The ability of a pathogen to sense and respond to its environment is important. In particular, because nitrogen sources available during infection are likely to be sub-optimal, the capacity to reprogramme metabolism in response to different nitrogen sources is known to be a crucial virulence determinant for Aspergillus fumigatus and other fungal pathogens. Although, the genetic response to different nitrogen sources, nitrogen catabolite repression (NCR), is very well understood in Saccharomyces cerevisiae, less in known in filamentous fungi, and some differences are apparent between different fungal species. A key area of interest is in determining whether the rapamycin-sensitive TORC1 complex is involved in NCR. TORC1 is a conserved kinase complex that regulates a broad range of genes and phenotypes in cells in response to growth and nutritional status. We investigated the link between TORC1 and regulation of genes involved in nitrogen metabolism in A. fumigatus. First, we carried out simple growth inhibition assays on agar plates to determine whether rapamycin inhibited the growth of an A. fumigatus colony is the same way as it did the non-pathogen A. nidulans and found that this was the case, confirming that TORC1 activity is required for growth of A. fumigatus. Second, we investigated whether S. cerevisiae could be used as a surrogate host to study the function of A. fumigatus genes. Using the A. fumigatus genome sequence, we identified TORC1/ NCR genes and cloned selected genes into yeast vectors to test whether they would complement yeast mutants. In this way, we showed that AfareA, Afure2, Afgap1, AfprA could all complement S. cerevisiae mutants lacking the orthologous genes. Third, we used RT-PCR to look at expression of TORC1 and NCR regulated genes in A. fumigatus and A. nidulans when growing under different nitrogen conditions and when TORC1 was inhibited by rapamycin. In agreement with previous workers, we did not see a link between TORC1 and NCR in A. nidulans, but we did find that TORC1 regulated, at least to some extent, NCR genes such as areA, mepA and gap1 in A. fumigatus. This indicates a significant difference in genetic and metabolic wiring between the non-pathogenic and pathogenic Aspergilli. Currently, we are carrying out whole genome analysis using A. fumigatus gene arrays to determine the full-spectrum of genes regulated by the TORC1 and NCR systems.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)