, Volume 158, Issue 1, pp 73–79

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

  • Emer P. Reeves
  • C.G.M. Messina
  • S. Doyle
  • K. Kavanagh


Aspergillus fumigatus is a pathogenic fungus capable of causing both allergic lung disease and invasive aspergillosis, a serious, life-threatening condition in neutropenic patients. Aspergilli express an array of mycotoxins and enzymes which may facilitate fungal colonisation of host tissue. In this study we investigated the possibility of using the insect, Galleria mellonella, for in vivo pathogenicity testing of Aspergillus species. Four clinical isolates of Aspergillus fumigatus and a single strain of Aspergillus niger were characterised for catalase and elastase activity and for the production of gliotoxin. Gliotoxin is an immunosuppressive agent previously implicated in assisting tissue penetration. Results illustrated a strain dependent difference in elastase activity but no significant difference in catalase activity. Gliotoxin production was detected in vitro and in vivo by Reversed Phase-High Performance Liquid Chromatography, with highest amounts being produced by A. fumigatus ATCC 26933 (350 ng/mg hyphae). Survival probability plots (Kaplan–Meier) of experimental groups infected with Aspergillus conidia indicate that G. mellonella is more susceptible to fungal infection by A. fumigatus ATCC 26933, implicating a critical role for gliotoxin production rather than growth rate or enzymatic activity in the virulence of A. fumigatus in this model.

gliotoxin Aspergillus fumigatus Galleria mellonella fungal infection immunosuppression 


