Phenotypic analysis of a family of transcriptional regulators, the zinc cluster proteins, in the human fungal pathogen Candida glabrata

Abstract

Candida glabrata is the second most important human fungal pathogen. Despite its formal name, C. glabrata is in fact more closely related to the nonpathogenic budding yeast Saccharomyces cerevisiae. However, less is known about the biology of this pathogen. Zinc cluster proteins form a large family of transcriptional regulators involved in the regulation of numerous processes such as the control of the metabolism of sugars, amino acids, fatty acids, as well as drug resistance. The C. glabrata genome encodes 41 known or putative zinc cluster proteins, and the majority of them are uncharacterized. We have generated a panel of strains carrying individual deletions of zinc cluster genes. Using a novel approach relying on tetracycline for conditional expression in C. glabrata at the translational level, we show that only two zinc cluster genes are essential. We have performed phenotypic analysis of nonessential zinc cluster genes. Our results show that two deletion strains are thermosensitive whereas two strains are sensitive to caffeine, an inhibitor of the target of rapamycin pathway. Increased salt tolerance has been observed for eight deletion strains, whereas one strain showed reduced tolerance to salt. We have also identified a number of strains with increased susceptibility to the antifungal drugs fluconazole and ketoconazole. Interestingly, one deletion strain showed decreased susceptibility to the antifungal micafungin. In summary, we have assigned phenotypes to more than half of the zinc cluster genes in C. glabrata. Our study provides a resource that will be useful to better understand the biological role of these transcription factors.

Keywords: Candida glabrata; drug resistance; phenotypic analysis; transcriptional regulators; zinc cluster proteins.

Figure 1

Alignment of the cysteine-rich motif of S. cerevisiae Gal4 with C. glabrata zinc cluster proteins. C. glabrata zinc cluster proteins were identified by BLAST searches of the C. glabrata genome using S. cerevisiae Gal4 and other zinc cluster proteins as queries and were named CgZCF1 to 41 (C. glabrata Zinc Cluster Factor). The cysteines residues (in yellow) of the 41 putative or known zinc cluster proteins were aligned using Gal4 as a reference. A consensus sequence is shown on top of the figure. Some residues (located between the second and third cysteines) are involved in DNA recognition by Gal4 and are shown in turquoise. Conserved or alternate residues found in other S. cerevisiae zinc cluster proteins are shown in green. Systematic and gene names are listed in Table 1.

Figure 2

Strategy used to generate cassettes for deletion of zinc cluster genes in C. glabrata. Fragments corresponding to sequences flanking an open reading frame (ORF) of interest were amplified by polymerase chain reaction (PCR). Oligos were designed so that they contain 45 bp of homology to the plasmid pRS-URA3 containing the S. cerevisiae URA3 gene. pRS-URA3 was digested with SmaI and transformed along with the two PCR products into S. cerevisiae. A quadruple recombination between the plasmid backbone, the PCR products and the URA3 marker allows the generation of a plasmid which can be recovered and amplified in E. coli. After digestion with SmaI, the DNA is then transformed into C. glabrata.

Figure 3

The genes CgZCF11, CgZCF13, and CgTOP2 are essential. Strains (as listed on the left) were grown overnight in rich medium containing 100 µM tetracycline. Cells were then serially diluted and spotted on plates containing tetracycline at concentrations indicated on the top of the Figure and plates were incubated at 30° for 24 h. Spotting experiments were performed with two independent clones for the genes tested. As a control, a C. glabrata ortholog of the essential S. cerevisiae gene TOP2 (encoding topoisomerase II) was used. It is not clear whether CgZCF5 is essential or not because partial growth inhibition could be due, for example, to incomplete translational inhibition.

Figure 4

Strains Cg∆zcf7 and Cg∆zcf20 are thermosensitive whereas strain Cg∆zcf24 is sensitive to oxidative stress. Strains were grown overnight in rich medium, serially diluted, and spotted on plates as described in the section Material and Methods. (A) Two independent clones of deletion strains Cg∆zcf7 and Cg∆zcf20 were tested and are Ura⁺. Cg∆zcf7-1A + ZCF7 and Cg∆zcf20-1A + ZCF20 are deletion strains were a wild-type allele was introduced and the strains are Ura^-. B) Two independent clones of deletion strain Cg∆zcf24 were tested and are Ura⁺. Cg∆zcf24-1A + ZCF24 is a deletion strain were a wild-type allele was introduced and the strain is Ura⁻.

Figure 5

Sensitivity of deletion strains to caffeine. Strains were grown overnight in rich medium, serially diluted and spotted on plates as described in the section Material and Methods. Two independent clones of deletion strains Cg∆zcf7 and Cg∆zcf20 were tested and are Ura⁺. Cg∆zcf7-1A + ZCF7, Cg∆zcf7-1B + ZCF7, Cg∆zcf20-1A + ZCF20, and Cg∆zcf20-1B + ZCF20 are deletion strains were a wild-type allele was introduced and the strains are Ura⁻.

Figure 6

Strains Cg∆zcf26 and Cg∆zcf37 show increased tolerance to salt stress. Strains were grown overnight in rich medium, serially diluted and spotted on plates as described in the section Material and Methods. Two independent clones of deletion strains Cg∆zcf26 and Cg∆zcf37 were tested and are Ura⁺. Cg∆zcf26-1A + ZCF26, Cg∆zcf26-1B + ZCF26, and Cg∆zcf37-1A + ZCF37 are deletion strains were a wild-type allele was introduced and the strains are Ura⁻.

Figure 7

Increased susceptibility of deletion strains Cg∆zcf4 and Cg∆zcf37 to azoles. Strains were grown overnight in rich medium, serially diluted, and spotted on plates with or without drugs as indicated in the figure. Two independent clones of deletion strains Cg∆zcf4 and Cg∆zcf37 were tested and are Ura⁺. Cg∆zcf4-2A + ZCF4, Cg∆zcf4-2B + ZCF4, Cg∆zcf37-2A + ZCF37 and Cg∆zcf37-2B + ZCF37 are deletion strains were a wild-type allele was introduced and the strains are Ura⁻.

Figure 8

Strain Cg∆zcf6 shows reduced susceptibility to micafungin. Strains were grown overnight in rich medium, serially diluted, and spotted on plates as described in the section Material and Methods. Two independent clones of deletion strains Cg∆zcf6 were tested and are Ura⁺. Cg∆zcf6-1A + ZCF6 is a deletion strain were a wild-type allele was introduced and the strain is Ura^-. MIC values are given at the bottom of the figure.