Characterization of Aspergillus fumigatus secretome during sublethal infection of Galleria mellonella larvae

Abstract

Introduction. The fungal pathogen Aspergillus fumigatus can induce prolonged colonization of the lungs of susceptible patients, resulting in conditions such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis.Hypothesis. Analysis of the A. fumigatus secretome released during sub-lethal infection of G. mellonella larvae may give an insight into products released during prolonged human colonisation.Methodology. Galleria mellonella larvae were infected with A. fumigatus, and the metabolism of host carbohydrate and proteins and production of fungal virulence factors were analysed. Label-free qualitative proteomic analysis was performed to identify fungal proteins in larvae at 96 hours post-infection and also to identify changes in the Galleria proteome as a result of infection.Results. Infected larvae demonstrated increasing concentrations of gliotoxin and siderophore and displayed reduced amounts of haemolymph carbohydrate and protein. Fungal proteins (399) were detected by qualitative proteomic analysis in cell-free haemolymph at 96 hours and could be categorized into seven groups, including virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular development and cell cycle (n = 53). Analysis of the Gallerial proteome at 96 hours post-infection revealed changes in the abundance of proteins associated with immune function, metabolism, cellular structure, insect development, transcription/translation and detoxification.Conclusion. Characterizing the impact of the fungal secretome on the host may provide an insight into how A. fumigatus damages tissue and suppresses the immune response during long-term pulmonary colonization.

Keywords: Aspergillus; Galleria mellonella; fungal–host interactions; gliotoxin; proteomics.

Fig. 1.. Dose-dependent survival of G. mellonella larvae infected with A. fumigatus, demonstrating a significant reduction in survival at 96 hours at concentrations of 1×10⁶ and 1×10⁷ (***P < 0.001) determined by log-rank (Mantel–Cox) test.

Fig. 2.. FIMTrack analysis of larval movement over time following infection with 1×10⁵ conidia, demonstrating a significant reduction in larval movement at all timepoints post 48 hours (P < 0.05) determined by Dunnett’s multiple comparisons test.

Fig. 3.. Assessment of haemolymph carbohydrate content post-infection with 1×10⁵ A. fumigatus conidia. Significant reduction in host carbohydrate content 48 hours post-infection (P = 0.0003) determined by unpaired t-test (n = 7–15 individual larvae per group). The experiment was conducted in three independent replicates.

Fig. 4.. Assessment of haemolymph protein content post-infection with 1×10⁵ A. fumigatus conidia. Significant reduction in host protein content 96 hours post-infection (P = 0.0001) determined by unpaired t-test (n = 7–15 individual larvae per group). The experiment was conducted in three independent replicates.

Fig. 5.. Quantification of gliotoxin in Galleria infected with 1×10⁵ A. fumigatus conidia, demonstrating significant increase production at 72 (**P = 0.02) and 96 (***P = 0.0006) hours post-infection when compared to the initial detection of 24 hours post-infection determined by Dunnett’s multiple comparisons test.

Fig. 6.. Quantification of total siderophore concentration detected in Galleria haemolymph infected with 1×10⁵ A. fumigatus conidia, demonstrating significantly increased production at 48 hours (P = 0.009) and all subsequent timepoints post-infection as determined by Dunnett’s multiple comparisons test.

Fig. 7.. Pie chart summarizing high-confidence qualitative A. fumigatus protein detections categorized into seven subcategories: virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular cycle and development (n = 53) proteins.

Fig. 8.. Bar graph summarizing (a) high-confidence qualitative G. mellonella protein detections categorized into seven subcategories in control larvae and in larvae at 96 hours post-infection. (b) Immune proteins further subdivided into 11 subcategories, and the greatest differences in protein abundance between the control and infected larvae were AMP (antimicrobial peptides), inflammation, nutrient reservoir and pathogen binding.