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  1. 1.
    Daly P, Kavanagh K. Pulmonary aspergillosis: clinical presentation, diagnosis and therapy. B Biomed Sci 2001; 58: 197–205.Google Scholar
  2. 2.
    Saha DT, Larsen B. Clinical isolates of yeast produce a gliotoxin like substance. Mycopathologia 1991; 116: 203–208.Google Scholar
  3. 3.
    Richard J, DeBey M, Chermette R, Pier A, Hasegawa A, Lund A, Bratberg A, Padhye A, Connole M. Advances in veterinary mycology. J Med Vet Mycol 1994; 32: 169–187.Google Scholar
  4. 4.
    Waring P, Beaver J. Gliotoxin and Related Epipolythiodioxopiperazine. Gen Pharmac 1996; 27: 1311–1316.Google Scholar
  5. 5.
    Braithwaite AW, Eichner RD, Waring P, Müllbacher A. The immunomodulating agent gliotoxin causes genomic DNA fragmentation. Mol Immunol 1987; 24: 47–55.Google Scholar
  6. 6.
    Müllbacher A, Eichner RD. Immunosuppression in vitroby a metabolite of a human pathogenic fungi. Proc Natl Acad Sci USA 1984; 81: 3835–3837.Google Scholar
  7. 7.
    Waring P, Eichner RD, Müllbacher A, Sjaarda A. Gliotoxin induces apoptosis in macrophages unrelated to its antiphagocytic properties. J Biol Chem 1988; 263: 18493–18499.Google Scholar
  8. 8.
    Boutibonnes P, Auffray Y, Malherbe C, Kogbo W, Marais C. Proprietes antibacteriennes et genotoxiques de 33 mycotoxines. Mycopathologia 1984; 87: 43–49.Google Scholar
  9. 9.
    Larin NM, Copping MP, Herbst-Laier RH, Roberts B, Wenham RMM. Antiviral activity of gliotoxin. Chemotheraphy 1965; 10: 14–25.Google Scholar
  10. 10.
    Richard JL, Dvorak TJ, Ross PF. Natural occurrence of gliotoxin in turkeys infected with Aspergillus fumigatus, Fresenius. Mycopathologia 1996; 134: 167–170.Google Scholar
  11. 11.
    Mondon P, De Champs C, Donadille A, Ambroise-Thomas P, Grillot R. Variation in virulence of Aspergillus fumigatusstrains in a murine model of invasive pulmonary aspergillosis. J Med Microbiol 1995; 45: 186–191.Google Scholar
  12. 12.
    Kang D, Liu G, Lundstrom A, Gelius E, Steiner H. A peptigoglycan recognition protein in innate immunity conserved from insects to humans. Proc Natl Acad Sci USA 1998; 95: 10078–10082.Google Scholar
  13. 13.
    Yoshimura A, Lien E, Ingalls RR, Tuomanen E, Dziarski R, Golenbock D. Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Tol-like receptor 2. J Immunol 1999; 163: 1–5.Google Scholar
  14. 14.
    Vilmos P, Kurucz E. Insect Immunity: evolutionary roots of the mammalian innate immune system. Immunol Letts 1998; 62: 59–66.Google Scholar
  15. 15.
    Slepneva IA, Glupov VV, Sergeeva SV, Khramtsov VV. EPR detection of reactive oxygen species in hemolymph of Galleria mellonellaand Dendrolimus superans sibiricus(Lepidoptera) larvae. Biochem Biophys Res Commun 1999; 264: 212–215.Google Scholar
  16. 16.
    Irving P, Troxler L, Heuer TS, Belvin M, Kopczynski C, Reichhart JM, Hoffmann JA, Hetru C. A genome-wide analysis of immune responses in Drosophila. Proc Natl Acad SciUSA 2001; 98: 15119–15124.Google Scholar
  17. 17.
    Cotter G, Doyle S, Kavanagh K. Development of an insect model for the in vivopathogenicity testing of yeast. FEMS Immunol Med Microbiol 2000; 27: 163–169.Google Scholar
  18. 18.
    Jander G, Rahme LG, Ausubel FM. Positive correlation between virulence of Pseudomonas aeruginosamutants in mice and insects. J Bacteriol 2000; 182: 3843–3845.Google Scholar
  19. 19.
    Dunphy G, Morton D, Kropinski A, Chadwick J. Patogenicity of lipopolysaccharide mutants of Pseudomonas aeruginosafor larvae of Galleria mellonella: bacterial properties associated with virulence. J Invert Pathol 1986; 47: 48–55.Google Scholar
  20. 20.
    Brennan M, Thomas DY, Whiteway M, Kavanagh K. Correlation between virulence of Candida albicansmutants in mice and Galleria mellonellalarvae. FEMS Immunol Med Microbiol 2002; 341: 153–157.Google Scholar
  21. 21.
    Kettle AJ, Winterbourn CC. A kinetic analysis of the catalase activity of myeloperoxidase. Biochemistry 2001; 40: 10204–10212.Google Scholar
  22. 22.
    Borger P, Koeter GH, Timmerman JAB, Vellenga E, Tomee JFC, Kauffman HF. Proteases from Aspergillus fumigatus. J Infect Dis 1999; 180: 1267–1274.Google Scholar
  23. 23.
    Nieminen SM, Maki-Paakkanen J, Hirvonen M-R, Roponen M, von Wright A. Genotoxicity of gliotoxin, a secondary metabolite of Aspergillus fumigatus, in a battery of short-term systems. Mut Res 2002; 520: 161–170.Google Scholar
  24. 24.
    Sutton P, Waring P, Müllbacher A. Exacerbation of invasive aspergillosis by the immunosuppressive fungal metabolite, gliotoxin. Immunol Cell Biol 1996; 74: 318–322.Google Scholar
  25. 25.
    Richard JL, Lyon Rl, Fichtner RE, Ross PF. Use of thin layer chromatography for dection and high performance liquid chromatography for quantitating gliotoxin from rice cultures of Aspergillus fumigatus. Mycopathologia 1989; 107: 145–151.Google Scholar
  26. 26.
    Reeves EP, Hui L, Lortat Jacobs H, Messina CGM, Bolsover S, Gabella G, Potma EO, Warley A, Roes J, Segal AW. Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 2002; 416: 291–297.Google Scholar
  27. 27.
    Washburn RG, Gallin JI, Bennett JE. Oxidative killing of Aspergillus fumigatusproceeds by parallel myeloperoxidasedependent and-independent pathways. Infect Immun 1987; 55: 2088–2092.Google Scholar
  28. 28.
    Odeberg H, Olsson I. Mechanisms for the microbicidal activity of cationic proteins of human granulocytes. Infect Immun 1976; 14: 1269–1275.Google Scholar
  29. 29.
    Eichner RD, Al Salami M, Wood PR, Müllbacher A. The effect of gliotoxin upon macrophage function. Int J Immunopharmacol 1986; 8: 789–797.Google Scholar
  30. 30.
    Leitz SM, Diamond RD. Mechanisms of resistance of Aspergillus fumigatusconidia to killing by neutrophils in vitro. J Infect Dis 1985; 152: 33–42.Google Scholar
  31. 31.
    Shimizu K, Kondoh Y, Tanaka K. Proteinase production and pathogenicity of Candida albicans. Microbiol Immunol 1987; 31: 1045–1060.Google Scholar
  32. 32.
    Kolattukudy PE, Lee JD, Rogers LM, Zimmerman P, Ceselski S, Fox B, Stein B, Copelan EA. Evidence for possible involvement of an elastolytic serine protease in aspergillosis. InfectImmun 1993; 61: 2357–2368.Google Scholar
  33. 33.
    Taylor A. The toxicology of sporodesim and other epipolydioxopiperasine. In: Kadis S, Ciegler A, Ajl SJ, eds.Microbiol. Toxins. Vol. 7. New York: Academic Press, 1971: 370–376.Google Scholar
  34. 34.
    Bertout S, Badoc C, Mallie M, Giaimis J, Bastide J-M. Spore diffusate isolated from some strains of Aspergillus fumigatusinhibits phagocytosis by murine alveolar macrophages. FEMS Immunol Med Microbiol 2002;33:101–106.Google Scholar
  35. 35.
    Sutton P, Newcombe NR, Waring P, Müllbacher A. In vivoimmunosuppressive activity of gliotoxin, a metabolite produced by human pathogenic Fungi. Infect Immun 1994; 62: 1192–1198.Google Scholar
  36. 36.
    Aratani Y, Koyama H, Nyui S-I, Suzuki K, Kura F, Maeda N. Severe impairment in early host defence against Candida albicansin mice deficient in myeloperoxidase. Infect Immun 1999; 67: 1828–1836.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Emer P. Reeves
    • 1
  • C.G.M. Messina
    • 2
  • S. Doyle
    • 1
  • K. Kavanagh
    • 1
  1. 1.Department of Biology, National Institute for Cellular BiotechnologyNational University of Ireland MaynoothCounty KildareIreland
  2. 2.Laboratory of Haematology and OncologyInstituto Superiore di SanitaRomaItaly

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