Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells


Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Arnér ESJ, Holmgren A (2000) Physiological functions of thioredoxin and thioredoxin reductase. Eur J Biochem 267:6102–6109

  2. Bexfield A, Nigam Y, Thomas S, Ratcliffe NA (2004) Detection and partial characterisation of two antibacterial factors from the excretions/secretions of the medicinal maggot Lucilia sericata and their activity against methicillin-resistant Staphylococcus aureus. Microbes Infect 6:1297–1304

  3. Choi CS, Lee IH, Kim E, Kim HR (2000) Antibacterial properties and partial cDNA sequences of secropin-like antibacterial peptides from the common cutworm, Spodoptera litura. Comp Biochem Physiol C Toxicol Pharmacol 125:287–297

  4. Doverskog M, Han L, Häggström L (1998) Cystine/cysteine metabolism in cultured Sf9 cells: influence of cell physiology on biosynthesis, amino acid uptake and growth. Cytotechnology 26:91–102

  5. Hancock REW, Diamond G (2000) The roles of cationic antimicrobial peptides in innate host defences. Trends Microbiol 8:402–410

  6. Henriksson H, Deniman SE, Campuzano IDG, Ademark P, Master ER, Teeri TT, Brumer H (2003) N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglyc endotransglycosylase 16A. Biochem J 375:61–73

  7. Holme T, Arvidsson S, Lindholm B, Pavlu B (1970) Enzymes: laboratory-scale production. Process Biochem 5:62–66

  8. Jin Z, Melaragno MG, Liao DF, Yan C, Haendeler J, Suh Y (2000) Cyclophilin A is a secreted growth factor induced by oxidative stress. Circ Res 87:789–796

  9. Kang D, Liu G, Gunne H, Steiner H (1996) PCR differential display of immune gene expression in Trichoplusia ni. Insect Biochem Mol Biol 26:177–184

  10. Kieffer A, Goumon Y, Ruh O, Chasserot-Golaz S, Nullans G, Gasnier C, Aunis D, Metz-Boutigue M (2003) The N- and C-terminal fragments of ubiquitin are important for the antimicrobial activities. FASEB J 17

  11. Kim HS, Yoon H, Minn Il, Park CB, Lee WT, Zasloff M, Kim SC (2000) Pepsin-mediated processing of the cytoplasmic histone H2A to strong antimicrobial peptide buforin I. J Immunol 165:3268–3274

  12. Kragol G, Lovas S, Varadi G, Condie BA, Hoffman R, Otvos L (2001) The antimibacterial pyrrhocoricin inhibits the ATPase action of DnaK and prevents chaperon-assisted protein folding. Biochemistry 40:3016–3026

  13. Lundström A, Liu G, Kang D, Berzins K, Steiner H (2002) Trichoplusia ni gloverin, an inducable immune gene encoding an antibacterial insect protein. Insect Biochem Mol Biol 32:795–801

  14. Mangoni ML, Papo N, Barra D, Simmaco M, Bozzi A, Di Giulio A, Rinaldi AC (2004) Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability and viability of Escherichia coli. Biochem J 380:859–865

  15. Marshall SH, Arenas G (2003) Antimicrobial peptides: a natural alternative to chemical antibiotics and a potential for applied biotechnology. Electron J Biotechnol 6:2

  16. Master E, Rudsander UJ, Zhou W, Henriksson H, Divne C, Denman S, Wilson DB, Teeri TT (2004) Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides. Biochemistry 43:10080–10089

  17. Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038

  18. Mendes MA, de Souza BM, Marques MR, Palma MS (2004) Structural and biological characterization of two novel peptides from the venom of the neotropical social wasp Agelaia pallipes pallipes. Toxicon 44:67–74

  19. Motobu M, Amer S, Yamada M, Nakamura K, Saido-Sakanaka H, Asaoka A, Yamakawa M, Hirota Y (2003) Effects of antimicrobial peptides derived from the beetle Allomyrina dichotoma defensin on mouse peritoneal macrophages stimulated with lipopolysaccharide. J Vet Med Sci 66:319–322

  20. Park CB, Kim HS, Kim SC (1998) Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochem Biophys Res 244:253–257

  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

  22. Volkoff A, Rocher J, d’Alencon E, Bouton M, Landais I, Quesada-Moraga E, Vey A, Fournier P, Mita K, Devauchelle G (2003) Characterization and transcriptional profiles of three Spodoptera frugiperda genes encoding cysteine-rich peptides. A new class of defensin-like genes from lepidopteran insects? Gene 319:43–53

  23. Wang Y, Griffiths WJ, Jörnvall H, Agerberth B, Johansson J (2002) Antibacterial peptides in stimulated human granulocytes. Characterization of ubiquinated histone H1A. Eur J Biochem 269:512–518

  24. Winder D, Günzburg WH, Erfle V, Salmons B (1998) Expression of antimicrobial peptides has an antitumour effect in human cells. Biochem Bioph Res 242:608–612

Download references


This work was supported by a grant from the Swedish Center for Bioprocess Technology, VINNOVA and KaroBio AB. We thank Dr. Harry Brumer (KTH Biotechnology) for help with experimental design and assistance with manuscript preparation. We also want to thank Åsa Nossed for her greatly appreciated assistance in this study.

Author information

Correspondence to Lena Häggström.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Svensson, I., Calles, K., Lindskog, E. et al. Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells. Appl Microbiol Biotechnol 69, 92–98 (2005). https://doi.org/10.1007/s00253-005-1958-6

Download citation


  • Antimicrobial Activity
  • Conditioned Medium
  • Antimicrobial Peptide
  • Insect Cell Culture
  • OD600 Unit