lakey J. H. 2015; Effect of divalent cation removal on the structure of gram-negative bacterial outer membrane models. Langmuir 31:404–412 [View Article][PubMed]
    [Google Scholar]
  7. Dashper S. G., O'Brien-Simpson N. M., Cross K. J., Paolini R. A., Hoffmann B., Catmull D. V., Malkoski M., Reynolds E. C. 2005; Divalent metal cations increase the activity of the antimicrobial peptide kappacin. Antimicrob Agents Chemother (Bethesda) 49:2322–2328 [View Article]
    [Google Scholar]
  8. Deslouches B., Phadke S. M., Lazarevic V., Cascio M., Islam K., Montelaro R. C., Mietzner T. A. 2005; De novo generation of cationic antimicrobial peptides: Influence of length and tryptophan substitution on antimicrobial activity. Antimicrob Agents Chemother 49:316–322 [View Article][PubMed]
    [Google Scholar]
  9. Deslouches B., Steckbeck J. D., Craigo J. K., Doi Y., Burns J. L., Montelaro R. C. 2015; Engineered cationic antimicrobial peptides to overcome multidrug resistance by ESKAPE pathogens. Antimicrob Agents Chemother (Bethesda) 59:1329–1333 [View Article]
    [Google Scholar]
  10. Deslouches B., Steckbeck J. D., Craigo J. K., Doi Y., Mietzner T. A., Montelaro R. C. 2013; Rational design of engineered cationic antimicrobial peptides consisting exclusively of arginine and tryptophan, and their activity against multidrug-resistant pathogens. Antimicrob Agents Chemother (Bethesda) 57:2511–2521 [View Article]
    [Google Scholar]
  11. Ding L., Yang L., Weiss T. M., Waring A. J., Lehrer R. I., Huang H. W. 2003; Interaction of antimicrobial peptides with lipopolysaccharides. Biochemistry 42:12251–12259 [View Article][PubMed]
    [Google Scholar]
  12. Georgescu J., Munhoz V. H., Bechinger B. 2010; NMR structures of the histidine-rich peptide LAH4 in micellar environments: Membrane insertion, ph-dependent mode of antimicrobial action, and DNA transfection. Biophys J 99:2507–2515 [View Article][PubMed]
    [Google Scholar]
  13. Hadnagy W., Seemayer N. H., Happel A., Kiell A. 1993; Human monocyte-derived macrophage cultures: An alternative test system for the detection of pulmonary toxicity induced by inhaled particulate pollutants. Toxicol in Vitro 7:365–371 [View Article][PubMed]
    [Google Scholar]
  14. Hale J. D., Hancock R. E. 2007; Alternative mechanisms of action of cationic antimicrobial peptides on bacteria. Expert Rev Anti Infect Ther 5:951–959 [View Article][PubMed]
    [Google Scholar]
  15. Hancock R. E. 2001; Cationic peptides: Effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 1:156–164 [View Article][PubMed]
    [Google Scholar]
  16. Hancock R. E., Brown K. L., Mookherjee N. 2006; Host defence peptides from invertebrates--emerging antimicrobial strategies. Immunobiology 211:315–322 [View Article][PubMed]
    [Google Scholar]
  17. Hell E., Giske C. G., Nelson A., Römling U., Marchini G. 2010; Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of staphylococcus epidermidis. Lett Appl Microbiol 50:211–215 [View Article][PubMed]
    [Google Scholar]
  18. Ho J. Y., Cira N. J., Crooks J. A., Baeza J., Weibel D. B. 2012; Rapid identification of ESKAPE bacterial strains using an autonomous microfluidic device. PLoS One 7:e41245 [View Article][PubMed]
    [Google Scholar]
  19. Jean-François F., Elezgaray J., Berson P., Vacher P., Dufourc E. J. 2008; Pore formation induced by an antimicrobial peptide: Electrostatic effects. Biophys J 95:5748–5756 [View Article][PubMed]
    [Google Scholar]
  20. Jelokhani-Niaraki M., Nakashima K., Kodama H., Kondo M. 1998; Interaction and orientation of an alpha-aminoisobutyric acid- and tryptophan-containing short helical peptide pore-former in phospholipid vesicles, as revealed by fluorescence spectroscopy. J Biochem 123:790–797 [View Article][PubMed]
    [Google Scholar]
  21. Kalia V., Sarkar S., Gupta P., Montelaro R. C. 2003; Rational site-directed mutations of the LLP-1 and LLP-2 lentivirus lytic peptide domains in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41 indicate common functions in cell-cell fusion but distinct roles in virion envelope incorporation. J Virol 77:3634–3646[PubMed] [CrossRef]
