Ste18p Is a Positive Control Element in the Mating Process of Candida albicans

Abstract

bHeterotrimeric G proteins are an important class of eukaryotic signaling molecules that have been identified as central elements in the pheromone response pathways of many fungi. In the fungal pathogen Candida albicans, the STE18 gene (ORF19.6551.1) encodes a potential ␥ subunit of a heterotrimeric G protein; this protein contains the C-terminal CAAX box characteristic of ␥ subunits and has sequence similarity to ␥ subunits implicated in the mating pathways of a variety of fungi. Disruption of this gene was shown to cause sterility of MTLa mating cells and to block pheromone-induced gene expression and shmoo formation; deletion of just the CAAX box residues is sufficient to inactivate Ste18 function in the mating process. Intriguingly, ectopic expression behind the strong ACT1 promoter of either the G␣ or the G␤ subunit of the heterotrimeric G protein is able to suppress the mating defect caused by deletion of the G␥ subunit and restore both pheromone-induced gene expression and morphology changes.

Deletion of a Yci1 Domain Protein of Candida albicans Allows Homothallic Mating in MTL Heterozygous Cells

“…OFR1 was deleted by standard two-step disruptions using PCR products ( 45 ). The markers HIS1 and URA3 were amplified by PCR from plasmids pFA-CaHIS1 and pFA-CaURA3 using primers that provided homology to the flanking regions of OFR1 .…”

“…Cells were grown at RT until the culture reached an OD 600 between 0.8 and 1.2, harvested, and stored at −80°C until RNA extraction. Total RNA was isolated by the hot phenol method as described elsewhere ( 45 ). mRNA was purified using the New England Biolabs polyA Spin mRNA isolation kit, and then reverse transcription for cDNA production was followed by indirect cDNA labeling with aminoallyl-dUTP for dye addition.…”

“…mRNA was purified using the New England Biolabs polyA Spin mRNA isolation kit, and then reverse transcription for cDNA production was followed by indirect cDNA labeling with aminoallyl-dUTP for dye addition. Arrays were obtained from the NRC-BRI Microarray Facility; hybridization protocols were as described previously ( 45 ).…”

“…Cells were then centrifuged, and tester and experimental strains were mixed in 5 ml fresh YCB-GlcNAc liquid medium in 50-ml Falcon tubes at a final concentration of each strain of 1 × 10 7 cells/ml. Cells were incubated at RT with shaking at 220 rpm for 48 h before being plated onto YCB-glucose medium for prototrophic selection; selection plates were incubated at 30°C for 3 days before the colonies were counted ( 45 ). The mating frequency is calculated based on the number of prototrophic mating product colonies (on the Trp − Lys − Arg − plates) divided by the limiting value for the input of tester cells (detected on the Arg − plates) or the experimental strain cells (detected on the Trp − Lys − plates).…”

Fungal G-protein-coupled receptors: mediators of pathogenesis and targets for disease control

“…Additionally, the CaSte2 and CaSte3 receptors also function in non-mating white cells, which respond to opaque-cell derived pheromones by increasing adhesion and biofilm formation, establishing a pheromone gradient that allows two opaque cells of opposite mating type to find each other in a thick, white cell-produced biofilm 64 . Both cell-types utilise the same MAPK pathway, but activate distinct transcription factors 65 . Finally, a parasexual cycle, which occurs instead of meiosis, results in concerted chromosome loss from the tetraploid form, generating diploid and aneuploid progeny without forming immunogenic spores, while increasing genetic variation and promoting antifungal resistance 66,67 .…”