Characterisation of the cellular and proteomic response of Galleria mellonella larvae to the development of invasive aspergillosis

Abstract

BackgroundGalleria mellonella larvae were infected with conidia of Aspergillus fumigatus and the cellular and humoral immune responses of larvae to the pathogen were characterized as invasive aspergillosis developed.ResultsAt 2 h post-infection there was an increase in hemocyte density to 7.43 ± 0.50 × 106/ml from 0.98 ± 0.08 × 106/ml at 0 h. Hemocytes from larvae immune primed for 6 h with heat killed A. fumigatus conidia displayed superior anti-fungal activity. Examination of the spread of the fungus by Cryo-imaging and fluorescent microscopy revealed dissemination of the fungus through the larvae by 6 h and the formation of distinct nodules in tissue. By 24 h a range of nodules were visible at the site of infection and at sites distant from that indicating invasion of tissue. Proteomic analysis of larvae infected with viable conidia for 6 h demonstrated an increase in the abundance of gustatory receptor candidate 25 (37 fold), gloverin-like protein (14 fold), cecropin-A (11 fold). At 24 h post-infection gustatory receptor candidate 25 (126 fold), moricin-like peptide D (33 fold) and muscle protein 20-like protein (12 fold) were increased in abundance. Proteins decreased in abundance included fibrohexamerin (13 fold) and dimeric dihydrodiol dehydrogenase (8 fold).ConclusionThe results presented here indicate that G. mellonella larvae may be a convenient model for studying the stages in the development of invasive aspergillosis and may offer an insight into this process in mammals.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1208-6) contains supplementary material, which is available to authorized users.

Candida albicans increases the pathogenicity of Staphylococcus aureus during polymicrobial infection of Galleria mellonella larvae

“…G. mellonella larvae were co-infected with C. albicans (1×10 5 larva −1 ) and S. aureus (2×10 4 larva −1 ) and incubated at 37 °C for 0, 6, 24 and 48 h. Larvae ( n =3) were embedded in Cryo-Imaging Embedding Compound (Bioinvision), flash-frozen in liquid nitrogen and mounted on a stage for sectioning [34, 40]. Sectioning and imaging was carried out every 10 µm using a Cryoviz (Bioinvision, Inc., Cleveland, OH, USA) cryo-imaging system.…”

Proteomic analysis of the processes leading to Madurella mycetomatis grain formation in Galleria mellonella larvae

“…Proteins were analyzed by using label-free quantitative LC-MS/MS by standardised protein purification procedures as described [20]. In summary, 0.75 μg of peptide mix was eluted onto a Q-Exactive (ThermoFisher Scientific, U.S.A) high resolution accurate mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system.…”

“…Results processing, statistical analyses and graphics generation were conducted using Perseus v. 1.5.5.3 as described [20]. Proteins that had non-existent values (indicative of absence or very low abundance in a sample) were also included in statistical analysis of the total differentially expressed group following imputation of the zero values using a number close to the lowest value of the range of proteins plus or minus the standard deviation.…”

“…Recently, we demonstrated that grains can also be formed in the invertebrate Galleria mellonella [11]. The invertebrate G. mellonella is a recognized in vivo system to assess the virulence of fungal species, assess the toxicity and efficacy of novel anti-fungal drugs and more recently to study the response of the innate immune response towards an invading fungal pathogen [14][15][16][17][18][19][20]. The wide spread acceptance of this model is due in part to the lack of legal and ethical considerations associated with larvae, their ease of use (inoculation, low cost, ability to generate results within 24-48 hours) and the fact that results correlate closely with those obtained using mice [21].…”

Cryptic species of Aspergillus section Terrei display essential physiological features to cause infection and are similar in their virulence potential in Galleria mellonella

“…Further, their function is repressed by secondary metabolites produced by A. fumigatus, gliotoxin and fumagillin, in a similar manner than the function of neutrophils [44,45]. A recent study highlighted significant parallels between the larval and mammalian response to aspergillosis, such as (a) an increase in immune cells after infection, (b) the ability to discriminate between the developmental fungal stages (resting spores, germinated spores, hyphae), (c) increased response to fungal pathogens when challenged with dead A. fumigatus conidia in an ex vivo assay, (d) similar capacity of tissue invasion and (e) increase in antimicrobial peptides [46]. Limitations of this alternative model are: the unavailability of an adaptive immune system, lacking understanding in the immune system and immune response of the larvae and the lack of standardized Galleria lines and experimental protocols [40].…”