Applied Microbiology and Biotechnology

, Volume 103, Issue 13, pp 5193–5213 | Cite as

A recombinant fungal defensin-like peptide-P2 combats multidrug-resistant Staphylococcus aureus and biofilms

  • Na Yang
  • Da Teng
  • Ruoyu Mao
  • Ya Hao
  • Xiao Wang
  • Zhenlong Wang
  • Xiumin WangEmail author
  • Jianhua WangEmail author
Biotechnological products and process engineering


There is an urgent need to discover new active drugs to combat methicillin-resistant Staphylococcus aureus, which is a serious threat to humans and animals and incompletely eliminated by antibiotics due to its intracellular accumulation in host cells, production of biofilms, and persisters. Fungal defensin-like peptides (DLPs) are emerging as a potential source of new antibacterial drugs due to their potent antibacterial activity. In this study, nine novel fungal DLPs were firstly identified by querying against UniProt databases and expressed in Pichia pastoris, and their antibacterial and anti-biofilm ability were tested against multidrug-resistant (MDR) S. aureus. Results showed that among them, P2, the highest activity and expression level, showed low toxicity, no resistance, and high stability. Minimal inhibitory concentrations (MICs) of P2 against Gram-positive bacteria were < 2 μg/mL. P2 exhibited the potent activity against intracellular MDR S. aureus (bacterial reduction in 80–97%) in RAW264.7 macrophages. P2 bound to/disrupted bacterial DNA, wrinkled outer membranes and permeabilized cytoplasmic membranes, but maintained the integrity of bacterial cells. P2 inhibited/eradicated the biofilm and killed 99% persister bacteria, which were resistant to 100× MIC vancomycin. P2 upregulated the anti-inflammatory cytokine (IL-10) and downregulated pro-inflammatory cytokines (TNF-α/IL-1β) and chemokine (MCP-1) levels in RAW 264.7 macrophages and in mice, respectively. Five milligram per kilogram P2 enhanced the survival of S. aureus-infected mice (100%), superior to vancomycin (30 mg/kg), inhibited the bacterial translocation, and alleviated multiple-organ injuries (liver, spleen, kidney, and lung). These data suggest that P2 may be a candidate for novel antimicrobial agents against MDR staphylococcal infections.


Recombinant Defensin-like peptides S. aureus Intracellular activity Anti-biofilm ability Mechanism of action 


Authors’ contributions

Na Yang, Xiumin Wang, Ruoyu Mao, Da Teng, and Jianhua Wang conceived and designed experiments. Na Yang carried out all the experiments. Na Yang, Xiumin Wang, and Jianhua Wang contributed in writing. Jianhua Wang contributed in funding acquisition. Ya Hao and Xiao Wang contributed to materials and reagents. Zhenlong Wang contributed in modifying figure.

Funding information

This study was supported by the National Natural Science Foundation of China (Grants 31672456, 31572444, 31572445, and 31372346), the Project of the National Support Program for Science and Technology in China (Grant 2013BAD10B02), the AMP Direction of the National Innovation Program of Agricultural Science and Technology in CAAS (Grant CAAS-ASTIP-2013-FRI-02), and its Key Project of Alternatives to Antibiotics for Feed Usages (Grant CAAS-ZDXT2018008).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical statement

The mouse experiment was performed according to the Animal Care and Use Committee of the Feed Research Institute of Chinese Academy of Agricultural Sciences (CAAS) and approved by the Laboratory Animal Ethical Committee and its Inspection of the Feed Research Institute of CAAS (AEC-CAAS-20090609).

