Chemical-Genetic Approaches for Exploring Mode of Action of Antifungal Compounds in the Fungal Pathogen Candida albicans

Abstract

Candida albicans is a prevalent fungal pathogen of humans that can cause both superficial and life-threatening disease, primarily in immunocompromised populations. Currently, antifungal drug classes available to treat fungal infections remain limited and the emergence of drug-resistant strains threatens antifungal efficacy, necessitating the discovery and development of additional therapeutics. The construction of the C. albicans double-barcoded heterozygous deletion collection (DBC) enables the rapid and systematic assessment of haploinsufficiency phenotypes in a pooled format. Specifically, this functional genomics resource can be used to identify heterozygous deletion mutants that are hypersensitive to compounds in order to define putative cellular targets and/or other modifiers of compound activity. Here, we describe protocols to characterize the mode of action of small molecules using the C. albicans DBC, including how to prepare compound-treated cultures, isolate genomic DNA, amplify strain-specific barcodes, and prepare DNA libraries for high-throughput sequencing. This technique provides a powerful approach to elucidate the compound mechanism of action in order to bolster the antifungal pipeline.

Keywords: Antifungal; Candida albicans; Chemical genomics; Haploinsufficiency profiling; High-throughput sequencing; Molecular barcodes; PCR.

Figure 1:

Haploinsufficiency profiling (HIP) identifies putative compound targets. In C. albicans HIP employs the C. albicans double-barcoded heterozygous deletion collection (DBC), which encompasses both essential and non-essential genes. Individual strains are tagged with two unique DNA barcodes that permit simultaneous analysis within a single pool. The DBC is grown competitively in the absence and presence of a compound of interest. Genomic DNA is isolated after a duration of pooled growth, and PCR amplification of strain-identifying barcodes is performed using universal primers for the upstream or downstream barcodes. High-throughput barcode sequencing and normalization to the untreated pool is used to quantify strain representation.

Figure 2:

The C. albicans double-barcoded heterozygous deletion collection (DBC) was constructed by systematically transforming a wild-type C. albicans strain with a PCR-generated disruption cassette containing a HIS3 selectable marker flanked with appropriate homologous sequence to precisely replace one allele of the target gene. Two distinct barcodes (UP, ‘UPTAG’ and DOWN, ‘DNTAG’) were introduced into the cassette during PCR amplification. Two primer pairs that anneal to the common arms flanking each ‘UPTAG’ (pink) and ‘DNTAG’ (green), enable PCR amplification of the strain-identifying barcodes

Figure 3:

Example of plate layout for genomic DNA quantification using the PicoGreen DNA quantification kit. Red numbers indicate standard DNA concentrations in ng/mL. Coloured wells indicate those containing gDNA samples with blue representing one replicate and green a second replicate for the same gDNA sample.

Figure 4:

Schematic of strain-specific PCR fragments generated during protocol. A) Universal primers are paired with distinct indexed primers to amplify strain-specific molecular barcodes. Primers anneal to conserved sequences that flank the unique molecular barcodes (pink for UPTAG and green for DOWNTAG). These primers also contain Illumina adapter sequences. B) The resultant PCR fragments that are generated along with a general schematic of where the sequencing primers anneal. C) A more detailed view of the UPTAG PCR amplicon that is generated and where sequencing primers anneal in order to analyze the pooled samples.

Figure 5:

Agarose and polyacrylamide gels generated throughout the protocol to visualize amplified barcodes. A) Example of a 2% agarose 1X TAE gel used to visualize 8 individual UPTAG barcodes. “–CON” indicates negative (water only) control that did not contain gDNA. B) Example of 5% polyacrylamide gel containing UPTAG and DOWNTAG pooled samples. Suggested excision boundary is boxed in red. C) Example of a 2% agarose 1X TAE gel used to visualize purified UPTAG and DOWNTAG pooled barcode samples after purification. * indicates 150 base pairs.

Figure 6:

Scatterplot depicting the raw barcode reads obtained with a genomic DNA preparation that biases extraction efficiency based on chromosomal location.