Cool Tools 1: Development and Application of a Candida albicans Two-Hybrid System

Bram Stynen; Patrick Van Dijck; Hélène Tournu

doi:10.1128/9781555817176.ch30

Chapter 30 : Cool Tools 1: Development and Application of a Candida albicans Two-Hybrid System

Authors: Bram Stynen¹, Patrick Van Dijck², Hélène Tournu³

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Affiliations: 1: VIB Department of Molecular Microbiology, K.U. Leuven Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, B-3001 Leuven, Belgium; 2: VIB Department of Molecular Microbiology, K.U. Leuven Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, B-3001 Leuven, Belgium; 3: VIB Department of Molecular Microbiology, K.U. Leuven Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, B-3001 Leuven, Belgium

Content Type: Monograph

Publication Year: 2012

Category: Fungi and Fungal Pathogenesis; Clinical Microbiology

Book DOI: 10.1128/9781555817176

Chapter DOI: 10.1128/9781555817176.ch30

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Abstract:

Interactions between proteins lie at the core of many crucial processes in the cell, including signal transduction, translation, transcription, DNA replication, and metabolic pathways. As a genetic approach, the yeast two-hybrid system has been highly successful in confirming and discovering protein-protein interactions. The principle of this method is based on the creation of two hybrid proteins, one fused to a DNA-binding domain (DBD; the "bait") and the other one fused to a transcription activation domain (AD; "prey"). Recently, an alternative Candida albicans two-hybrid system was developed, called the vesicle capture assay. This method is based upon the construction of two hybrid proteins, one protein that is fused to the vesicle targeted protein Vps32 and one protein fused to green fluorescent protein (GFP). This chapter describes the C. albicans two-hybrid system based upon the classic approach of transcription factor complementation. To develop a two-hybrid system suitable for C. albicans, all components of a yeast two-hybrid system needed to be included, with the requirement that each component was functional in C. albicans. In conclusion, this C. albicans two-hybrid system can be used to enhance one's knowledge of protein-protein interactions in C. albicans. Although only one-to-one interactions were tested until now, the method should be compatible for screening experiments due to the selective step involved.

Citation: Stynen B, Van Dijck P, Tournu H. 2012. Cool Tools 1: Development and Application of a Candida albicans Two-Hybrid System, p 483-487. In Calderone R, Clancy C (ed), Candida and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch30

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Cool Tools 1: Development and Application of a Candida albicans Two-Hybrid System

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FIGURE 1

Overview of the C. albicans two-hybrid system. (A) Bait and prey constructs. Bait and prey genes are expressed under the control of an inducible MET3 promoter. The bait fusion protein includes S. aureus LexA as DNA binding domain, and the prey protein comprises a codon-optimized VP16 activation domain. Both constructs contain SV40 NLSs and epitope tags (HA tag for bait and FLAG [FL] tag for prey). (B) Interaction leading to reporter gene expression. The bait protein is located at the promoter region of reporter genes S. thermophilus lacZ and C. albicans HIS1 by the binding of S. aureus LexA with its target LexA operator (LexAOp) sequences. When proteins X and Y interact, the VP16 activation domain is brought in close proximity to the reporter genes, resulting in their transcription. Analysis of an interaction is performed on selective medium (HIS1) or by a galactosidase assay (lacZ). doi:10.1128/9781555817176.ch30.f1

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FIGURE 1

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FIGURE 2

(A) The two-hybrid strain SC2H3 contains two integrated reporter constructs. Plasmid pC2H-HIS1 includes reporter gene HIS1 in front of a basal ADH1 promoter and five LexA operator (LexAOp) sequences for binding of the bait protein. Plasmid pC2H-LACZ contains reporter gene lacZ preceded by a basal ADH1 promoter sequence and five LexAOp sequences for binding of the bait protein. The complete plasmid is flanked by restriction sites used for integration. Linearized pC2H-HIS1 integrates at the HpaI site located between genes PGA59 and PGA62 on chromosome 4, while pC2H-LACZ integrates at the RPS1 locus on chromosome 1, at the NcoI site. ADH1b, basal promoter of ADH1; ampR, ampicillin resistance gene; Ori, origin of replication; t, terminator; p, promoter. (B) Bait and prey plasmids, pC2HB and pC2HP. A bait gene of interest (left panel) is cloned into the multiple cloning site downstream of the DNA-binding protein SalexA and under the control of the MET3 promoter. The pC2HB plasmid is integrated between loci XOG1 and HOL1 on chromosome 1 after linearization at restriction site NotI, and selection is obtained with the auxotrophic marker LEU2 from Candida maltosa. For the prey plasmid pC2HP (right panel), a prey gene of interest is cloned into the multiple cloning site, downstream of AD VP16. The MET3 promoter controls expression of the prey gene. An integration sequence, situated in the intergenic region between genes RXT3 and ORF19.3569 on chromosome 2, makes insertion in the genome possible after linearization of the plasmid at restriction site NotI. C. dubliniensis ARG4 is the auxotrophic marker for transformation. Reprinted from Nucleic Acids Research ( 24 ) with permission of the publisher. doi:10.1128/9781555817176.ch30.f2

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FIGURE 2

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FIGURE 3

(A) Two-hybrid interaction of C. albicans Kis1 with Snf4. Kis1 and Snf4 interact with each other as shown by growth of SC2H3 on SC-HIS and a high galactosidase activity. For the HIS1 reporter assay, cells were incubated for up to 2 days. (B) Coimmunoprecipitation of Kis1 and Snf4. The interaction between bait LexA-HA-Kis1 (73 kDa) and prey VP16-FLAG-Snf4 (47 kDa) is confirmed in a coimmunoprecipitation experiment. Total protein concentrations were equal for each sample. Lane 1, loading control of Kis1; lane 2, immunoprecipitation of Kis1 with anti-HA antibodies; lane 3, coimmunoprecipitation of Kis1 with anti-FLAG antibodies; lane 4, negative control of Kis1 immunoprecipitation without antibodies; lane 5, loading control of Snf4; lane 6, coimmunoprecipitation of Snf4 with anti-HA antibodies; lane 7, immunoprecipitation of Snf4 with anti-FLAG antibodies; lane 8, negative control of Snf4 immunoprecipitation without antibodies. The antibodies used for Western blotting are indicated on the right side of each blot, and the antibodies for immunoprecipitation are shown below the blot. IP-AB, antibody used for immunoprecipitation; LC, loading control; αFL, anti-FLAG antibody; αHA, anti-HA antibody. Reprinted from Nucleic Acids Research ( 24 ) with permission of the publisher. doi:10.1128/9781555817176.ch30.f3

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FIGURE 3

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