A toolbox for epitope-tagging and genome-wide location analysis in Candida albicans

<p>Abstract</p> <p>Background</p> <p><it>Candida albicans </it>is a diploid pathogenic fungus not yet amenable to routine genetic investigations. Understanding aspects of the regulation of its biological functions and the assembly of its protein complexes would lead to further insight into the biology of this common disease-causing microbial agent.</p> <p>Results</p> <p>We have developed a toolbox allowing <it>in vivo </it>protein tagging by PCR-mediated homologous recombination with TAP, HA and MYC tags. The transformation cassettes were designed to accommodate a common set of integration primers. The tagged proteins can be used to perform tandem affinity purification (TAP) or chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP). Tandem affinity purification of <it>C. albicans </it>Nop1 revealed the high conservation of the small processome composition in yeasts. Data obtained with <it>in vivo </it>TAP-tagged Tbf1, Cbf1 and Mcm1 recapitulates previously published genome-wide location profiling by ChIP-CHIP. We also designed a new reporter system for <it>in vivo </it>analysis of transcriptional activity of gene <it>loci </it>in <it>C. albicans</it>.</p> <p>Conclusion</p> <p>This toolbox provides a basic setup to perform purification of protein complexes and increase the number of annotated transcriptional regulators and genetic circuits in <it>C. albicans</it>.</p

Transcriptional Regulation of Carbohydrate Metabolism in the Human Pathogen Candida albicans

Glycolysis is a metabolic pathway that is central to the assimilation of carbon for either respiration or fermentation and therefore is critical for the growth of all organisms. Consequently, glycolytic transcriptional regulation is important for the metabolic flexibility of pathogens in their attempts to colonize diverse niches. We investigated the transcriptional control of carbohydrate metabolism in the human fungal pathogen Candida albicans and identified two factors, Tye7p and Gal4p, as key regulators of glycolysis. When respiration was inhibited or oxygen was limited, a gal4tye7 C. albicans strain showed a severe growth defect when cultured on glucose, fructose or mannose as carbon sources. The gal4tye7 strain displayed attenuated virulence in both Galleria and mouse models as well, supporting the connection between pathogenicity and metabolism. Chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP) and transcription profiling revealed that Tye7p bound the promoter sequences of the glycolytic genes and activated their expression during growth on either fermentable or non-fermentable carbon sources. Gal4p also bound the glycolytic promoter sequences and activated the genes although to a lesser extent than Tye7p. Intriguingly, binding and activation by Gal4p was carbon source-dependent and much stronger during growth on media containing fermentable sugars than on glycerol. Furthermore, Tye7p and Gal4p were responsible for the complete induction of the glycolytic genes under hypoxic growth conditions. Tye7p and Gal4p also regulated unique sets of carbohydrate metabolic genes; Tye7p bound and activated genes involved in trehalose, glycogen, and glycerol metabolism, while Gal4p regulated the pyruvate dehydrogenase complex. This suggests that Tye7p represents the key transcriptional regulator of carbohydrate metabolism in C. albicans and Gal4p provides a carbon source-dependent fine-tuning of gene expression while regulating the metabolic flux between respiration and fermentation pathways

A Candida albicans early stage biofilm detachment event in rich medium

<p>Abstract</p> <p>Background</p> <p>Dispersal from <it>Candida albicans </it>biofilms that colonize catheters is implicated as a primary factor in the link between contaminated catheters and life threatening blood stream infections (BSI). Appropriate in vitro <it>C. albicans </it>biofilm models are needed to probe factors that induce detachment events.</p> <p>Results</p> <p>Using a flow through system to culture <it>C. albicans </it>biofilms we characterized a detachment process which culminates in dissociation of an entire early stage biofilm from a silicone elastomer surface. We analyzed the transcriptome response at time points that bracketed an abrupt transition in which a strong adhesive association with the surface is weakened in the initial stages of the process, and also compared batch and biofilm cultures at relevant time points. K means analysis of the time course array data revealed categories of genes with similar patterns of expression that were associated with adhesion, biofilm formation and glycoprotein biosynthesis. Compared to batch cultures the biofilm showed a pattern of expression of metabolic genes that was similar to the <it>C. albicans </it>response to hypoxia. However, the loss of strong adhesion was not obviously influenced by either the availability of oxygen in the medium or at the silicone elastomer surface. The detachment phenotype of mutant strains in which selected genes were either deleted or overexpressed was characterized. The microarray data indicated that changes associated with the detachment process were complex and, consistent with this assessment, we were unable to demonstrate that transcriptional regulation of any single gene was essential for loss of the strong adhesive association.</p> <p>Conclusion</p> <p>The massive dispersal of the early stage biofilm from a biomaterial surface that we observed is not orchestrated at the level of transcriptional regulation in an obvious manner, or is only regulated at this level by a small subpopulation of cells that mediate adhesion to the surface.</p

Widespread occurrence of chromosomal aneuploidy following the routine production of Candida albicans mutants

