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. 2010 Oct;38(19):e184.
doi: 10.1093/nar/gkq725. Epub 2010 Aug 17.

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

Bram Stynen  1 , Patrick Van Dijck, Hélène Tournu
Affiliations

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

Bram Stynen et al. Nucleic Acids Res. 2010 Oct.

Abstract

The genetics of the most common human pathogenic fungus Candida albicans has several unique characteristics. Most notably, C. albicans does not follow the universal genetic code, by translating the CUG codon into serine instead of leucine. Consequently, the use of Saccharomyces cerevisiae as a host for yeast two-hybrid experiments with C. albicans proteins is limited due to erroneous translation caused by the aberrant codon usage of C. albicans. To circumvent the need for heterologous expression and codon optimalization of C. albicans genes we constructed a two-hybrid system with C. albicans itself as the host with components that are compatible for use in this organism. The functionality of this two-hybrid system was shown by successful interaction assays with the protein pairs Kis1-Snf4 and Ino4-Ino2. We further confirmed interactions between components of the filamentation/mating MAP kinase pathway, including the unsuspected interaction between the MAP kinases Cek2 and Cek1. We conclude that this system can be used to enhance our knowledge of protein-protein interactions in C. albicans.

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Figures

Figure 1.
Figure 1.
(A) Influence of LexAOp sequences on reporter gene activation. Strains SC2H0, SC2H1 and SC2H2, carrying respectively no, one and five LexAOp sequences upstream of HIS1 reporter gene were compared in their ability to grow on SC medium lacking histidine. While the presence of at least one LexAOp sequence is necessary for HIS1 expression, the hybrid transcription factor LexA–VP16 clearly takes advantage of the presence of multiple LexAOp sequences. Two independent strains for each of the constructs are shown and grown for 1 day. (B) 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 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 its 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.
Figure 2.
Figure 2.
(A) 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 C. maltosa. In the case of 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. (B) Comparison of activation strength of five different ADs fused to the binding domain LexA in a one-hybrid experiment. Bacterial B42, C. albicans Gcn4, C. albicans Ino2, C. albicans Tac1 hyperactive AD and viral protein VP16 were tested for their ability to stimulate HIS1 and lacZ expression in strain SC2H3. VP16 was chosen to act as the AD in the prey plasmid. kanR: kanamycin resistance gene; Ori: origin of replication; NLS: nuclear localization sequence.
Figure 3.
Figure 3.
(A) Two-hybrid interaction of C. albicans Ino4 with Ino2. Ino4 and Ino2 interact strongly, shown by growth of strain SC2H3 on SC–HIS after one day with Ino4 as bait and Ino2 as prey. This interaction is confirmed in the galactosidase assay where the activity measured (in Miller units) was 2.5-fold the background activity. (B) Two-hybrid interaction of C. albicans Kis1 with Snf4. Kis1 and Snf4, two proteins of the Snf1 complex, 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. (C) Co-immunoprecipitation of Kis1 and Snf4. The interaction between bait LexA–HA–Kis1 (73 kDa) and prey VP16–FLAG–Snf4 (47 kDa) is confirmed in a co-IP experiment. Total protein concentrations were equal for each sample. Lane 1: loading control of Kis1; lane 2: IP of Kis1 with anti-HA antibodies; lane 3: co-IP of Kis1 with anti-FLAG antibodies; lane 4: negative control of Kis1 immunoprecipitation without antibodies; lane 5: loading control of Snf4; lane 6: co-IP of Snf4 with anti-HA antibodies; lane 7: IP 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.
Figure 4.
Figure 4.
Interactions between downstream targets of the mating/filamentation MAP kinase pathway. (A) All pair-wise combinations of interactions between the MAPKK Hst7, the MAP kinases Cek2 and Cek1, and the transcription factor Cph1 were tested in a two-hybrid assay. Interactions between the proteins pairs Hst7 and Cek1, Cek2 and Cek1, Cek1 and Cph1, and Cek2 and Cph1 were detected. In all cases, a representative transformant is shown for growth for up to 5 days on SC–HIS. At least two independent transformants were used for the galactosidase assay. (B) The interaction between bait LexA–HA–Hst7 (91 kDa) and prey VP16–FLAG–Cek1 (57 kDa) is confirmed in a co-immunoprecipitation (co-IP) experiment. Total protein concentrations were equal for each sample. Lane 1: loading control of Hst7; lane 2: IP of Hst7 with anti-HA antibodies; lane 3: co-IP of Hst7 with anti-FLAG antibodies, visualized with anti-HA antibodies; lane 4: negative control of Hst7 with beads but without antibodies; lane 5: loading control of Cek1; lane 6: IP of Cek1 with anti-FLAG antibodies. IP-AB: antibody used for immunoprecipitation; LC: loading control; αFL: anti-FLAG antibody; αHA: anti-HA antibody. (C) Yeast two-hybrid experiment with Hst7 and Cek1 after 7 days on selective medium. The interacting S. cerevisiae protein pair Bcy1 with Tpk1 was used as positive control. (D) Comparison of pair-wise interactions of C. albicans proteins as shown by a C. albicans two-hybrid experiment (A) and of S. cerevisiae proteins demonstrated in the yeast two-hybrid assay (48,54–56). Arrows are directed from the bait protein to the prey protein.
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