An alternative insect pathogenic strategy in an Aspergillus flavus auxotroph

In order to study fungal pathogen evolution, we used a model system whereby the opportunistic fungus Aspergillus flavus was serially propagated through the insect (Galleria mellonella) larvae, yielding a cysteine/methionine auxotroph of A. flavus with properties of an obligate insect pathogen. The auxotroph exhibited insect host restriction but did not show any difference in virulence when compared with the wild-type (Scully LR, Bidochka MJ, 2006. Microbiology 152, 223-232). Here, we report that on 1% insect cuticle medium and synthetic Galleria medium, the auxotroph displayed increased extracellular protease production, a virulence factor necessary for insect pathogenesis. In the wild-type strain, protease production was deregulated during carbon (glucose), nitrogen (nitrate), or sulphate deprivation. If all three were present, protease production was vastly reduced. However, in the cysteine/methionine auxotroph, protease production was deregulated in complete medium. We suggest that the deficiency in sulphate assimilation in the auxotroph resulted in deregulation of protease production. The auxotroph exhibited delayed germination and slower hyphal growth when compared to the wild-type but there were no differences in virulence or cuticle penetration, suggesting a shift in pathogenic strategy that compensated decreased growth with increased virulence factor (extracellular protease) production. We concluded that the biosynthetic deficiency that mediated insect host restriction also increased protease production in the slow-growing auxotroph, resulting in an alternate, more host-specific pathogenic strategy. However, we argue that transmission is not necessarily correlated with virulence as competition bioassays in insect larvae showed that the wild-type generally out-competed the auxotroph by producing the majority of the conidia on the sporulating cadavers. This is one of the few examples that highlight the effect of genome decay on nutrition acquisition, virulence, and transmission in fungal pathogen evolution.