It has come to our attention that approximately 35% of >100 published microarray datasets, where transcript levels were compared between two different strains, exhibit some form of chromosome-specific bias. While some of these arose from the use of strains whose aneuploidies were not known at the time, in a worrisome number of cases the recombinant strains have acquired additional aneuploidies that were not initially present in the parental strain. The aneuploidies often affected a different chromosome than the one harboring the insertion site. The affected strains originated from either CAI-4, RM1000, BWP17 or SN95 and were produced through a variety of strategies. These observations suggest that aneuploidies frequently occur during the production of recombinant strains and have an effect on global transcript profiles outside of the afflicted chromosome(s), thus raising the possibility of unintended phenotypic consequences. Thus, we propose that all Candida albicans mutants and strains should be tested for aneuploidy before being used in further studies. To this end, we describe a new rapid testing method, based on a multiplex quantitative PCR assay, that produces eight bands of distinct sizes from either the left or right arms of each C. albicans chromosome

The BioGRID interaction database: 2013 update

The Biological General Repository for Interaction Datasets (BioGRID: http//thebiogrid.org) is an open access archive of genetic and protein interactions that are curated from the primary biomedical literature for all major model organism species. As of September 2012, BioGRID houses more than 500 000 manually annotated interactions from more than 30 model organisms. BioGRID maintains complete curation coverage of the literature for the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and the model plant Arabidopsis thaliana. A number of themed curation projects in areas of biomedical importance are also supported. BioGRID has established collaborations and/or shares data records for the annotation of interactions and phenotypes with most major model organism databases, including Saccharomyces Genome Database, PomBase, WormBase, FlyBase and The Arabidopsis Information Resource. BioGRID also actively engages with the text-mining community to benchmark and deploy automated tools to expedite curation workflows. BioGRID data are freely accessible through both a user-defined interactive interface and in batch downloads in a wide variety of formats, including PSI-MI2.5 and tab-delimited files. BioGRID records can also be interrogated and analyzed with a series of new bioinformatics tools, which include a post-translational modification viewer, a graphical viewer, a REST service and a Cytoscape plugin

The BioGRID interaction database: 2015 update

The Biological General Repository for Interaction Datasets (BioGRID: http://thebiogrid.org) is an open access database that houses genetic and protein interactions curated from the primary biomedical literature for all major model organism species and humans. As of September 2014, the BioGRID contains 749 912 interactions as drawn from 43 149 publications that represent 30 model organisms. This interaction count represents a 50% increase compared to our previous 2013 BioGRID update. BioGRID data are freely distributed through partner model organism databases and meta-databases and are directly downloadable in a variety of formats. In addition to general curation of the published literature for the major model species, BioGRID undertakes themed curation projects in areas of particular relevance for biomedical sciences, such as the ubiquitin-proteasome system and various human disease-associated interaction networks. BioGRID curation is coordinated through an Interaction Management System (IMS) that facilitates the compilation interaction records through structured evidence codes, phenotype ontologies, and gene annotation. The BioGRID architecture has been improved in order to support a broader range of interaction and post-translational modification types, to allow the representation of more complex multi-gene/protein interactions, to account for cellular phenotypes through structured ontologies, to expedite curation through semi-automated text-mining approaches, and to enhance curation quality control

Etude du déterminisme moléculaire du pouvoir pathogène d'Alternaria brassicicola, l'agent du black spot des crucifères

