A High-Throughput <i>Candida albicans</i> Two-Hybrid System

Abstract

<i>Candida albicans</i> is a human fungal pathogen that does not follow the universal codon usage, as it translates the CUG codon into serine rather than leucine. This makes it difficult to study protein-protein interactions using the standard yeast two-hybrid (Y2H) system in the model organism <i>Saccharomyces cerevisiae</i> Due to the lack of adapted tools, only a small number of protein-protein interactions (PPIs) have been detected or studied using <i>C. albicans</i>-optimized tools despite the importance of PPIs to understand cell biology. However, with the sequencing of the whole genome of <i>C. albicans</i>, the availability of an ORFeome collection containing 5,099 open reading frames (ORFs) in Gateway-adapted donor vectors, and the creation of a Gateway-compatible <i>C. albicans</i>-specific two-hybrid (C2H) system, it became possible to study protein-protein interactions on a larger scale using <i>C. albicans</i> itself as the model organism. Erroneous translations are hereby eliminated compared to using the <i>S. cerevisiae</i> Y2H system. Here, we describe the technical adaptations and the first application of the C2H system for a high-throughput screen, thus making it possible to screen thousands of PPIs at once in <i>C. albicans</i> itself. This first, small-scale high-throughput screen, using Pho85 as a bait protein against 1,646 random prey proteins, yielded one interacting partner (Pcl5). The interaction found with the high-throughput setup was further confirmed with a low-throughput C2H experiment and with a coimmunoprecipitation (co-IP) experiment.<b>IMPORTANCE</b><i>Candida albicans</i> is a major fungal pathogen, and due to the rise of fungal infections and emerging resistance to the limited antifungals available, it is important to develop novel and more specific antifungals. Protein-protein interactions (PPIs) can be applied as very specific drug targets. However, because of the aberrant codon usage of <i>C. albicans</i>, the traditional yeast two-hybrid system in <i>Saccharomyces cerevisiae</i> is difficult to use, and only a limited number of PPIs have been described in <i>C. albicans</i> To overcome this, a <i>C. albicans</i> two-hybrid (C2H) system was developed in 2010. The current work describes, for the first time, the application of the C2H system in a high-throughput setup. We hereby show the usefulness of the C2H system to investigate and detect PPIs in <i>C. albicans</i>, making it possible to further elucidate protein networks in <i>C. albicans</i>, which has the potential to lead to the development of novel antifungals which specifically disrupt PPIs important for virulence.