Generating genomic platforms to study Candida albicans pathogenesis

Abstract

The advent of the genomic era has made elucidating gene function on a large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources toward this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5099 ORFs cloned in a Gateway™ donor vector, representing 83% of the currently annotated coding sequences of C. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database at http://candidaorfeome.eu. We also engineered 49 expression vectors with a choice of promoters, tags and selection markers and demonstrated their applicability to the study of target ORFs transferred from the C. albicans ORFeome. In addition, the use of the ORFeome in the detection of protein–protein interaction was demonstrated. Mating-compatible strains as well as Gateway™-compatible two-hybrid vectors were engineered, validated and used in a proof of concept experiment. These unique and valuable resources should greatly facilitate future functional studies in C. albicans and the elucidation of mechanisms that underlie its pathogenicity.

Recent advances in understanding Candida albicans hyphal growth

“…These studies yielded important information on gene essentiality and azole resistance in this fungal pathogen. Extensive effort was also invested in generating a genomic platform centered on an ORFeome collection representing the majority of open reading frames (ORFs) in Gateway donor vectors, together with a wide range of expression vectors 18 facilitating genome-wide overexpression analyses and protein–protein interaction studies 16, 20, 34 . Together, these new technologies have facilitated recent advances in hyphal growth signaling, host interactions, and membrane traffic.…”

Systematic gene overexpression inCandida albicansidentifies a regulator of early adaptation to the mammalian gut

“…The resulting strain was transformed with plasmid CIp10 to generate the prototrophic strain CEC4425 (Table S11) that we used as a BFPlabelled control for our 1:1 competitive assays (Figure 3c,d). Strains overexpressing CRZ2 from the constitutive TDH3 promoter were generated by transferring the CRZ2 ORF from the respective pDONR207 derivative to the Gateway-compatible CIp10-P TDH3 -GTW plasmid (Legrand et al 2018) followed by transformation of strain BWP17AH (CEC161; Chauvel et al, 2012) to generate three independent P TDH3driven CRZ2-overexpression clones (C26, C27, and C28; Table S11).…”

Protein-Protein Interactions in Candida albicans

“…In the most recent paper using the C2H system, Wangsanut et al (2018) tried to demonstrate the interaction between transcription factors Grf10 and Bas1 but without success (Wangsanut et al, 2018, 2019). The C2H system had been limited to small-scale studies until recently when it was adapted to a high-throughput setup (Legrand et al, 2018; Schoeters et al, 2018).…”

“…It is a diploid organism without a complete sexual cycle (no meiosis found so far) and has a non-canonical codon usage (CUG translated as serine rather than leucine), in addition, it lacks stable episomal plasmids and has a low transformation efficiency (Datta et al, 1990; Murad et al, 2000; Noble and Johnson, 2007; Stynen et al, 2010; Hickman et al, 2015). In recent years, as novel research tools have been developed, the whole genome has been sequenced and the ORFs have been made available for the community in Gateway TM -adapted vectors (Jones et al, 2004; Kaplanek et al, 2006; Muzzey et al, 2013; Legrand et al, 2018).…”