Variation among biosynthetic gene clusters, secondary metabolite profiles, and cards of virulence across Aspergillus species

<i>Aspergillus fumigatus </i>is a major human pathogen. In contrast, <i>Aspergillus</i> <i>fischeri</i> and the recently described <i>Aspergillus</i> <i>oerlinghausenensis</i>, the two species most closely related to <i>A. fumigatus</i>, are not known to be pathogenic. Some of the genetic determinants of virulence (or “cards of virulence”) that <i>A. fumigatus </i>possesses are secondary metabolites that impair the host immune system, protect from host immune cell attacks, or acquire key nutrients. To examine whether secondary metabolism-associated cards of virulence vary between these species, we conducted extensive genomic and secondary metabolite profiling analyses of multiple <i>A. fumigatus</i>, one <i>A. oerlinghausenensis</i>, and multiple<i> A. fischeri</i> strains. We identified two cards of virulence (gliotoxin and fumitremorgin) shared by all three species and three cards of virulence (trypacidin, pseurotin, and fumagillin) that are variable. For example, we found that all species and strains examined biosynthesized gliotoxin, which is known to contribute to virulence, consistent with the conservation of the gliotoxin biosynthetic gene cluster (BGC) across genomes. For other secondary metabolites, such as fumitremorgin, a modulator of host biology, we found that all species produced the metabolite but that there was strain heterogeneity in its production within species. Finally, species differed in their biosynthesis of fumagillin and pseurotin, both contributors to host tissue damage during invasive aspergillosis. <i>A. fumigatus</i> biosynthesized fumagillin and pseurotin, while <i>A. oerlinghausenensis</i> biosynthesized fumagillin and <i>A. fischeri</i> biosynthesized neither. These biochemical differences were reflected in sequence divergence of the intertwined fumagillin/pseurotin BGCs across genomes. These results delineate the similarities and differences in secondary metabolism-associated cards of virulence between a major fungal pathogen and its nonpathogenic closest relatives, shedding light onto the genetic and phenotypic changes associated with the evolution of fungal pathogenicity.