In Vivo Systematic Analysis of Candida albicans Zn2-Cys6 Transcription Factors Mutants for Mice Organ Colonization

The incidence of fungal infections in immuno-compromised patients increased considerably over the last 30 years. New treatments are therefore needed against pathogenic fungi. With Candida albicans as a model, study of host-fungal pathogen interactions might reveal new sources of therapies. Transcription factors (TF) are of interest since they integrate signals from the host environment and participate in an adapted microbial response. TFs of the Zn2-Cys6 class are specific to fungi and are important regulators of fungal metabolism. This work analyzed the importance of the C. albicans Zn2-Cys6 TF for mice kidney colonization. For this purpose, 77 Zn2-Cys6 TF mutants were screened in a systemic mice model of infection by pools of 10 mutants. We developed a simple barcoding strategy to specifically detect each mutant DNA from mice kidney by quantitative PCR. Among the 77 TF mutant strains tested, eight showed a decreased colonization including mutants for orf19.3405, orf19.255, orf19.5133, RGT1, UGA3, orf19.6182, SEF1 and orf19.2646, and four an increased colonization including mutants for orf19.4166, ZFU2, orf19.1685 and UPC2 as compared to the isogenic wild type strain. Our approach was validated by comparable results obtained with the same animal model using a single mutant and the revertant for an ORF (orf19.2646) with still unknown functions. In an attempt to identify putative involvement of such TFs in already known C. albicans virulence mechanisms, we determined their in vitro susceptibility to pH, heat and oxidative stresses, as well as ability to produce hyphae and invade agar. A poor correlation was found between in vitro and in vivo assays, thus suggesting that TFs needed for mice kidney colonization may involve still unknown mechanisms. This large-scale analysis of mice organ colonization by C. albicans can now be extended to other mutant libraries since our in vivo screening strategy can be adapted to any preexisting mutants

Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p

CAP1 encodes a basic region-leucine zipper (bZip) transcriptional regulatory protein that is required for oxidative stress tolerance in Candida albicans. Cap1p is a homologue of a Saccharomyces cerevisiae bZip transcription factor designated Yap1p that is both required for oxidative stress tolerance and localized to the nucleus in response to the presence of oxidants. Oxidant-regulated localization of Yap1p to the nucleus requires the presence of a carboxy-terminal cysteine residue (C629) that is conserved in Cap1p as C477. To examine the role of this conserved cysteine residue, C477 was replaced with an alanine residue. This mutant protein, C477A Cap1p, was analysed for its behaviour both in S. cerevisiae and C. albicans. Wild type and C477A Cap1p were able to complement the oxidant hypersensitivity of a Deltayap1 S. cerevisiae strain. Whereas a Yap1p-responsive lacZ fusion gene was oxidant inducible in the presence of YAP1, the C. albicans Cap1p derivatives were not oxidant responsive in S. cerevisiae. Introduction of wild type and C477A Cap1p-expressing plasmids into C. albicans produced differential resistance to oxidants. Glutathione reductase activity was found to be inducible by oxidants in the presence of Cap1p but was constitutively elevated in the presence of C477A Cap1p. Western blot assays indicate Cap1p is post-translationally regulated by oxidants. Green fluorescent protein fusions to CAP1 showed that this protein is localized to the nucleus only in the presence of oxidants while C477A Cap1p is constitutively nuclear localized. Directly analogous to S. cerevisiae Yap1p, regulated nuclear localization of C. albicans Cap1p is crucial for its normal function

Transcriptional control of the yeast PDR5 gene by the PDR3 gene product.

Saccharomyces cerevisiae cells possess the ability to simultaneously acquire resistance to an array of drugs with different cytotoxic activities. The genes involved in this acquisition are referred to as pleiotropic drug resistant (PDR) genes. Several semidominant, drug resistance-encoding PDR mutations have been found that map near the centromere on chromosome II, including PDR3-1 and PDR4-1. DNA sequencing of chromosome II identified a potential open reading frame, designated YBL03-23, that has the potential to encode a protein with strong sequence similarity to the product of the PDR1 gene, a zinc finger-containing transcription factor. Here we show that YBL03-23 is allelic with PDR3. The presence of a functional copy of either PDR1 or PDR3 is essential for drug resistance and expression of a putative membrane transporter-encoding gene, PDR5. Deletion mapping of the PDR5 promoter identified a region from -360 to -112 that is essential for expression of this gene. DNase I footprinting analysis using bacterially expressed Pdr3p showed specific recognition by this protein of at least one site in the -360/-112 interval in the PDR5 promoter. A high-copy-number plasmid carrying the PDR3 gene elevated resistance to both oligomycin and cycloheximide. Increasing the number of PDR3 gene copies in a delta pdr5 strain increased oligomycin resistance but was not able to correct the cycloheximide hypersensitivity that results from loss of PDR5. These data are consistent with the notion that PDR3 acts to increase cycloheximide resistance by elevating the level of PDR5 transcription, while PDR3-mediated oligomycin resistance acts through some other target gene