A CUG codon adapted two-hybrid system for the pathogenic fungus Candida albicans

Stynen, Bram; Dijck, Patrick Van; Tournu, HÃ©lÃ ̈ne

doi:10.1093/nar/gkq725

Cited by 34 publications

(58 citation statements)

References 55 publications

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“…This system relies on the construction of an artificial locus on Ch4 in the intergenic region between the PGA59 and PGA62 open reading frames (ORFs) (36). This 9-kb region has been used previously as a platform for the integration of C. albicans two-hybrid plasmids (37), and its modification is thought to be neutral to C. albicans biology, as its deletion did not result in any phenotype (36). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

et al. 2015

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f Loss of heterozygosity (LOH) plays important roles in genome dynamics, notably, during tumorigenesis. In the fungal pathogen Candida albicans, LOH contributes to the acquisition of antifungal resistance. In order to investigate the mechanisms that regulate LOH in C. albicans, we have established a novel method combining an artificial heterozygous locus harboring the blue fluorescent protein and green fluorescent protein markers and flow cytometry to detect LOH events at the single-cell level. Using this fluorescence-based method, we have confirmed that elevated temperature, treatment with methyl methanesulfonate, and inactivation of the Mec1 DNA damage checkpoint kinase triggered an increase in the frequency of LOH. Taking advantage of this system, we have searched for C. albicans genes whose overexpression triggered an increase in LOH and identified four candidates, some of which are known regulators of genome dynamics with human homologues contributing to cancer progression. Hence, the approach presented here will allow the implementation of new screens to identify genes that are important for genome stability in C. albicans and more generally in eukaryotic cells.N ormally found as a harmless commensal organism, Candida albicans is also a major fungal pathogen of humans and is capable of causing serious and even life-threatening diseases when the immune system of the host is compromised (1). Although C. albicans is found mostly as a diploid organism, haploid and tetraploid forms have been observed in the laboratory and upon the passage of C. albicans in animal models of infection (2-4). The formation of tetraploids results from mating between diploids of opposite mating types that have undergone the so-called whiteopaque phenotypic switch (5). Meiosis is thought to have been lost in C. albicans, and the formation of haploids from diploids or diploids from tetraploids results from concerted chromosome loss (2, 6). The C. albicans genome is highly plastic, undergoing a number of important genome rearrangements, such as loss-ofheterozygosity (LOH) events, aneuploidies, and the formation of isochromosomes (7). In particular, LOH has been shown to occur during commensal carriage (8) and during systemic infection in a mouse model (9) and to contribute to the acquisition of antifungal resistance (10, 11). Stressful conditions such as high temperature, oxidative stress, or azole antifungal treatment trigger an increase in the frequency of LOH, as well as changes in the mechanisms leading to LOH; while azole treatment and temperature cause an increase in LOH due to chromosome loss and reduplication, oxidative stress results in an increase in gene conversion events (12). Yet, little is known about the molecular mechanisms that control LOH in C. albicans, despite the apparent importance of LOH in the biology of this species. Overall, it has been shown that knockout mutations in genes involved in base excision repair, nucleotide excision repair, and mismatch repair had little impact on the frequency of LOH in C. albican...

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Section: Resultsmentioning

confidence: 99%

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

et al. 2015

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“…Recent contributions include the adaptation of yeast two-hybrid techniques (15) and tandem-affinity purification tag techniques (16,17), as well as the establishment of a vesicle targeting method (18), for C. albicans. However, amino acids represent the smallest building blocks of a protein that can be potentially substituted by unnatural amino acids, which offer a wider scope to add new biochemical properties to a protein of interest or to improve or manipulate its function.…”