Supplementary material

253_2019_9785_MOESM1_ESM.pdf (629 kb)
ESM 1 (PDF 628 kb)


  1. Ahn KB, Baik JE, Yun CH, Han SH (2018) Lipoteichoic acid inhibits Staphylococcus aureus biofilm formation. Front Microbiol 9:327Google Scholar
  2. Anitha P, Anbarasu A, Ramaiah S (2016) Gene network analysis reveals the association of important functional partners involved in antibiotic resistance: a report on an important pathogenic bacterium Staphylococcus aureus. Gene 575(2 Pt 1):253–263Google Scholar
  3. Batta G, Barna T, Gáspári Z, Sándor S, Kövér KE, Binder U, Sarg B, Kaiserer L, Chhillar AK, Eigentler A, Leiter E, Hegedüs N, Pócsi I, Lindner H, Marx F (2009) Functional aspects of the solution structure and dynamics of PAF-a highly-stable antifungal protein from Penicillium chrysogenum. FEBS J 276(10):2875–2890Google Scholar
  4. Beenken KE, Blevins JS, Smeltzer MS (2003) Mutation of sarA in Staphylococcus aureus limits biofilm formation. Infect Immun 71(7):4206–4211Google Scholar
  5. Brinch KS, Sandberg A, Baudoux P, Van Bambeke F, Tulkens PM, Frimodt-Møller N, Høiby N, Kristensen HH (2009) Plectasin shows intracellular activity against Staphylococcus aureus in human THP-1 monocytes and in a mouse peritonitis model. Antimicrob Agents Chemother 53(11):4801–4808Google Scholar
  6. Bulmer MS, Crozier RH (2004) Duplication and diversifying selection among termite antifungal peptides. Mol Biol Evol 21(12):2256–2264Google Scholar
  7. Cameron DR, Howden BP, Peleg AY (2011) The interface between antibiotic resistance and virulence in Staphylococcus aureus and its impact upon clinical outcomes. Clin Infect Dis 53(6):576–582Google Scholar
  8. Chen H, Mao R, Teng D, Wang X, Hao Y, Feng X, Wang J (2017) Design and pharmacodynamics of recombinant NZ2114 histidine mutants with improved activity against methicillin-resistant Staphylococcus aureus. AMB Express 7(1):46Google Scholar
  9. Cociancich S, Ghazi A, Hetru C, Hoffmann JA, Letellier L (1993) Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J Biol Chem 268(26):19239–19245Google Scholar
  10. Cornut I, Büttner K, Dasseux JL, Dufourcq J (1994) The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin. FEBS Lett 349(1):29–33Google Scholar
  11. Davies D (2003) Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov 2(2):114–122Google Scholar
  12. de Breij A, Riool M, Kwakman PH, de Boer L, Cordfunke RA, Drijfhout JW, Cohen O, Emanuel N, Zaat SA, Nibbering PH, Moriarty TF (2016) Prevention of Staphylococcus aureus biomaterial-associated infections using a polymer-lipid coating containing the antimicrobial peptide OP-145. J Control Release 222:1–8Google Scholar
  13. de Breij A, Riool M, Cordfunke RA, Malanovic N, de Boer L, Koning RI, Ravensbergen E, Franken M, van der Heijde T, Boekema BK, Kwakman PHS, Kamp N, El Ghalbzouri A, Lohner K, Zaat SAJ, Drijfhout JW, Nibbering PH (2018) The antimicrobial peptide SAAP-148 combats drug-resistant bacteria and biofilms. Sci Transl Med 10(423):pii:eaan4044Google Scholar
  14. Foster TJ, Geoghegan JA, Ganesh VK, Höök M (2014) Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus. Nat Rev Microbiol 12(1):49–62Google Scholar
  15. Glukhov E, Burrows LL, Deber CM (2008) Membrane interactions of designed cationic antimicrobial peptides: the two thresholds. Biopolymers 89(5):360–371Google Scholar
  16. Guérillot R, Gonçalves da Silva A, Monk I, Giulieri S, Tomita T, Alison E, Porter J, Pidot S, Gao W, Peleg AY, Seemann T, Stinear TP, Howden BP (2018) Convergent evolution driven by rifampin exacerbates the global burden of drug-resistant Staphylococcus aureus. Msphere 3(1) pii:e00550-00517Google Scholar