    [Google Scholar]
  22. Khatami M. H., Bromberek M., Saika-Voivod I., Booth V. 2014; Molecular dynamics simulations of histidine-containing cod antimicrobial peptide paralogs in self-assembled bilayers. Biochimica Et Biophysica Acta (BBA) - Biomembranes 1838:2778–2787 [View Article]
    [Google Scholar]
  23. Kulkarni M. M., Karafova A., Kamysz W., McGwire B. S. 2014; Design of protease-resistant pexiganan enhances antileishmanial activity. Parasitol Res 113:1971–1976 [View Article][PubMed]
    [Google Scholar]
  24. Kuster D. J., Liu C., Fang Z., Ponder J. W., Marshall G. R. 2015a; High-resolution crystal structures of protein helices reconciled with three-centered hydrogen bonds and multipole electrostatics. PLoS One 10:e0123146 [View Article]
    [Google Scholar]
  25. Kuster D. W., Govindan S., Springer T. I., Martin J. L., Finley N. L., Sadayappan S. 2015b; A hypertrophic cardiomyopathy-associated MYBPC3 mutation common in populations of South Asian descent causes contractile dysfunction. J Biol Chem 290:5855–5867 [CrossRef]
    [Google Scholar]
  26. Lai Y., Adhikarakunnathu S., Bhardwaj K., Ranjith-Kumar C. T., Wen Y., Jordan J. L., Wu L. H., Dragnea B., San Mateo L., Kao C. C. 2011; LL37 and cationic peptides enhance TLR3 signaling by viral double-stranded RNAs. PLoS One 6:e26632 [View Article][PubMed]
    [Google Scholar]
  27. Lamb H. M., Wiseman L. R. 1998; Pexiganan acetate. Drugs 56:1047–1052 [View Article][PubMed]
    [Google Scholar]
  28. Lequin O., Ladram A., Chabbert L., Bruston F., Convert O., Vanhoye D., Chassaing G., Nicolas P, Amiche M.. 2006; Dermaseptin S9, an alpha-helical antimicrobial peptide with a hydrophobic core and cationic termini. Biochemistry 45:468–480 [View Article][PubMed]
    [Google Scholar]
  29. Lipsky B. A., Holroyd K. J., Zasloff M. 2008; Topical versus systemic antimicrobial therapy for treating mildly infected diabetic foot ulcers: A randomized, controlled, double-blinded, multicenter trial of pexiganan cream. Clin Infect Dis 47:1537–1545 [View Article][PubMed]
    [Google Scholar]
  30. Magiorakos A. P., Srinivasan A., Carey R. B., Carmeli Y., Falagas M. E., Giske C. G., Harbarth S., Hindler J. F., Kahlmeter G, other authers. 2012; Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268–281 [View Article][PubMed]
    [Google Scholar]
  31. Mason A. J., Bertani P., Moulay G., Marquette A., Perrone B., Drake A. F., Kichler A., Bechinger B. 2007; Membrane interaction of chrysophsin-1, a histidine-rich antimicrobial peptide from red sea bream. Biochemistry 46:15175–15187 [View Article][PubMed]
    [Google Scholar]
  32. Mihajlovic M., Lazaridis T, Lazaridis. 2010; Antimicrobial peptides in toroidal and cylindrical pores. Biochim Biophys Acta 1798:1485–1493 [View Article][PubMed]
    [Google Scholar]
  33. Mitchell D. J., Kim D. T., Steinman L., Fathman C. G., Rothbard J. B, Steinman. 2000; Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 56:318–325 [View Article][PubMed]
    [Google Scholar]
  34. Ozcan A. V., Demir M., Onem G., Goksin I., Baltalarli A., Topkara V. K., Kaleli I. 2006; Topical versus systemic vancomycin for deep sternal wound infection caused by methicillin-resistant staphylococcus aureus in a rodent experimental model. Tex Heart Inst J 33:107–110[PubMed]
    [Google Scholar]
  35. Park S. C., Kim M. H., Hossain M. A., Shin S. Y., Kim Y., Stella L., Wade J. D., Park Y., Hahm K. S.. 2008; Amphipathic alpha-helical peptide, HP (2-20), and its analogues derived from helicobacter pylori: Pore formation mechanism in various lipid compositions. Biochim Biophys Acta 1778:229–241 [View Article][PubMed]
    [Google Scholar]
  36. Park Y., Lee D. G., Jang S. H., Woo E. R., Jeong H. G., Choi C. H., Hahm K. S. 2003; A leu-lys-rich antimicrobial peptide: Activity and mechanism. Biochim Biophys Acta 1645:172–182 [View Article][PubMed]
