FEBS advanced lecture course on human fungal pathogens edition:2 location:La Colle sur Loup, France date:11-16 May 2007
The research on the human pathogenic fungus Candida albicans has some major restricions, for example C. albicans has a diploid genome and lacks a sexual cycle. Moreover, C. albicans has an alternative codon usage in which the codon CUG is translated into serine and not into leucine like the majority of organisms. Because of this alternative codon usage, the application of the yeast two-hybrid system for screening genes from C. albicans is not optimal. Many C. albicans proteins expressed in yeast will have a wrong amino acid sequence and this will influence the interactions observed in a screening.
The development of a two-hybrid system for the use in C. albicans itself overcomes this problem. Components of this two-hybrid system will have to be functional in C. albicans, like LexA from Staphylococcus aureus as DNA binding protein and LacZ from Streptococcus thermophilus as reporter protein (Russell and Brown, 2005; Uhl and Johnson, 2001). We will try out different strategies, like changing the promoters, activation domains and reporter genes to provide an optimal system that can be used as a better alternative to the existing yeast two-hybrid system. This new technology should be useful both for the confirmation of expected interactions as for the discovery of new interactions by screening with a gDNA/cDNA library.
After finishing the development we will apply the C. albicans two-hybrid system looking for interacting partners of the signal transduction proteins Ras1 and Cdc25. Both proteins will be inserted separately into the bait plasmid. A prey gDNA library will be used to screen for interaction partners.
Russell, C.L., Brown, A.J. (2005). Expression of one-hybrid fusions with Staphylococcus aureus lexA in Candida albicans confirms that Nrg1 is a transcriptional repressor and that Gcn4 is a transcriptional activator. Fungal Genet. Biol., 42(8):676-683.
Uhl, M.A., Johnson, A.D. (2001). Development of Streptococcus thermophilus lacZ as a reporter gene for Candida albicans. Microbiology, 147(5):1189-1195.