Isogenic Strain Construction and Gene Targeting in Candida dubliniensis

Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of C. albicans and C. dubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to C. albicans, no auxotrophic C. dubliniensis strains are available for genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensis wild-type isolate by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPA(R)-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The URA3 gene from C. albicans (CaURA3) was then used as a selection marker for targeted integration of a fusion between the C. dubliniensis MDR1 (CdMDR1) promoter and a C. albicans-adapted GFP reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent ura3-negative parent strains had correctly integrated the reporter gene fusion into the CdMDR1 locus, demonstrating that the CaURA3 gene can be used for efficient and specific targeting of recombinant DNA into the C. dubliniensis genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that CdMDR1 is not significantly expressed under these conditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that the CdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs

The Lower Saxony research network design of environments for ageing : towards interdisciplinary research on information and communication technologies in ageing societies

Worldwide, ageing societies are bringing challenges for independent living and healthcare. Health-enabling technologies for pervasive healthcare and sensor-enhanced health information systems offer new opportunities for care. In order to identify, implement and assess such new information and communication technologies (ICT) the 'Lower Saxony Research Network Design of Environments for Ageing' (GAL) has been launched in 2008 as interdisciplinary research project. In this publication, we inform about the goals and structure of GAL, including first outcomes, as well as to discuss the potentials and possible barriers of such highly interdisciplinary research projects in the field of health-enabling technologies for pervasive healthcare. Although GAL's high interdisciplinarity at the beginning slowed down the speed of research progress, we can now work on problems, which can hardly be solved by one or few disciplines alone. Interdisciplinary research projects on ICT in ageing societies are needed and recommended