Extensive sequence divergence of non-coding regions between Aspergillus fumigatus, a major fungal pathogen of humans, and its relatives

Invasive aspergillosis is a deadly fungal disease; more than 400,000 patients are infected worldwide each year and the mortality rate can be as high as 50-95%. Of the [~]450 species in the genus Aspergillus only a few are known to be clinically relevant, with the major pathogen Aspergillus fumigatus being responsible for [~]50% of all invasive mold infections. Genomic comparisons of A. fumigatus to other Aspergillus species have historically focused on protein-coding regions. However, most A. fumigatus genes, including those that modulate its virulence, are also present in non-pathogenic close relatives of A. fumigatus. Our hypothesis is that differential gene regulation - mediated through the non-coding regions upstream of genes transcription start sites - contributes to A. fumigatus pathogenicity. To begin testing this, we compared non-coding regions up to 500 base pairs upstream of the first codon of single-copy orthologous genes from the two A. fumigatus reference strains Af293 and A1163 and eight closely related Aspergillus section Fumigati species. We found that non-coding regions showed extensive sequence variation and lack of homology across species. By examining the evolutionary rates of both protein-coding and non-coding regions in a subset of orthologous genes with highly conserved non-coding regions across the phylogeny, we identified 418 genes, including 25 genes known to modulate A. fumigatus virulence, whose non-coding regions exhibit a different rate of evolution in A. fumigatus. Examination of sequence alignments of these non-coding regions revealed numerous instances of insertions, deletions, and other types of mutations of at least a few nucleotides in A. fumigatus compared to its close relatives. These results show that closely related Aspergillus species that vary greatly in their pathogenicity exhibit extensive non-coding sequence variation and identify numerous changes in non-coding regions of A. fumigatus genes known to contribute to virulence.