Conidiation Color Mutants of Aspergillus fumigatus Are Highly Pathogenic to the Heterologous Insect Host Galleria mellonella

Abstract

The greater wax moth Galleria mellonella has been widely used as a heterologous host for a number of fungal pathogens including Candida albicans and Cryptococcus neoformans. A positive correlation in pathogenicity of these yeasts in this insect model and animal models has been observed. However, very few studies have evaluated the possibility of applying this heterologous insect model to investigate virulence traits of the filamentous fungal pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. Here, we have examined the impact of mutations in genes involved in melanin biosynthesis on the pathogenicity of A. fumigatus in the G. mellonella model. Melanization in A. fumigatus confers bluish-grey color to conidia and is a known virulence factor in mammal models. Surprisingly, conidial color mutants in B5233 background that have deletions in the defined six-gene cluster required for DHN-melanin biosynthesis caused enhanced insect mortality compared to the parent strain. To further examine and confirm the relationship between melanization defects and enhanced virulence in the wax moth model, we performed random insertional mutagenesis in the Af293 genetic background to isolate mutants producing altered conidia colors. Strains producing conidia of previously identified colors and of novel colors were isolated. Interestingly, these color mutants displayed a higher level of pathogenicity in the insect model compared to the wild type. Although some of the more virulent color mutants showed increased resistance to hydrogen peroxide, overall phenotypic characterizations including secondary metabolite production, metalloproteinase activity, and germination rate did not reveal a general mechanism accountable for the enhanced virulence of these color mutants observed in the insect model. Our observations indicate instead, that exacerbated immune response of the wax moth induced by increased exposure of PAMPs (pathogen-associated molecular patterns) may cause self-damage that results in increased mortality of larvae infected with the color mutants. The current study underscores the limitations of using this insect model for inferring the pathogenic potential of A. fumigatus strains in mammals, but also points to the importance of understanding the innate immunity of the insect host in providing insights into the pathogenicity level of different fungal strains in this model. Additionally, our observations that melanization defective color mutants demonstrate increased virulence in the insect wax moth, suggest the potential of using melanization defective mutants of native insect fungal pathogens in the biological control of insect populations.

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

“…15,25 Dose-dependent mortality observed in this study was expected, as it has also been shown in studies carried out with A. fumigatus. 25 This, plus the obvious development of hyphae inside the larval body, indicates that A. terreus kills larvae through an active infection mechanism rather than a merely physical effect of the injected conidia. Similar to what has been shown in 2 different mouse models and embryonated chicken eggs, higher doses of A.terreus conidia were necessary to cause same lethality compared to A. fumigatus in larvae.…”

“…In contrast, investigating putative differences in virulence potential of A. fumigatus color mutants did not reveal any correlation between faster germination and higher pathogenicity. 25 Different outcome of infection is also linked to variable response of immune cells, such as killing of intruding pathogens by phagocytic cells, as shown in this study. Reasons therefore could be explained by variations in the outer layers of the isolates or to a difference in resistance to killing by reactive oxygen species.…”

Genetic Relatedness versus Biological Compatibility between Aspergillus fumigatus and Related Species

“…Galleria mellonella larvae in the final larval stage (Vanderhorst Wholesale, Inc., St. Marys, OH) were inoculated on the same day of their arrival. Larvae (15 per strain) were inoculated with 5 l of conidial suspension (2 ϫ 10 7 conidia/ml) as described elsewhere (28,29) and incubated at 37°C. Mortality was scored based on color change (from pale beige to dark brown or black) and unresponsiveness to tactile stimulus.…”

Involvement of Enterococcus faecalis Small RNAs in Stress Response and Virulence

“…First, we compared the survival of larvae of G. mellonella infected with the sRNA mutant and the corresponding complemented strains with those infected with the wild type. Infection of G. mellonella worms has emerged as a reliable model system to study the pathogenesis of numerous human pathogens (60)(61)(62)(63)(64)(65)(66). Indeed, the growth of the larvae at 37°C and the high degree of structural and functional homology between the innate immune systems of Galleria and mammals make the model pertinent (67,68).…”