  17. Hajji M, Jellouli K, Hmidet N, Balti R, Sellami-Kamoun A, Nasri M (2010) A highly thermostable antimicrobial peptide from Aspergillus clavatus ES1: biochemical and molecular characterization. J Ind Microbiol Biotechnol 37(8):805–813Google Scholar
  18. Hara S, Mukae H, Sakamoto N, Ishimoto H, Amenomori M, Fujita H, Ishimatsu Y, Yanagihara K, Kohno S (2008) Plectasin has antibacterial activity and no affect on cell viability or IL-8 production. Biochem Biophys Res Commun 374(4):709–713Google Scholar
  19. Hirakura Y, Kobayashi S, Matsuzaki K (2002) Specific interactions of the antimicrobial peptide cyclic beta-sheet tachyplesin I with lipopolysaccharides. Biochim Biophys Acta 1562(1–2):32–36Google Scholar
  20. Jones SM, Morgan M, Humphrey TJ, Lappin-Scott H (2001) Effect of vancomycin and rifampicin on meticillin-resistant Staphylococcus aureus biofilms. Lancet 357(9249):40–41Google Scholar
  21. Klein K, Grønnemose RB, Alm M, Brinch KS, Kolmos HJ, Andersen TE (2017) Controlled release of plectasin NZ2114 from a hybrid silicone-hydrogel material for inhibition of Staphylococcus aureus biofilm. Antimicrob Agents Chemother 61(7):e00604–e00617Google Scholar
  22. Landis RF, Li CH, Gupta A, Lee YW, Yazdani M, Ngernyuang N, Altinbasak I, Mansoor S, Khichi MAS, Sanyal A, Rotello VM (2018) Biodegradable nanocomposite antimicrobials for the eradication of multidrug-resistant bacterial biofilms without accumulated resistance. J Am Chem Soc 140(19):6176–6182Google Scholar
  23. Laxminarayan R, Heymann DL (2012) Challenges of drug resistance in the developing world. BMJ 344:e1567Google Scholar
  24. Lee JH, Park JH, Cho HS, Joo SW, Cho MH, Lee J (2013) Anti-biofilm activities of quercetin and tannic acid against Staphylococcus aureus. Biofouling 29(5):491–499Google Scholar
  25. Lee JK, Seo CH, Luchian T, Park Y (2015) Antimicrobial peptide CMA3 derived from the CA-MA hybrid peptide: antibacterial and anti-inflammatory activities with low cytotoxicity and mechanism of action in Escherichia coli. Antimicrob Agents Chemother 60(1):495–506Google Scholar
  26. Li J, Jiang N, Ke Y, Feßler AT, Wang Y, Schwarz S, Wu C (2017a) Characterization of pig-associated methicillin-resistant Staphylococcus aureus. Vet Microbiol 201:183–187Google Scholar
  27. Li ZZ, Wang XM, Wang X, Teng D, Mao RY, Hao Y, Wang JH (2017b) Research advances on plectasin and its derivatives as new potential antimicrobial candidates. Process Biochem 56:62–70Google Scholar
  28. Li Z, Mao R, Teng D, Hao Y, Chen H, Wang X, Wang X, Yang N, Wang J (2017c) Antibacterial and immunomodulatory activities of insect defensins-DLP2 and DLP4 against multidrug-resistant Staphylococcus aureus. Sci Rep 7(1):12124Google Scholar
  29. Lister JL, Horswill AR (2014) Staphylococcus aureus biofilms: recent developments in biofilm dispersal. Front Cell Infect Microbiol 4:178Google Scholar
  30. Morrisett JD, David JS, Pownall HJ, Jr GA (1973) Interaction of an apolipoprotein (apoLP-alanine) with phosphatidylcholine. Biochemistry 12(7):1290–1299Google Scholar
  31. Mygind PH, Fischer RL, Schnorr KM, Hansen MT, Sönksen CP, Ludvigsen S, Raventós D, Buskov S, Christensen B, De Maria L, Taboureau O, Yaver D, Elvig-Jørgensen SG, Sørensen MV, Christensen BE, Kjaerulff S, Frimodt-Moller N, Lehrer RI, Zasloff M, Kristensen HH (2005) Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature 437(7061):975–980Google Scholar
