Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

Abstract

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.

A Novel Hybrid Iron Regulation Network Combines Features from Pathogenic and Nonpathogenic Yeasts

“…To identify factors important for iron homeostasis, an extensive deletion mutant library (Schwarzmüller et al [25] and mutants generated in this work) was screened for iron-dependent growth defects. In total, 649 strains were analyzed for their growth behavior under conditions of different iron concentrations, and 100 of them displayed an iron-dependent phenotype under at least one set of conditions.…”

“…The mutants screened in this study comprised the strains described in Schwarzmüller et al (25) as well as C. glabrata gene knockout strains generated in this study, constructed in the ATCC 2001 wild-type strain using the same gene deletion strategy. The latter included orthologs of S. cerevisiae or C. albicans high- and low-affinity iron uptake systems ( fet3 Δ, ftr1 Δ, and fet4 Δ mutants) and transcriptional regulators ( aft1 Δ, sef1 Δ, hap4 Δ, and hap5 Δ mutants) (Table 1).…”

Beyond Candida albicans: Mechanisms of immunity to non-albicans Candida species

“…As the Candida field has largely focused on C. albicans , a wealth of genetic mutants exist, such as morphotype-locked mutants, fluorophore-expressing reporters (e.g., GFP, Luciferase), and epitope-tagged strains for in vivo tracking [138, 166]. A parallel collection of genetic mutants does not yet exist for NAC species, although progress is being made for C. glabrata [167]. …”

Saccharomyces cerevisiae single-copy plasmids for auxotrophy compensation, multiple marker selection, and for designing metabolically cooperating communities

“…These libraries include gene deletion mutants 5, 10, 11 , genetically introduced GFP, GST, and TAP fusions 12– 14 , transposon insertion mutants 15 , decreased abundance by mRNA perturbation (DAmP) mutants 16 , Tet-promoter controlled expression 17 and the ts-alleles for essential genes 18, 19 . Furthermore, systematic strain collections of other fungal species including Schizosaccharomyces pombe 20, 21 and the pathogens Candida glabrata 22 , Candida albicans 23 or Neurospora crassa 24 , all involve use of auxotrophic markers as well. As a result, auxotrophic backgrounds are omnipresent in a large number of functional genomic experiments, and have been used in a countless number of small-scale experiments, resulting in their ubiquity across yeast molecular biology literature.…”