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.

Figure 1.

Statistics on the Candida albicans ORFeome. (A) Percentage of successfully cloned ORFs that are identical to the reference sequence (No SNPs, no DIPs) or that contain SNPs and/or DIPs. Only SNPs that do not introduce a STOP codon and DIPs multiple of 3 bp have been accepted. (B) Haplotypes distribution. Each cloned ORF has been assigned to HapA or HapB when there are differences between the two alleles of the ORF, and HapA/B when the two alleles of the ORF are identical (as defined in Assembly22). (C) Success rate on each chromosome. The graphs represent the number of reference ORFs and the number of successfully cloned ORFs on each chromosome, as well as the overall percentage of success for each chromosome. (D) Percentage of success based on ORF size.

Figure 2.

Snapshot of the CandidaOrfDB interface. An example of an ORF page is shown. In the ‘ORF details’ box, the different ID names, the length and the chromosome location of the ORF of interest are displayed. The haplotype assigned to the cloned ORF is noted (A, B or A/B if there is no allelic differences or equal number of differences against both haplotypes). The coordinates of introns are indicated when present. The summary results of the sequence analysis against the reference sequence are presented. The ‘SNP(s)’ box shows a table that lists the sequence differences between the cloned ORF and the reference sequences (Haplotypes A and B from Assembly22, and Assembly21 sequences). The ‘Nucleotide and Protein sequences’ boxes display the sequences with a color code for synonymous and non-synonymous SNPs. All sequences can be downloaded. The ‘Resources’ box displays links towards information that is relevant to each resource, i.e. oligonucleotide sequences for the BP clones, barcode sequence for the overexpression plasmids and the C. albicans overexpression strains, position of the clone in the different plates of the collection, plasmid sequences. The ‘Restrictions on the cloned sequence’ box indicates the existence of restriction sites, as well as the size of the expected fragments for enzymes that are used in regards to subsequent applications of these donor plasmids.

Figure 3.

Structure of the destination vectors for use with the Candida albicans ORFeome. Schematic view of the 49 destination vectors, each designated with a standardized nomenclature, pCA-DESTijkl, whereby i stands for the transformation marker (1, URA3; 2, NAT1); j stands for the promoter (1, P_TET; 2, P_PCK1; 3, P_TDH3; 4, P_ACT1); k stands for N-terminal tagging (0, no tag; 1, 3xHA; 2, GFP; 3, TAP); and l stands for C-terminal tagging (0, no tag; 1, 3xHA; 2, GFP; 3, TAP). The spacer sequence (SP), represented by the yellow box, is intended to facilitate subsequent Illumina-based barcode sequencing. The Gateway cassette (GTW), represented by the green box, requires working with ccdB resistant Escherichia coli strains. Integration of StuI-linearized plasmids (or at the nearby I-SceI site if the cloned ORF contains StuI sites) is targeted at the RPS1 locus.

Figure 4.

Ume6-driven filamentation validates the primary set of 15 destination vectors. Inducible (A) and constitutive (B) overexpression of UME6 triggers filamentation. Isolates were grown in rich medium in presence or absence of ATc3 for 2–4 h at 30°c. Cells were observed with a Leica DM RXA microscope (Leica Microsystems) with an x40 oil-immersion objective.

Figure 5.

Detection of 3xHA- or TAP-tagged Ume6 protein by western blot. Production of 3xHA-tagged (A) and TAP-tagged (B) Ume6 proteins. Candida albicans strains harboring the P_TET and P_TDH3 constructions were grown in YPD ± ATc3 for 2 and 4 h, respectively. Whole cell extracts were separated by SDS-PAGE and probed with a peroxidase-coupled antibody, allowing the detection of the 3xHA-tagged and TAP-tagged Ume6 protein. The tagged Ume6 proteins are indicated by an arrow along with their deduced sizes. M1: PageRuler Prestained Protein Ladder (Thermo Scientific) and M2: Precision Plus Protein™ Dual Color Standards (Bio-Rad).

Figure 6.

Proof of principle of two-hybrid based PPI detection via mating in Candida albicans. (A) Schematic representation of the concept. Diploid opaque MTLa bait-expressing cells were mixed with opaque MTLα prey-expressing cells to obtain tetraploids, as selected on leucine and arginine-free medium. Detection of protein-protein interaction was observed on medium lacking histidine as an indicator of expression of the two-hybrid readout marker. (B) Proof of principle using Hst7 as a bait and Cek1 as a prey, previously shown to interact (31). Cells of each type were spotted in a dilution series and growth was monitored on SC-leu-arg, which allowed growth of the tetraploid and diploid offspring and on SC-met-his, which allowed detection of PPI, only in those cells that are expressing both bait and prey proteins. As negative controls, tetraploids derived from the crossing of bait-expressing strains with empty prey vector transformed strains, and from the crossing of prey-expressing strains with strains expressing the empty bait vector grew on SC-leu-arg but not on SC-met-his.