Caenorhabditis elegans- Based Aspergillus fumigatus Infection Model for Evaluating Pathogenicity and Drug Efficacy

Abstract

Aspergillus fumigatus is the most reported causative pathogen associated with the increasing global incidences of aspergilloses, with the health of immunocompromised individuals mostly at risk. Monitoring the pathogenicity of A. fumigatus strains to identify virulence factors and evaluating the efficacy of potent active agents against this fungus in animal models are indispensable in current research effort. Caenorhabditis elegans has been successfully utilized as an infection model for bacterial and dimorphic fungal pathogens because of the advantages of being time-efficient, and less costly. However, application of this model to the filamentous fungus A. fumigatus is less investigated. In this study, we developed and optimized a stable and reliable C. elegans model for A. fumigatus infection, and demonstrated the infection process with a fluorescent strain. Virulence results of several mutant strains in our nematode model demonstrated high consistency with the already reported pathogenicity pattern in other models. Furthermore, this C. elegans-A. fumigatus infection model was optimized for evaluating the efficacy of current antifungal drugs. Interestingly, the azole drugs in nematode model prevented conidial germination to a higher extent than amphotericin B. Overall, our established C. elegans infection model for A. fumigatus has potential applications in pathogenicity evaluation, antifungal agents screening, drug efficacy evaluation as well as host-pathogen interaction studies.

Keywords: Aspergillus fumigatus; Caenorhabditis elegans; hyphal filamentation; infection model; pathogenicity.

Figure 1

Schematic procedure of C. elegans-based A. fumigatus infection model. Synchronized L4 stage worms were put into NGM plates containing A. fumigatus spores at the four edges for 16 h pre-infection. Then extensive washing step was applied to remove conidia that were not ingested by worms. Finally the washed worms were added to BHI medium for killing assay.

Figure 2

Survival rate comparison in C. elegans hosts. (A), Kaplan-Meier survival plots of fem-3(q96) fed by E.coli OP50 or conidia of indicated A. fumigatus strains. (B), Kaplan-Meier survival plots of glp-4(bn2); sek-1(km4) fed by E.coli OP50 or conidia of indicated A. fumigatus strains. Compared to OP50 and dead conidia the Af293-dsRed and KU80Δ strains exhibited significant pathogenicity [P < 0.0001, Log-rank (Mantel-Cox) test] in both fem-3(q96) and glp-4(bn2); sek-1(km4) worms. Three biological repeats (each with triplicates) were conducted for each strain.

Figure 3

Infection and progression stages of Af293-dsRed in infection to glp-4(bn2); sek-1(km4) worms. Images were taken under DIC and TRITC channels at killing time of 0, 24, 48, and 72 h. Scale bar is 200 μm.

Figure 4

Survival curve and hyphal filamentation rate of A. fumigatus mutant strains in C. elegans model. (A), Kaplan-Meier survival plots of glp-4(bn2); sek-1(km4) on solid BHI plates after 16 h pre-infection with conidia of indicate A. fumigatus mutant strains. (B), Hyphal filamentation rates resulting from infection by indicated A. fumigatus mutant strains at 24 h in killing assay. Three biological repeats (each with triplicates) were conducted for each strain. One-way ANOVA was used for statistical analysis of filamentation rate, where p > 0.05 showing not significant, ns; p ≤ 0.0001 showing highly significant, ****, compared to the KU80Δ strain.

Figure 5

Kaplan-Meier survival plots of C. elegans infected by KU80Δ in the presence of antifungal drugs. The glp-4(bn2); sek-1(km4) worms were pre-infected with KU80Δ for 8 h then transferred into liquid killing media containing different concentrations of AmB (A), VoZ (B), and ItrZ (C). Three biological repeats (each with triplicates) were conducted for each concentration.

Figure 6

Effect of antifungal treatment on Af293-dsRed infection to glp-4(bn2); sek-1(km4) worms. Images were taken under DIC and TRITC channels from DMSO treatment by 24 h, 1.5 μg/ml AmB treatment by 48 h, 2 μg/ml ItrZ treatment by 72 h and 0.5 μg/ml VoZ treatment by 72 h. Scale bar is 200 μm.