Purpose: Sequencing of the A. fumigatus genome has enabled the development of powerful molecular tools for the analysis of disease initiation. Whole genome microarrays have previously been constructed to investigate gene expression throughout spore germination, antifungal treatment and temperature stress in vitro. However, of considerable interest is transcriptional regulation during the initiation of mammalian infection. Methods: We have developed a methodology for stage-specific gene expression analysis of both host and pathogen transcriptomes throughout a time-course of murine aspergillosis. Groups of 24 immunosuppressed male CD1 mice were intranasally inoculated with 1 ?108 A. fumigatus spores. Pathogen RNA from invasive germlings was isolated by bronchoalveolar lavage sampling at 4, 8 and 14 hours post infection. Fungal RNA was pooled and extracted for 8 mice per time point, providing sufficient concentrations for amplification and microarray analysis. Murine lung RNA was also sampled at the respective time points for analysis by SuperArray qRT-PCR. We are currently using this methodology for probing the role of fungal secondary metabolites during invasive infection by comparing transcriptional regulation throughout murine infection between the Af293 clinical isolate and laeA-deleted strains. Results: The 916;laeA strain lacks a putative methlytransferase leading to inappropriate regulation of gene expression at multiple secondary metabolite gene clusters. The loss of this global regulator of secondary metabolite biosynthesis results in avirulence the murine model. We predict that secondary metabolite clusters inappropriately regulated during infection in the 916;laeA strain will identify gene clusters essential for virulence. Conclusions: The development of this methodology will enable future investigations of host and pathogen transcriptomes throughout early murine infection for members of the Aspergillus genus. We predict this may identify much needed targets for diagnostic design and therapeutic benefit.
Full conference title:
4th Advances Against Aspergillosis
- AAA 4th (2010)