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confidence: 99%

An Expanded Genetic Code in Candida albicans To Study Protein-Protein Interactions In Vivo

et al. 2013

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cFor novel insights into the pathogenicity of Candida albicans, studies on molecular interactions of central virulence factors are crucial. Since methods for the analysis of direct molecular interactions of proteins in vivo are scarce, we expanded the genetic code of C. albicans with the synthetic photo-cross-linking amino acid p-azido-L-phenylalanine (AzF). Interacting molecules in close proximity of this unnatural amino acid can be covalently linked by UV-induced photo-cross-link, which makes unknown interacting molecules available for downstream identification. Therefore, we applied an aminoacyltRNA synthetase and a suppressor tRNA pair (EcTyrtRNA CUA ) derived from Escherichia coli, which was previously reported to be orthogonal in Saccharomyces cerevisiae. We further optimized the aminoacyl-tRNA synthetase for AzF (AzF-RS) and EcTyrtRNA CUA for C. albicans and identified one AzF-RS with highest charging efficiency. Accordingly, incorporation of AzF into selected model proteins such as Tsa1p or Tup1p could be considerably enhanced. Immunologic detection of C-terminally tagged Tsa1p and Tup1p upon UV irradiation in a strain background containing suppressor tRNA and optimized AzF-RS revealed not only the mutant monomeric forms of these proteins but also higher-molecularweight complexes, strictly depending on the specific position of incorporated AzF and UV excitation. By Western blotting and tandem mass spectrometry, we could identify these higher-molecular-weight complexes as homodimers consisting of one mutant monomer and a differently tagged, wild-type version of Tsa1p or Tup1p, respectively, demonstrating that expanding the genetic code of C. albicans with the unnatural photo-cross-linker amino acid AzF and applying it for in vivo binary protein interaction analyses is feasible.

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“…The UPC2 overexpression strain used in this study is part of a transcription factor overexpression library constructed in our laboratory on the basis of the previously developed C. albicans two-hybrid system (27). Briefly, the nuclear localization sequence (NLS) was removed from the one-hybrid plasmid pC2HB, resulting in pC1H.…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, genes encoding known and putative transcription factors were successfully cloned in fusion with the DNA-binding domain of LexA in the plasmid to create the pC1H-PTF library. The resulting plasmids were integrated into the two-hybrid reporter strain S2CH3 (27) between XOG1 and HOL1 loci on chromosome 1 after linearization at the NotI restriction site. Selection was performed on CSMϪLeu medium.…”

Section: Methodsmentioning

confidence: 99%

Ascorbic Acid Inhibition of Candida albicans Hsp90-Mediated Morphogenesis Occurs via the Transcriptional Regulator Upc2

2014

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Morphogenetic transitions of the opportunistic fungal pathogen Candida albicans are influenced by temperature changes, with induction of filamentation upon a shift from 30 to 37°C. Hsp90 was identified as a major repressor of an elongated cell morphology at low temperatures, as treatment with specific inhibitors of Hsp90 results in elongated growth forms at 30°C. Elongated growth resulting from a compromised Hsp90 is considered neither hyphal nor pseudohyphal growth. It has been reported that ascorbic acid (vitamin C) interferes with the yeast-to-hypha transition in C. albicans. In the present study, we show that ascorbic acid also antagonizes the morphogenetic change caused by hampered Hsp90 function. Further analysis revealed that Upc2, a transcriptional regulator of genes involved in ergosterol biosynthesis, and Erg11, the target of azole antifungals, whose expression is in turn regulated by Upc2, are required for this antagonism. Ergosterol levels correlate with elongated growth and are reduced in cells treated with the Hsp90 inhibitor geldanamycin (GdA) and restored by cotreatment with ascorbic acid. In addition, we show that Upc2 appears to be required for ascorbic acid-mediated inhibition of the antifungal activity of fluconazole. These results identify Upc2 as a major regulator of ascorbic acid-induced effects in C. albicans and suggest an association between ergosterol content and elongated growth upon Hsp90 compromise.

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A CUG codon adapted two-hybrid system for the pathogenic fungus Candida albicans

Cited by 34 publications

References 55 publications

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

An Expanded Genetic Code in Candida albicans To Study Protein-Protein Interactions In Vivo

Ascorbic Acid Inhibition of Candida albicans Hsp90-Mediated Morphogenesis Occurs via the Transcriptional Regulator Upc2

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