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

Abstract

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.

Galleria mellonellaas a host model to studyAspergillus terreusvirulence and amphotericin B resistance

“…32,33 The production of gliotoxin and fumagillin was also proved in infected G. mellonella larvae. 31,34 A study by Lewis et al 35 detected gliotoxin in culture medium of A. terreus, but as we did not test gliotoxin production, or any other secondary metabolites, of the 5 different isolates we can only speculate its contribution to differences in virulence potential. The same effect may account for the variable production of proteases by the ATR and ATS strains.…”

“…A well studied example is A. fumigatus gliotoxin which is known to induce apoptosis and prevents NF-kB activation by inhibition of the proteasome and suppression of angiogenesis. [27][28][29][30][31] Additionally, fumagillin is also produced by A. fumigatus during hyphal growth and has been identified as an inhibitor of angiogenesis through a covalent interaction with methionine aminopeptidase-2 and suppresses tumor growth. 32,33 The production of gliotoxin and fumagillin was also proved in infected G. mellonella larvae.…”

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

“…Conventional RP-HPLC methods, which use UV absorbance for detecting gliotoxin, depend solely on the R T of gliotoxin in test specimens compared with that of a standard preparation [10,20]. While this approach is acceptable, the number of fungal metabolites which can be present in organic extracts, combined with the possibility of peptide generation due to unwanted proteolysis, demands a more rigorous confirmation of gliotoxin presence.…”

“…Gliotoxin detection is normally by reversed-phase high-performance-liquid chromatography (RP-HPLC) with UV absorbance detection (220-280 nm), gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry (LC-MS) analysis [10][11][12][13]. More specifically, gliotoxin detection following RP-HPLC separation is usually carried out at 254 nm [10]. The immunological detection of gliotoxin by ELISA has been demonstrated, and here, the limit of detection was 10 μg mL −1 [14].…”

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

“…Larvae of the insect Galleria mellonella have been used to differentiate between pathogenic and non-pathogenic yeast species [5] and a positive correlation between the immune response of G. mellonella and mice to mutants of Candida albicans has been established [6]. Recent work has established the role of gliotoxin in the virulence of the human pathogen Aspergillus fumigatus to Galleria larvae [7] and factors affecting the virulence of C. albicans mutants in Galleria larvae have been elucidated [8]. The use of Galleria larvae for quantifying the virulence of mutants of A. fumigatus by introducing conidia into the haemocoel via the last left pro-leg has been demonstrated [9].…”