Transformation of Candida albicans by electroporation

Backer, Marianne D. De; Maes, Dirk; Vandoninck, Sandy; Logghe, Marc; Contreras, Roland; Luyten, Walter H. M. L.

doi:10.1002/(sici)1097-0061(199911)15:15<1609::aid-yea485>3.3.co;2-p

Cited by 51 publications

(50 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…For the generation of strains that express cytoplasmic green fluorescent protein (GFP) constitutively under the control of the promoter of the gene for enolase (ENO1), we used the plasmid pENO1-yEGFP3-NAT (26) and transformation methods that have been previously described (27). To generate the cho1⌬/⌬::CHO1 reintegrant strain that expresses GFP, the SAT1 marker was flipped out of the CHO1 reintegration construct (pCHO1-SAT1-flipper) (strain SED022) and then transformed with the above-described GFP plasmid using nourseothricin selection at a concentration of 200 g/ml (WERNER BioAgents).…”

Section: Methodsmentioning

confidence: 99%

Masking of β(1-3)-Glucan in the Cell Wall of Candida albicans from Detection by Innate Immune Cells Depends on Phosphatidylserine

et al. 2014

View full text Add to dashboard Cite

The virulence of Candida albicans in a mouse model of invasive candidiasis is dependent on the phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE). Disruption of the PS synthase gene CHO1 (i.e., cho1⌬/⌬) eliminates PS and blocks the de novo pathway for PE biosynthesis. In addition, the cho1⌬/⌬ mutant's ability to cause invasive disease is severely compromised. The cho1⌬/⌬ mutant also exhibits cell wall defects, and in this study, it was determined that loss of PS results in decreased masking of cell wall ␤(1-3)-glucan from the immune system. In wild-type C. albicans, the outer mannan layer of the wall masks the inner layer of ␤(1-3)-glucan from exposure and detection by innate immune effector molecules like the C-type signaling lectin Dectin-1, which is found on macrophages, neutrophils, and dendritic cells. The cho1⌬/⌬ mutant exhibits increases in exposure of ␤(1-3)-glucan, which leads to greater binding by Dectin-1 in both yeast and hyphal forms. The unmasking of ␤(1-3)-glucan also results in increased elicitation of TNF-␣ from macrophages in a Dectin-1-dependent manner. The role of phospholipids in fungal pathogenesis is an emerging field, and this is the first study showing that loss of PS in C. albicans results in decreased masking of ␤(1-3)-glucan, which may contribute to our understanding of fungus-host interactions.

show abstract

Section: Methodsmentioning

confidence: 99%

Masking of β(1-3)-Glucan in the Cell Wall of Candida albicans from Detection by Innate Immune Cells Depends on Phosphatidylserine

et al. 2014

View full text Add to dashboard Cite

show abstract

“…The SAT1-2A fragment was then inserted into the SmaI-digested, dephosphorylated plasmid. This plasmid was digested with SacI plus SphI to generate the deletion cassette, which was then used to transform C. albicans strain P37005, P57072, or WO-1 by electroporation (De Backer et al, 1999). For each gene, two independent transformants were confirmed as heterozygous by both PCR and Southern analysis.…”

Section: Generation Of Null Mutantsmentioning

confidence: 99%

The Same Receptor, G Protein, and Mitogen-activated Protein Kinase Pathway Activate Different Downstream Regulators in the Alternative White and Opaque Pheromone Responses ofCandida albicans

