The SAT1 flipper, an optimized tool for gene disruption in Candida albicans

Reuß, Oliver; Vik, Åshild; Kolter, Roberto; Morschhäuser, Joachim

doi:10.1016/j.gene.2004.06.021

Cited by 653 publications

(617 citation statements)

References 36 publications

(34 reference statements)

Supporting

Mentioning

601

Contrasting

Unclassified

Order By: Relevance

“…One hundred to two hundred cells were spread on YPD plates containing 20 mg nourseothricin ml 21 (Werner Bioagents) and grown for 2 days at 30 uC. Nou S clones were identified by their small colony size and confirmed by restreaking on YPD plates containing 100 mg nourseothricin ml 21 as described previously (Reuß et al, 2004). To test for growth on BSA as the sole nitrogen source, strains were grown at 30 uC or 37 uC in YCB-BSA medium [23.4 g yeast carbon base, 4 g bovine serum albumin (Fraction V; Gerbu) per litre, pH 4.0].…”

Section: Methodsmentioning

confidence: 99%

“…Analogous constructs were generated for the deletion of SAP1 and SAP3-SAP6. ApaI-SalI fragments containing the upstream sequences of these genes were obtained from plasmids pSFL13, pSFL33, pSFL43, pSFL53 and pSFL63 (Staib et al, 2000) and ligated into ApaI/XhoIdigested pOPT1M3 (Reuß et al, 2004), resulting in plasmids pSAP1MS1, pSAP3MS1, pSAP4MS1, pSAP5MS1 and pSAP6MS1, respectively. The downstream regions of the SAP genes were then amplified using the primer pairs SAP1K/SAP1L, SAP3K/SAP3L, SAP4K/SAP4L, SAP5N/SAP5Q and SAP6N/SAP6Q (primer sequences are given in Table 2).…”

Section: Methodsmentioning

confidence: 99%

“…C. albicans strain SC5314 was transformed by electroporation (Köhler et al, 1997) with the gel-purified ApaI-SacI fragments from plasmids pSAP1MS2, pSAP2MS2, pSAP3MS2, pSAP4MS2, pSAP5MS2 and pSAP6MS2. Nourseothricinresistant transformants were selected on YPD agar plates containing 200 mg nourseothricin ml 21 as described previously (Reuß et al, 2004). Single-copy integration of all constructs was confirmed by Southern hybridization.…”

Section: Methodsmentioning

confidence: 99%

“…The sap mutants used in earlier studies were generated from the auxotrophic laboratory strain CAI4 using the Ura-blaster protocol Sanglard et al, 1997). As the use of the URA3 marker for mutant construction in C. albicans can sometimes cause problems in the interpretation of mutant phenotypes (Bain et al, 2001;Brand et al, 2004;Cheng et al, 2003;Lay et al, 1998;Sharkey et al, 2005), we constructed a new set of sap mutants from the prototrophic wild-type model strain SC5314 using the SAT1-flipping strategy (Reuß et al, 2004). For each of the SAP1-SAP6 genes, two independent series of homozygous deletion mutants were generated.…”

Section: Construction Of Mutants Of the C Albicans Wildtype Strain Smentioning

confidence: 99%

See 3 more Smart Citations

Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans

2008

Self Cite

View full text Add to dashboard Cite

A well-known virulence attribute of the human-pathogenic yeast Candida albicans is the secretion of aspartic proteases (Saps), which may contribute to colonization and infection of different host niches by degrading tissue barriers, destroying host defence molecules, or digesting proteins for nutrient supply. The role of individual Sap isoenzymes, which are encoded by a large gene family, for the pathogenicity of C. albicans has been investigated by assessing the virulence of mutants lacking specific SAP genes and by studying the expression pattern of the SAP genes in various models of superficial and systemic infections. We used a recombination-based genetic reporter system to detect the induction of the SAP1-SAP6 genes during infection of reconstituted human vaginal epithelium. Only SAP5, but none of the other tested SAP genes, was detectably activated in this in vitro infection model. To directly address the importance of the SAP1-SAP6 genes for invasion of reconstituted human epithelia (RHE), we constructed a set of mutants of the wild-type C. albicans model strain SC5314 in which either single or multiple SAP genes were specifically deleted. Even mutants lacking all of the SAP1-SAP3 or the SAP4-SAP6 genes displayed the same capacity to invade and damage both oral and vaginal RHE as their wild-type parental strain, in contrast to a nonfilamentous efg1D mutant that was avirulent under these conditions. We therefore conclude from these results that the secreted aspartic proteases Sap1p-Sap6p are not required for invasion of RHE by C. albicans.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Construction Of Mutants Of the C Albicans Wildtype Strain Smentioning

confidence: 99%

See 2 more Smart Citations

Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans

2008

Self Cite

View full text Add to dashboard Cite

show abstract

“…To delete the second copy of WOR1 or EFG1, we constructed plasmids pSFS2a-WOR1KO and pSFS2a-EFG1KO. Two fragments that are homologous to the 5 0 -and 3 0 -sequences of WOR1 were inserted into the ApaI/ XhoI and SacII/SacI sites of pSFS2A 45 to generate plasmid pSFS2a-WOR1KO. Two fragments that are homologous to the 5 0 -and 3 0 -sequences of EFG1 were inserted into the KpnI/XhoI and SacII/SacI sites to generate plasmid pSFS2a-EFG1KO.…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Discovery of the gray phenotype and white-gray-opaque tristable phenotypic transitions inCandida dubliniensis

Yue

Guan

et al. 2016

Virulence

View full text Add to dashboard Cite

Candida dubliniensis is closely related to Candida albicans, a major causative agent of candidiasis, and is primarily associated with oral colonization and infection in human immunodeficiency virus (HIV)-positive patients. Despite the high similarity of genomic and phenotypic features between the 2 species, C. dubliniensis is much less virulent and less prevalent than C. albicans. The ability to change morphological phenotypes is a striking feature of Candida species and is linked to virulence. In this study, we report a novel phenotype, the gray phenotype, in C. dubliniensis. Together with the previously reported white and opaque cell types, the gray phenotype forms a tristable phenotypic switching system in C. dubliniensis that is similar to the white-gray-opaque tristable switching system in C. albicans. Gray cells of C. dubliniensis are similar to their counterparts in C. albicans in terms of several biological aspects including cellular morphology, mating competence, and genetic regulatory mechanisms. However, the gray phenotypes of the 2 species have some distinguishing features. For example, the secreted aspartyl protease (Sap) activity is induced by bovine serum albumin (BSA) in gray cells of C. albicans, but not in gray cells of C. dubliniensis. Taken together, our results demonstrate that the biological features and regulatory mechanisms of white-gray-opaque tristable transitions are largely conserved in the 2 pathogenic Candida species.

show abstract

Monitoring Phenotypic Switching in Candida albicans and the Use of Next‐Gen Fluorescence Reporters

2019

View full text Add to dashboard Cite

Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white‐opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.

show abstract

The SAT1 flipper, an optimized tool for gene disruption in Candida albicans

Cited by 653 publications

References 36 publications

Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans

Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans

Discovery of the gray phenotype and white-gray-opaque tristable phenotypic transitions inCandida dubliniensis

Monitoring Phenotypic Switching in Candida albicans and the Use of Next‐Gen Fluorescence Reporters

Contact Info

Product

Resources

About