Despite
C. albicans being an important human fungal pathogen and its whole genome being sequenced, the majority of
C. albicans genes and proteins remain uncharacterized, largely limited by the aberrant codon usage (
16). Yet, a better comprehension of the regulation and function of proteins can lead to a better understanding of
C. albicans and its role in being both a commensal and pathogenic organism (
43,
44). This can be achieved by an in-depth study of proteins and PPIs. Besides offering information on the biology of an organism, PPIs can also be exploited as the target of drugs such as antifungals (
7,
9,
45,
46). Antifungals that can target the pathogen very specifically are increasingly needed with emerging fungal pathogens and rising resistance (
1,
47). The creation of these very specific antifungals is a significant challenge given the host-like eukaryotic nature of fungal pathogens. Perhaps, the answer to this problem lies in the development of small molecules targeting PPIs (
7,
47,
48). Several techniques are available to study PPIs, and since the first description of the Y2H system by Fields and Song (
49), this system has been used to study thousands of PPIs for hundreds of organisms (
50,
51). Originally, the Y2H technique was used in small-scale (low-throughput) studies in which specific interactions between often-known proteins were studied using
S. cerevisiae as the host organism (
52,
53). Since then, the system has been improved, changed, and adapted, leading to high-throughput screens and even genome-wide studies (
12,
50,
51,
54). The Y2H system is, however, a limited system when studying
C. albicans due to the differences in codon translation for the CUG codon. To overcome the difference in codon usage, the CUG codons are sometimes substituted when using the Y2H system (
55); however, this is not ideal. To overcome this problem, a C2H system was created in 2010 (
19). Here, we presented the first small-scale C2H high-throughput screening based on a mating approach. Using one specific bait, Pho85, we tested a total of 1,478 out of 1,646 potential (90%) interactions. The 168 potential interactions that could not be tested were caused by failed mating due to either the prey not growing on the original SC-Arg plate or the mating failing on Spider-plus-Dox plates. Current optimization in our lab has reduced this problem, and a mating efficiency up to 95% is achievable. To eliminate the problem of false positives, we applied a cautious two-step validation protocol. This reduced the potential interactions from 15 to 5 and finally yielded one interaction between the Pho85 and Pcl5 proteins. This interaction was further demonstrated in a low-scale (cotransformation) C2H setup and also by swapping bait and prey. Finally, it was validated by a co-IP experiment. The interaction was already described between the homologues in
S. cerevisiae, and the
C. albicans Pcl5 was further described in the work of Gildor et al. to be a putative Pho85 interactor (
40). Our results now demonstrate the physical interaction in
C. albicans between the cyclin-dependent kinase Pho85 and the cyclin Pcl5. Our lab is currently creating more prey- and bait-containing
C. albicans mating-compatible strains, and ongoing C2H experiments in our lab are performed to screen several other baits against a random selection of +1,600 preys and are already showing promising novel interactions. Our prey collection contains five orthologues of
S. cerevisiae proteins found to interact with
ScPho85. Only for one of them could we show a clear strong interaction in
C. albicans. Either this may reflect physiological differences between the two species, or the effect may also be caused by the technology itself. In
S. cerevisiae, the Y2H system is performed with a strong promoter coupled with multicopy plasmids, resulting is very strong overexpression of the hybrid proteins. This is not the case in
C. albicans, where each construct is integrated in the genome.
The two-step validation approach and the use of a manual 96-replicate pinner, however, currently limits the C2H system to smaller (manual) high-throughput screens. Although partly limited at the moment, it will be possible to scale up the C2H technique in the near future with adjustments so that an automated (robotic) approach can be used. Together with the completion of a genome-wide prey and bait collection, this will allow a genome-wide C2H screen of PPIs in
C. albicans. Jointly with other techniques, such as the vesicle capture interaction assay (
27), BiFC (
26), tandem affinity purification (
56,
57), and the “expanded genetic code” technique (
28), the C2H system will bring us another step closer to elucidating the
C. albicans protein interaction network and perhaps to the development of novel, very specific antifungals. The confirmed PPI found in this study will be uploaded to the BioGRID database (
58), making it possible to extend the protein-protein interaction network in
C. albicans. With the results presented here, we show the
Candida community the potential of the C2H system to investigate PPIs. C2H high-throughput screening can be applied to detect multiple novel interactions, while the low-scale C2H system can be used to study putative interactions.