  32. Oeemig JS, Lynggaard C, Knudsen DH, Hansen FT, Nørgaard KD, Schneider T, Vad BS, Sandvang DH, Nielsen LA, Neve S, Kristensen HH, Sahl HG, Otzen DE, Wimmer R (2012) Eurocin, a new fungal defensin. J Biol Chem 287(50):42361–42372Google Scholar
  33. Pantic JM, Mechkarska M, Lukic ML, Conlon JM (2014) Effects of tigerinin peptides on cytokine production by mouse peritoneal macrophages and spleen cells and by human peripheral blood mononuclear cells. Biochimie 101:83–92Google Scholar
  34. Park CB, Yi KS, Matsuzaki K, Kim MS, Kim SC (2000) Structure–activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline hinge is responsible for the cell-penetrating ability of buforin II. Proc Natl Acad Sci U S A 97(15):8245–8250Google Scholar
  35. Pivkin IV, Peng Z, Karniadakis GE, Buffet PA, Dao M, Suresh S (2016) Biomechanics of red blood cells in human spleen and consequences for physiology and disease. Proc Natl Acad Sci U S A 113(28):7804–7809Google Scholar
  36. Rughani RV, Schneider JP (2008) Molecular design of beta-hairpin peptides for material construction. MRS Bull 33(5):530–535Google Scholar
  37. Sagaram US, El-Mounadi K, Buchko GW, Berg HR, Kaur J, Pandurangi RS, Smith TJ, Shah DM (2013) Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: identification of an RGFRRR motif governing fungal cell entry. PLoS One 8(12):e82485Google Scholar
  38. Sarkar P, Acharyya S, Banerjee A, Patra A, Thankamani K, Koley H, Bag PK (2016) Intracellular, biofilm-inhibitory and membrane-damaging activities of nimbolide isolated from Azadirachta indica a. Juss (Maliaceae) against methicillin-resistant Staphylococcus aureus. J Med Microbiol 65(10):1205–1214Google Scholar
  39. Schneider T, Kruse T, Wimmer R, Wiedemann I, Sass V, Pag U, Jansen A, Nielsen AK, Mygind PH, Raventós DS, Neve S, Ravn B, Bonvin AM, De Maria L, Andersen AS, Gammelgaard LK, Sahl HG, Kristensen HH (2010) Plectasin, a fungal defensin, targets the bacterial cell wall precursor lipid II. Science 328(5982):1168–1172Google Scholar
  40. Stewart P, Costerton JW (2001) Antibiotic resistance of bacteria in biofilms. Lancet 358(9276):135–138Google Scholar
  41. Takahashi D, Shukla SK, Prakash O, Zhang G (2010) Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity. Biochimie 92(9):1236–1241Google Scholar
  42. Tiwari JG (2013) Trends in therapeutic and prevention strategies for management of bovine mastitis: an overview. J Vaccines Vaccin 4(2):1000176Google Scholar
  43. Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr (2015) Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 28(3):603–661Google Scholar
  44. Trotonda MP, Manna AC, Cheung AL, Lasa I, Penadés JR (2005) SarA positively controls bap-dependent biofilm formation in Staphylococcus aureus. J Bacteriol 187(16):5790–5798Google Scholar
  45. Uyterhoeven ET, Butler CH, Ko D, Elmore DE (2008) Investigating the nucleic acid interactions and antimicrobial mechanism of buforin II. FEBS Lett 582(12):1715–1718Google Scholar
  46. Valle J, Toledo-Arana A, Berasain C, Ghigo JM, Amorena B, Penadés JR, Lasa I (2003) SarA and not sigmaB is essential for biofilm development by Staphylococcus aureus. Mol Microbiol 48(4):1075–1087Google Scholar
  47. Wang X, Teng D, Mao R, Yang N, Hao Y, Wang J (2016) Combined systems approaches reveal a multistage mode of action of a marine antimicrobial peptide against pathogenic Escherichia coli and its protective effect against bacterial peritonitis and endotoxemia. AAC 61(1):1–20Google Scholar
  48. Wang X, Wang X, Teng D, Mao R, Hao Y, Yang N, Li Z, Wang J (2018) Increased intracellular activity of MP1102 and NZ2114 against Staphylococcus aureus in vitro and in vivo. Sci Rep 8(1):4204Google Scholar
