Agrobacterium Mediated Transformation and Splinkerette PCR for the Development and Characterization of an Aspergillus fumigatus Cell Wall Mutant Library

D. Samar, E. Mecklenburg, J. S. Klutts

Author address: 

Univ. of Iowa Carver Coll. of Med., Iowa City, IA.

Abstract: 

Background:

Aspergillus fumigatus is significant fungal pathogen, causing invasive infections in a variety of immunocompromised patients. These severe infections are associated with mortality rates as high as 75-85% in some patient populations, partially due to a lack of effective antifungals for treating invasive aspergillosis. 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.

Methods:

In a forward genetics approach, we utilized Agrobacterium tumefaciens mediated transformation and an innovative screening protocol to construct a library of A. fumigatus strains containing random disruptions of genes involved in cell wall synthesis. Mutagenesis involves A. tumefaciens mediated random insertion of a hygromycin resistance marker (hygR) into the A. fumigatus genome. This transformation results in a single insertion per cell. In order to identify A. fumigatus mutants that are more likely to have cell wall abnormalities, transformants were screened on hygromycin, and then hygromycin resistant colonies were screened in the presence of the cell wall stressors, Congo Red or Calcofluor White. Colonies that grew in the absence of cell wall stress but are unable to grow in the presence of such stress were chosen and the growth phenotypes were independently confirmed. As the sequence of hygR is known, we applied Splinkerette PCR and sequencing to identify the location of hygR insertion within the mutant A. fumigatus genome.

Results:

Using this methodology, more than 100 random insertional mutants have been identified that demonstrate this growth pattern and possess apparent cell wall defects. The gene disrupted in many of these transformants has been identified by Splinkerette PCR and sequencing.

Conclusions:

A new forward genetics methodology, along with novel screening and PCR techniques, has identified genes likely involved in A. fumigatus cell wall synthesis, and these will be studied in more detail in the future.

2011

abstract No: 

187

Full conference title: 

111th General Meeting American Society for Microbiology
    • ASM 111th (2011)