The potential of URA3 as a selectable reporter gene for in vivo expression technology (IVET) in Candida albicans

Candida albicans is a medically important opportunistic fungus in severely immunocompromised individuals. Since precise and coordinated control of gene expression is important for successful invasion of the host environment, identification of genes expressed in vivo could be instrumental in dissecting its pathogenic ability. In this report, a potential method of selecting in vivo expressed genes was evaluated in C. albicans using native URA3 as a reporter. URA3 encodes orotidine 5'monophosphate decarboxylase (OMPase), an enzyme required for pyrimidine synthesis. The URA3 open reading frame (ORF) was fused to the promoter of the pH-regulated gene PHR1 and the construct was introduced into a ura3 mutant. PHR1 is highly expressed in alkaline conditions and down regulated in acid conditions. The resulting strain did not exhibit the expected pH-dependent auxotrophy suggesting that basal expression was adequate for complementation. To circumvent this limitation, the reporter construct was redesigned to decrease the half-life of OMPase by fusing a ubiquitin (UBI)-arginine (R) complex to the N-terminal end of the OMPase. Integration of the PHR1 promoter-UBI-R-URA3 construct into the PHR1 locus of a ura3 null strain resulted in a pH-conditional growth requirement for uridine and pH-conditional production of OMPase. To determine whether the reporter would confer growth and survival in vivo, the strain was tested in animal models of systemic and vaginal candidiasis. The pH-conditional strain survived as well as the URA3 positive control in both infection models, and demonstrated that the reporter was adequately expressed in vivo.; IVET requires an integrating vector to provide a stable, single copy of the reporter. To satisfy this criteria, an efficient cloning vector lambda YES-Candida was constructed in which the UBI-R-URA3 reporter was placed 3' of a unique XhoI cloning site. This cloning vector should facilitate construction of a comprehensive genomic library.