  49. Wei W, Behloul N, Baha S, Liu Z, Aslam MS, Meng J (2018) Dimerization: a structural feature for the protection of hepatitis E virus capsid protein against trypsinization. Sci Rep 8(1):S786–S787Google Scholar
  50. Wiegand I, Hilpert K, Hancock REW (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3(2):163–175Google Scholar
  51. Wommack AJ, Robson SA, Wanniarachchi YA, Wan A, Turner CJ, Wagner G, Nolan EM (2012) NMR solution structure and condition-dependent oligomerization of the antimicrobial peptide human defensin 5. Biochem 51(48):9624–9637Google Scholar
  52. Woods AS, Huestis MA (2001) A study of peptide-peptide interaction by matrix-assisted laser desorption/ionization. J Am Soc Mass Spectrom 12(1):88–96Google Scholar
  53. Wu G, Ding J, Li H, Li L, Zhao R, Shen Z, Fan X, Xi T (2008) Effects of cations and pH on antimicrobial activity of thanatin and s-thanatin against Escherichia coli ATCC25922 and B. subtilis ATCC21332. Curr Microbiol 57(6):552–557Google Scholar
  54. Wu G, Wu P, Xue X, Yan X, Liu S, Zhang C, Shen Z, Xi T (2013) Application of S-thanatin, an antimicrobial peptide derived from thanatin, in mouse model of Klebsiella pneumoniae infection. Peptides 45:73–77Google Scholar
  55. Wu J, Gao B, Zhu S (2014) The fungal defensin family enlarged. Pharmaceuticals 7(8):866–880Google Scholar
  56. Yang N, Liu X, Teng D, Li Z, Wang X, Mao R, Wang X, Hao Y, Wang J (2017) Antibacterial and detoxifying activity of NZ17074 analogues with multi-layers of selective antimicrobial actions against Escherichia coli and Salmonella enteritidis. Sci Rep 7(1):3392Google Scholar
  57. Yin LM, Edwards MA, Li J, Yip CM, Deber CM (2012) Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions. J Biol Chem 287(10):7738–7745Google Scholar
  58. Yount NY, Yeaman MR, Kaback HR (2004) Multidimensional signatures in antimicrobial peptides. Proc Natl Acad Sci U S A 101(19):7363–7368Google Scholar
  59. Zhang J, Yang Y, Teng D, Tian Z, Wang S, Wang J (2011) Expression of plectasin in Pichia pastoris and its characterization as a new antimicrobial peptide against Staphyloccocus and Streptococcus. Protein Expr Purif 78(2):189–196Google Scholar
  60. Zhu S (2008) Discovery of six families of fungal defensin-like peptides provides insights into origin and evolution of the CSαβ defensins. Mol Immunol 45(3):828–838Google Scholar
  61. Zhu S, Gao B, Tytgat J (2005) Phylogenetic distribution, functional epitopes and evolution of the CSαβ superfamily. Cell Mol Life Sci 62(19–20):2257–2269Google Scholar
  62. Zhu S, Gao B, Harvey PJ, Craik DJ (2012) Dermatophytic defensin with antiinfective potential. Proc Natl Acad Sci U S A 109(22):8495–8500Google Scholar
  63. Zhu X, Zhang L, Wang J, Zhi M, Xu W, Li J, Shan AS (2015) Characterization of antimicrobial activity and mechanisms of low amphipathic peptides with different α-helical propensity. Acta Biomater 18(04):155–167Google Scholar
  64. Zielinska AK, Beenken KE, Mrak LN, Spencer HJ, Post GR, Skinner RA, Tackett AJ, Horswill AR, Smeltzer MS (2012) sarA-mediated repression of protease production plays a key role in the pathogenesis of Staphylococcus aureus USA300 isolates. Mol Microbiol 86(5):1183–1196Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Team of Alternatives to Antibiotics, Gene Engineering Laboratory, Feed Research InstituteChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  2. 2.Key Laboratory of Feed BiotechnologyMinistry of Agriculture and Rural AffairsBeijingPeople’s Republic of China

Personalised recommendations