Aspergillus fumigatus is currently the most common cause of invasive fungal infection among post-transplant patients. These very severe infections are associated with very high mortality rates, as high as 85% in some patient populations. One of the reasons that invasive aspergillosis (IA) is so lethal is a lack of effective antifungals for the treatment of IA. The specific mechanisms employed by fungi in constructing their cell walls are ideal drug target candidates, but these processes are poorly understood in A. fumigatus. We hypothesized that utilizing Agrobacterium tumefaciens mediated transformation and an innovative screening protocol would allow us to construct a library of A. fumigatus strains containing random disruptions of genes involved in cell wall synthesis. A. tumefaciens has recently been applied successfully in the experimental transformation of various fungi. The process of mutagenesis involves the A. tumefaciens mediated random insertion of a hygromycin resistance marker (hygR). This transformation results in a single, random insertion per cell, and since the sequence of the marker is known, identification of the disrupted gene in any particular transformant can be accomplished using inverse PCR. In order to identify A. fumigatus mutants that are more likely to have cell wall abnormalities, transformants were first screened on hygromycin, and then hygromycin resistant colonies were screened in the presence of the known cell wall stressors, Congo Red or Calcofluor White. Colonies that grew relatively well in the absence of cell wall stress but are unable to grow in the presence of such stress were transferred to individual cultures and the growth phenotypes were independently confirmed. Using this methodology, more than 50 random insertional mutants have been identified that demonstrate this growth pattern and possess apparent cell wall defects. Upon elucidation of the specific locations of the hygR insertions, this novel forward genetics and screening approach will highlight genes involved in A. fumigatus cell wall synthesis that can be studied further in efforts to elucidate the mechanisms involved in this vital cellular process.
Full conference title:
110th General Meeting American Society for Microbiology
- ASM 110th (2010)