Nous nous sommes intéressés à l étude des mécanismes de détournement des défenses chimiques des plantes hôtes (phytoanticipines et les phytoalexines) dans le but d identifier de potentiels déterminants du pouvoir pathogène chez Alternaria brassicicola. Les effets antimicrobiens des isothiocyanates (ITC) et des phytoalexines indoliques de crucifères sur différents paramètres de croissance de plusieurs souches d A. brassicicola ont tout d abord été étudiés. Les résultats obtenus confirment l effet inhibiteur des métabolites testés en particulier sur la germination des conidies et l élongation des hyphes germinatives. Ils,révèlent aussi une variabilité de réponses au sein cette espèce. La réponse transcriptomique d A. brassicicola à l exposition aux différents métabolites de défenses a été ensuite caractérisée. L analyse globale du transcriptome d A. brassicicola exposé aux ITC et à la camalexine a permis d identifier plusieurs gènes dont l expression est induite également in planta, suggérant un rôle potentiel dans le déterminisme du pouvoir pathogène du champignon. Cette étude a permis également de révéler pour la première fois le mode d action des ITC et de la camalexine sur A. brassicicola. Les ITC agissent en générant un stress oxydatif auquel le champignon répond en enclenchant une réponse mettant en jeu différentes pompes d efflux et l expression de plusieurs gènes codant des glutathion transférases (GSTs). L analyse fonctionnelle de l une de ces GSTs nommée AbGst1p suggère que cette protéine contribue activement à la détoxication des ITC par le pathogène durant l infection en catalysant leur conjugaison au GSH. Le mode d action de la camalexine serait similaire à celui décrit pour les bactéries et se traduirait par des perturbations au niveau de la membrane plasmique et de la paroi. En réponse à ce stress , une surexpression de plusieurs gènes codant des pompes d efflux est observée chez A. brassicicola. En parallèle, une approche ciblée, visant à caractériser deux gènes candidats AbAtrA et AbAtrB codant des ABC transporteurs, a été entreprise. Les résultats obtenus montrent que l expression de ces deux gènes est induite à la fois par différents métabolites antimicrobiens et in planta suggérant qu ils pourraient être potentiellement impliqués dans l expression du pouvoir pathogène d A. brassicicola.We have been interested in the study of the detoxification mechanisms toward host defense compounds (phytoanticipins and phytoalexins) in order to identify potential Alternaria brassicicola pathogeniciy determinants. This work started with the evaluation of the antimicrobial effects of ITC and crucifer indolic phytoalexins on various growth parameters of several A. brassicicola strains. The obtained results confirmed the antifungal effects of the tested metabolites mainly on conidia germination and germ-tube elongation, and reveal a response variability within the studied strains. The transcriptomic responses occurring in A. brassicicola germinating conidia treated with different host defense compounds were then investigated. The analysis of A. brassicicola whole transcriptome after exposure to ITC and camalexin, allowed us to identify several genes which are differentially expressed and up-regulated in planta suggesting a potential involvement during host infection. The data generated by this study throws light, for the first time, on the mode of action of ITC and camalexin. Following exposure to ITC, A. brassicola displays a response similar to that expressed during oxidative stress, by activating the expression of several GSTs and drug efflux genes. Functional analysis of one of the ITC-induced GST named AbGst1p, suggested that this protein may actively contribute to ITC detoxification by catalyzing their conjugation to GSH during the interaction. The mode of action of the phytoalexin camalexin was similar to that described for bacteria and is suggestive of alterations in the cell wall and plasma membrane. In response to this stress, several genes coding for drug efflux pumps were upregulated. At the same time, a targeted approach was also undertaken which consisted in cloning of two ABC transporters encoding genes, AbAtrA and AbAtrB. The obtained results showed that these two genes were upregulated by different crucifer antimicrobial compounds and also during host infection suggesting that these genes are potentially involved in A. brassicicola pathogenesis.ANGERS-BU Lettres et Sciences (490072106) / SudocSudocFranceF

Recent advances on Candida albicans biology and virulence [version 1; referees: 2 approved]

Candida albicans is an important human fungal pathogen, in terms of both its clinical significance and its use as an experimental model for scientific investigation. Although this opportunistic pathogen is a natural component of the human flora, it can cause life-threatening infections in immunosuppressed patients. There are currently a limited number of antifungal molecules and drug targets, and increasing resistance to the front-line therapeutics, demonstrating a clear need for new antifungal drugs. Understanding the biology of this pathogen is an important prerequisite for identifying new drug targets for antifungal therapeutics. In this review, we highlight some recent developments that help us to understand how virulence traits are regulated at the molecular level, in addition to technical advances that improve the ability of genome editing in C. albicans

pH-Dependant Antifungal Activity of Valproic Acid against the Human Fungal Pathogen Candida albicans

Current antifungal drugs suffer from limitations including toxicity, the emergence of resistance and decreased efficacy at low pH that are typical of human vaginal surfaces. Here, we have shown that the antipsychotic drug valproic acid (VPA) exhibited a strong antifungal activity against both sensitive and resistant Candida albicans in pH condition similar to that encountered in vagina. VPA exerted a strong anti-biofilm activity and attenuated damage of vaginal epithelial cells caused by C. albicans. We also showed that VPA synergizes with the allylamine antifungal, Terbinafine. We undertook a chemogenetic screen to delineate biological processes that underlies VPA-sensitivity in C. albicans and found that vacuole-related genes were required to tolerate VPA. Confocal fluorescence live-cell imaging revealed that VPA alters vacuole integrity and support a model where alteration of vacuoles contributes to the antifungal activity. Taken together, this study suggests that VPA could be used as an effective antifungal against vulvovaginal candidiasis

Role of Ndt80p in Sterol Metabolism Regulation and Azole Resistance in Candida albicans▿ †

The Ndt80p transcription factor modulates azole tolerance in Candida albicans by controlling the expression of the gene for the drug efflux pump Cdr1p. To date, the contribution of this transcriptional modulator to drug tolerance is not yet well understood. Here, we investigate the role of Ndt80p in mediating fluconazole tolerance by determining its genome-wide occupancy using chromatin immunoprecipitation coupled to high-density tiling arrays. Ndt80p was found to bind a large number of gene promoters with diverse biological functions. Gene ontology analysis of these Ndt80p targets revealed a significant enrichment in gene products related to the cell wall, carbohydrate metabolism, stress responses, hyphal development, multidrug transport, and the cell cycle. Ndt80p was found on the promoters of ergosterol biosynthesis genes, including on the azole target Erg11p. Additionally, expression profiling was used to identify fluconazole-responsive genes that require Ndt80p for their proper expression. We found that Ndt80p is crucial for the expression of numerous fluconazole-responsive genes, especially genes involved in ergosterol metabolism. Therefore, by combining genome-wide location and transcriptional profiling, we have characterized the Ndt80p fluconazole-dependent regulon and demonstrated the key role of this global transcriptional regulator in modulating sterol metabolism and drug resistance in C. albicans