Poster Presentation Lorne Infection and Immunity 2019

Acetamidase as a superior genetic marker in the fungal pathogen Cryptococcus neoformans (#124)

Paige E Erpf 1 2 , James A Fraser 1 2
  1. SCMB, School of Chemsitry and Molecular Biosciences, St Lucia, Queensland, Australia
  2. Australian Infectious Disease Research Centre, St Lucia, Queensland, Australia

The human fungal pathogen Cryptoccocus neoformans is responsible for 180 000 deaths annually, with mortality rates reaching 75% in Sub-Saharan Africa. Despite the importance of this disease, the molecular genetic tools available in C. neoformans are extremely limited. To enable an unprecedented level of control over manipulation of the C. neoformans genome we have developed the first dominant recyclable marker in the species by expanding on the classic work of renowned Australian fungal geneticist Michael J. Hynes. The filamentous ascomycete Aspergillus nidulans employs the acetamidase gene amdS to hydrolyse acetamide to ammonium and acetate, which serve as a nitrogen and carbon source, respectively. None of the members of the phylum Basidiomycota, including C. neoformans, have been reported to have this metabolic activity. We have therefore created a heterologous expression constructs for amdS, regulated by native C. neoformans promoters and terminators, and successfully demonstrated that acetamide can be utilized as a nitrogen or carbon source in this species. Growth on acetamide allows amdS to be used as a dominant marker, while growth on its toxic analogue fluoroacetamide enables counterselection. Exploiting these features, we have found amdS as a reliable tool in C. neoformans that enables repeated marker use by recycling, successfully deleting multiple genes in the one strain.