    [Google Scholar]
  37. Perfetto S. P., Chattopadhyay P. K., Lamoreaux L., Nguyen R., Ambrozak D., Koup R. A., Roederer M. 2006; Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry. J Immunol Methods 313:199–208 [View Article][PubMed]
    [Google Scholar]
  38. Peschel A., Sahl H. G. 2006; The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nat Rev Microbiol 4:529–536 [View Article][PubMed]
    [Google Scholar]
  39. Pezzulo A. A., Tang X. X., Hoegger M. J., Abou Alaiwa M. H., Ramachandran S., Moninger T. O., Karp P. H., Wohlford-Lenane C. L., Haagsman H. P., other authors. 2012; Reduced airway surface ph impairs bacterial killing in the porcine cystic fibrosis lung. Nat New Biol 487:109–113 [CrossRef]
    [Google Scholar]
  40. Pfaller M. A., Boyken L., Hollis R. J., Messer S. A., Tendolkar S., Diekema D. J. 2005; In vitro susceptibilities of clinical isolates of candida species, cryptococcus neoformans, and aspergillus species to itraconazole: Global survey of 9,359 isolates tested by clinical and laboratory standards institute broth microdilution methods. J Clin Microbiol 43:3807–3810 [View Article][PubMed]
    [Google Scholar]
  41. Phadke S. M., Deslouches B., Hileman S. E., Montelaro R. C., Wiesenfeld H. C., Mietzner T. A. 2005; Antimicrobial peptides in mucosal secretions: The importance of local secretions in mitigating infection. J Nutr 135:1289–1293[PubMed]
    [Google Scholar]
  42. Phadke S. M., Islam K., Deslouches B., Kapoor S. A., Beer Stolz D., Watkins S. C., Montelaro R. C., Pilewski J. M., Mietzner T. A. 2003; Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model. Peptides 24:1099–1107 [View Article][PubMed]
    [Google Scholar]
  43. Phadke S. M., Lazarevic V., Bahr C. C., Islam K., Stolz D. B., Watkins S., Tencza S. B., Vogel H. J., Montelaro R. C., Mietzner T. A. 2002; Lentivirus lytic peptide 1 perturbs both outer and inner membranes of serratia marcescens. Antimicrob Agents Chemother 46:2041–2045 [View Article]
    [Google Scholar]
  44. Qiu S., Yi H., Hu J., Cao Z., Wu Y., Li W. 2012; The binding mode of fusion inhibitor T20 onto HIV-1 gp41 and relevant t20-resistant mechanisms explored by computational study. Curr HIV Res 10:182–194 [View Article][PubMed]
    [Google Scholar]
  45. Sader H. S., Ferraro M. J., Reller L. B., Schreckenberger P. C., Swenson J. M., Jones R. N. 2007; Reevaluation of clinical and laboratory standards institute disk diffusion breakpoints for tetracyclines for testing enterobacteriaceae. J Clin Microbiol 45:1640–1643 [View Article][PubMed]
    [Google Scholar]
  46. Schibli D. J., Epand R. F., Vogel H. J., Epand R. M. 2002; Tryptophan-rich antimicrobial peptides: Comparative properties and membrane interactions. Biochem Cell Biol 80:667–677 [View Article][PubMed]
    [Google Scholar]
  47. Sekimata M., Homma Y, Wang Z., Wang G.. 2004; Sequence-specific transcriptional repression by an mbd2-interacting zinc finger protein MIZF. Nucleic Acids Res 32:590–592 (Database issue), D [View Article][PubMed]
    [Google Scholar]
  48. Seshadri Sundararajan V., Gabere M. N., Pretorius A., Adam S., Christoffels A., Lehväslaiho M., Archer J. A., Bajic V. B. 2012; DAMPD: A manually curated antimicrobial peptide database. Nucleic Acids Res 40:D1108–D1112 [View Article][PubMed]
    [Google Scholar]
  49. Shanmugam G., Polavarapu P. L., Gopinath D., Jayakumar R. 2005; The structure of antimicrobial pexiganan peptide in solution probed by fourier transform infrared absorption, vibrational circular dichroism, and electronic circular dichroism spectroscopy. Biopolymers 80:636–642 [View Article][PubMed]
    [Google Scholar]
  50. Shi S. H., Kong H. S., Xu J., Zhang W. J., Jia C. K., Wang W. L., Shen Y., Zhang M., Zheng S. S. 2009; Multidrug resistant gram-negative bacilli as predominant bacteremic pathogens in liver transplant recipients. Transpl Infect Dis 11:405–412 [View Article][PubMed]
    [Google Scholar]