Song

Sahni

Daniels

et al. 2008

MBoC

251

View full text Add to dashboard Cite

Candida albicans must undergo a switch from white to opaque to mate. Opaque cells then release mating type-specific pheromones that induce mating responses in opaque cells. Uniquely in C. albicans, the same pheromones induce mating-incompetent white cells to become cohesive, form an adhesive basal layer of cells on a surface, and then generate a thicker biofilm that, in vitro, facilitates mating between minority opaque cells. Through mutant analysis, it is demonstrated that the pathways regulating the white and opaque cell responses to the same pheromone share the same upstream components, including receptors, heterotrimeric G protein, and mitogen-activated protein kinase cascade, but they use different downstream transcription factors that regulate the expression of genes specific to the alternative responses. This configuration, although common in higher, multicellular systems, is not common in fungi, and it has not been reported in Saccharomyces cerevisiae. The implications in the evolution of multicellularity in higher eukaryotes are discussed. INTRODUCTIONBoth single cell organisms and the individual cells of multicellular organisms must respond to a variety of environmental signals (Dorsky et al., 2000;Balázsi and Oltvai, 2005). In addition, in higher eukaryotes different cell types frequently respond to the same signal in unique ways (Rincón and Pedraza-Alva, 2003;Bacci et al., 2005;Dailey et al., 2005). In most cases, different signals interact with unique surface receptors that activate different signal transduction pathways, as has been demonstrated in Saccharomyces cerevisiae (Leberer et al., 1997;Gustin et al., 1998;Madhani and Fink, 1998;Elion, 2000). The mitogen-activated protein (MAP) kinase pathways have evolved as highly efficient, multipurpose signal transduction systems. S. cerevisiae uses multiple MAP kinase pathways, each one for a distinct signaling system, including the mating process, the filamentation process, cell wall integrity, ascospore formation, and osmoregulation (Levin and Errede, 1995;Gustin et al., 1998;Saito and Tatebayashi, 2004;Chen and Thorner, 2007). Several of these pathways share a limited number of components, but all are presumed to use different receptors to elicit very different responses. In the mating process of S. cerevisiae, a cells release a-factor that interacts with the a-receptor on ␣ cells, and ␣ cells release ␣-factor that interacts with the ␣-receptor on a cells. These alternative signals are then transduced through the same heterotrimeric G protein to activate the same MAP kinase pathway, which in turn activates the same downstream regulators that elicit similar mating responses, including G1 arrest, polarization, and shmooing (Sprague et al., 1983;Bender and Sprague, 1986;Leberer et al., 1997). Other fungi, including Magnaporthe grisea (Dixon et al., 1999;Zhao et al., 2005b) Neurospora crassa (Li et al., 2005), and Cryptococcus neoformans (Davidson et al., 2003;Kraus et al., 2003;Bahn et al., 2005) also use MAP kinase pathways for a variety of responses...

show abstract

“…albicans transformation. Competent C. albicans cells were electroporated according to previously described methods (6,26). One to five micrograms of a PCR product was used in each transformation.…”

Section: Strainsmentioning

confidence: 99%

Role of Candida albicans Transcription Factor Upc2p in Drug Resistance and Sterol Metabolism

2004

View full text Add to dashboard Cite

In Candida albicans, drug resistance to clinically important antifungal drugs may be regulated through the action of transcription factors in a manner that may or may not be similar to regulation in Saccharomyces cerevisiae. A search of the C. albicans genome identified a single homolog of the S. cerevisiae transcription factor genes UPC2 (ScUPC2) and ECM22 (ScECM22) that have been associated with regulation of ergosterol biosynthesis. Sequence analysis of this C. albicans UPC2 (CaUPC2) gene identifies two domains, an anchoring transmembrane domain and a transcription factor region containing multiple nuclear localization signals and a fungal Zn(2)-Cys(6) binuclear cluster domain. Heterozygous deletion, homozygous deletion, and reconstructed strains of CaUPC2 as well as the parental strain were tested against several antifungal drugs, including ergosterol biosynthesis inhibitors. The CaUPC2 homozygous deletion strain showed marked hypersusceptibility to most drugs, compared to the parental and reconstructed strains. The deletion strains accumulate significantly less radiolabeled cholesterol, suggesting reduced ergosterol scavenging in those strains. When grown under azole drug pressure, the parental, heterozygous deletion and reconstructed strains of CaUPC2 upregulate the ERG2 and ERG11 ergosterol biosynthesis genes, while the homozygous deletion strain shows no such upregulation. Consistent with these results, CaUPC2 deletion strains show reduced ergosterol levels, which may explain the increased susceptibilities of the CaUPC2 deletion strains. Thus, it appears that CaUPC2 acts as a transcription factor involved in the regulation of ergosterol biosynthetic genes and as a regulator of sterol uptake across the plasma membrane.

show abstract

Transformation of Candida albicans by electroporation

Cited by 51 publications

References 17 publications

Masking of β(1-3)-Glucan in the Cell Wall of Candida albicans from Detection by Innate Immune Cells Depends on Phosphatidylserine

Masking of β(1-3)-Glucan in the Cell Wall of Candida albicans from Detection by Innate Immune Cells Depends on Phosphatidylserine

The Same Receptor, G Protein, and Mitogen-activated Protein Kinase Pathway Activate Different Downstream Regulators in the Alternative White and Opaque Pheromone Responses ofCandida albicans

Role of Candida albicans Transcription Factor Upc2p in Drug Resistance and Sterol Metabolism

Contact Info

Product

Resources

About