  51. Skinner M. C., Kiselev A. O., Isaacs C. E., Mietzner T. A., Montelaro R. C., Lampe M. F. 2010; Evaluation of WLBU2 peptide and 3-o-octyl-sn-glycerol lipid as active ingredients for a topical microbicide formulation targeting chlamydia trachomatis. Antimicrob Agents Chemother (Bethesda) 54:627–636 [View Article]
    [Google Scholar]
  52. Steckbeck J. D., Deslouches B., Montelaro R. C. 2014; Antimicrobial peptides: New drugs for bad bugs?. Expert Opin Biol Ther 14:11–14 [View Article][PubMed]
    [Google Scholar]
  53. Straus S. K., Hancock R. E. W. 2006; Mode of action of the new antibiotic for gram-positive pathogens daptomycin: Comparison with cationic antimicrobial peptides and lipopeptides. Biochimica Et Biophysica Acta (BBA) - Biomembranes 1758:1215–1223 [View Article]
    [Google Scholar]
  54. Su Y., Doherty T., Waring A. J., Ruchala P., Hong M. 2009; Roles of arginine and lysine residues in the translocation of a cell-penetrating peptide from (13)C, (31)P, and (19)F solid-state NMR. Biochemistry 48:4587–4595 [View Article][PubMed]
    [Google Scholar]
  55. Sugiarto H., Yu P. L. 2007; Effects of cations on antimicrobial activity of ostricacins-1 and 2 on E. coli O157:H7 and S. aureus 1056MRSA. Curr Microbiol 55:36–41 [View Article][PubMed]
    [Google Scholar]
  56. Swoboda J. G., Campbell J., Meredith T. C., Walker S, Campbell. 2010; Wall teichoic acid function, biosynthesis, and inhibition. Chembiochem 11:35–45 [View Article][PubMed]
    [Google Scholar]
  57. Tencza S. B., Creighton D. J., Yuan T., Vogel H. J., Montelaro R. C., Mietzner T. A. 1999; Lentivirus-derived antimicrobial peptides: Increased potency by sequence engineering and dimerization. J Antimicrob Chemother 44:33–41 [View Article][PubMed]
    [Google Scholar]
  58. Tencza S. B., Douglass J. P., Creighton D. J., Montelaro R. C., Mietzner T. A. 1997; Novel antimicrobial peptides derived from human immunodeficiency virus type 1 and other lentivirus transmembrane proteins. Antimicrob Agents Chemother 41:2394–2398[PubMed]
    [Google Scholar]
  59. Tencza S. B., Miller M. A., Islam K., Mietzner T. A., Montelaro R. C. 1995; Effect of amino acid substitutions on calmodulin binding and cytolytic properties of the LLP-1 peptide segment of human immunodeficiency virus type 1 transmembrane protein. J Virol 69:5199–5202[PubMed]
    [Google Scholar]
  60. Thomas K. J., Rice C. V. 2014; Revised model of calcium and magnesium binding to the bacterial cell wall. Biometals 27:1361–1370 [View Article][PubMed]
    [Google Scholar]
  61. Tossi A., Sandri L., Giangaspero A. 2000; Amphipathic, alpha-helical antimicrobial peptides. Biopolymers 55:4–30 [View Article][PubMed]
    [Google Scholar]
  62. Varney M. L., Olsen K. J., Mosley R. L., Bucana C. D., Talmadge J. E., Singh R. K. 2002; Monocyte/macrophage recruitment, activation and differentiation modulate interleukin-8 production: A paracrine role of tumor-associated macrophages in tumor angiogenesis. In Vivo 16:471– 4710
    [Google Scholar]
  63. Wang G. 2008; Structures of human host defense cathelicidin LL-37 and its smallest antimicrobial peptide KR-12 in lipid micelles. J Biol Chem 283:32637–32643 [View Article][PubMed]
    [Google Scholar]
  64. Wang G., Li X., Wang Z, Li X., Wang Z.. 2009; APD2: The updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Res 37:D933–D937 (Database issue), D [View Article]
    [Google Scholar]
  65. Zhang L., Parente J., Harris S. M., Woods D. E., Hancock R. E. W., Falla T. J. 2005; Antimicrobial peptide therapeutics for cystic fibrosis. Antimicrob Agents Chemother (Bethesda) 49:2921–2927 [View Article]
    [Google Scholar]
  66. Zhang Y., Shi W., Tang S., Li J., Yin S., Gao X., Wang L., Zou L., Zhao J., other authors. 2013; The influence of cathelicidin LL37 in human anti-neutrophils cytoplasmic antibody (anca)-associated vasculitis. Arthritis Res & Ther 15:R161 [View Article][PubMed]
    [Google Scholar]
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