Identification of Caspofungin-hypersensitive Transcription Factor Mutants
To find regulators of the cell wall damage response, we attempted to create homozygous insertion mutants for 67 genes that were related to the transcription process (). We were unable to create mutants in 34 of these genes, some of which may be essential. We note that the S. cerevisiae orthologues of 13 of these genes are essential, but homozygous C. albicans mutants for another six of these genes have been made previously by other methods. We screened the mutants we recovered in 33 genes for altered growth on caspofungin medium and found a caspofungin-sensitive strain with an insertion in SKO1 ().
C. albicans insertion mutant summary
Sko1 is orthologous to the S. cerevisiae transcription factor ScSko1, which functions in the osmotic stress response. To verify that Sko1 governs caspofungin sensitivity in C. albicans, we constructed a sko1Δ/Δ deletion mutant. Growth of the sko1Δ/Δ mutant was drastically reduced on caspofungin plates compared with nutrient YPD plates (). Similar results were observed using another independent sko1Δ/Δ mutant (derived from an independent heterozygote; data not shown). The caspofungin-hypersensitive phenotype of both mutants was complemented by introduction of a wt copy of SKO1 ( and data not shown), indicating that the sko1Δ mutation is the cause of caspofungin hypersensitivity. These findings show that SKO1 is required for normal caspofungin sensitivity.
Figure 1. Caspofungin sensitivity assays. Overnight cultures of prototrophic C. albicans strains were serially diluted and spotted onto nutrient YPD medium or YPD supplemented with caspofungin (125 ng/ml). The wild-type C. albicans reference strain (DAY185), null (more ...)
Regulation of SKO1 Expression by Cell Wall Damage
Transcription factors are often induced under conditions that require their biological activity. Thus, we hypothesized that caspofungin treatment may induce SKO1 expression. We measured SKO1 transcript levels by RT-PCR after caspofungin treatment. SKO1 was up-regulated sixfold in wt cells treated with caspofungin (A). SKO1 expression was not detected in the sko1Δ/Δ deletion mutant, thus confirming primer specificity, and was restored to wt levels in the sko1Δ/Δ/+-complemented strain (B). To monitor Sko1 protein levels, we constructed a strain carrying a functional epitope-tagged Sko1-V5 (). Consistent with our gene expression results, Western blotting analysis showed that there was an increase in the amount of Sko1-V5 protein levels after caspofungin treatment (C). We conclude that caspofungin induces SKO1 gene expression and protein accumulation.
Figure 2. SKO1 Expression Analysis. (A) SKO1 expression was monitored using real-time (RT) PCR analysis in the reference strain DAY185 and prototrophic hog1Δ/Δ strain (JMR114) with or without 125 ng caspofungin. (B) RT-PCR analysis of SKO1 expression (more ...)
Role of SKO1 in the Transcriptional Response to Cell Wall Damage
We considered the possibility that Sko1 may be required for expression of caspofungin-responsive genes. Alternatively, Sko1 may be required for expression of osmotic stress response genes that promote survival after cell wall damage. To test these hypotheses, we monitored expression of the caspofungin-responsive gene PGA13
and the osmotic stress response genes RHR2
and GPD2. PGA13
specifies a cell wall protein and is induced in response to cell wall damage (Bruno et al., 2006
) but not in response to osmotic stress (Enjalbert et al., 2006
). Rhr2 and Gpd2 catalyze the synthesis of glycerol, which is critical in adaptation to osmotic stress (Fan et al., 2005
; Enjalbert et al., 2006
). We observed that PGA13
was induced in the wt and sko1
Δ/Δ/+-complemented strains, but not in the sko1
Δ/Δ mutant (A). On the other hand, GPD2
expression was similar in the wt strain and sko1
Δ/Δ mutant (, B and C). Therefore, although caspofungin treatment induces two osmotic stress-responsive genes, this response is independent of Sko1 function. In contrast, induction of the cell wall protein gene PGA13
depends on Sko1 function.
Figure 3. Gene expression response to caspofungin in wt and sko1Δ/Δ strains. Kinetic analysis of PGA13 (A), GPD2 (B), and RHR2 (C) expression after caspofungin treatment in reference strain DAY185 strain (solid line with black squares), the sko1 (more ...)
To define Sko1-dependent genes in broader terms, we performed microarray comparisons of the wt strain and sko1Δ/Δ mutant treated with caspofungin (Supplementary Dataset 1, Worksheet 1 and 2). We found that Sko1 regulates 79 caspofungin-responsive genes, including several cell wall biogenesis genes (Supplemental Dataset 1, Worksheet 3). RT-PCR analysis confirmed the reduced expression of cell wall biogenesis genes CRH11, MNN2, and SKN1 in the sko1Δ/Δ mutant treated with caspofungin (, A–C). Gene expression levels were restored to wt in the sko1Δ/Δ/+-complemented strain (, A–C). Therefore, Sko1 is necessary for expression of many caspofungin-responsive genes.
Figure 4. Verification of Sko1 target genes identified through microarray analysis. RT-PCR expression analysis of SKO1 array target genes CRH11 (A), MNN2 (B), SKN1 (C), and HGT6 (D) with or without caspofungin treatment in reference strain DAY185, the sko1Δ/Δ (more ...)
We noted that carbohydrate metabolic genes, such as the glucose transporter gene HGT6, were significantly overexpressed in the sko1Δ/Δ mutant (Supplementary Table S1, Worksheets 1 and 2). These genes are not induced by caspofungin. RT-PCR assays showed that HGT6 is overexpressed in the sko1Δ/Δ mutant with or without caspofungin treatment (D). These findings indicate that Sko1 is a negative regulator of carbon metabolic genes.
Identification of Upstream Regulators of SKO1 Expression
To identify upstream regulators of Sko1 activity, we first considered the S. cerevisiae
paradigm. The protein kinase ScHog1 activates ScSko1 by phosphorylation in response to osmotic shock, thereby causing a change in ScSko1 electrophoretic mobility (Proft et al., 2001
). Thus, we considered that C. albicans
Hog1 may be a regulator of Sko1 in response to caspofungin treatment. Prior studies have shown that the C. albicans
the HOG pathway is important for cell wall biosynthesis and stability (Eisman et al., 2006
; Enjalbert et al., 2006
; Munro et al., 2007
). However, we observed that a hog1
Δ/Δ mutant was only slightly hypersensitive to caspofungin compared with the sko1
Δ/Δ mutant (), and it expressed SKO1
normally (A). Protein analysis from wt cells treated with caspofungin showed that Sko1 does not undergo an electrophoretic shift (C). On the other hand, we observed a Sko1 electrophoretic shift after osmotic shock in wt cells but not in the hog1
Δ/Δ mutant strain (A). The Sko1 electrophoretic shift was sensitive to phosphatase treatment (B). These results suggest that Hog1 phosphorylates Sko1 after osmotic stress, but argue that the HOG pathway does not regulate Sko1 after caspofungin-induced cell wall damage.
Figure 5. Hog1-dependent phosphorylation of Sko1 after osmotic stress. (A) Sko1-V5 was visualized on an immunoblot of wt cells (strain JMR143) or hog1Δ/Δ cells, with or without 1.5 M NaCl treatment for 10 min. (B) Total protein extracts were collected (more ...)
We have recently identified insertion mutants in several protein kinase–related genes that are hypersensitive to caspofungin (Blankenship, Fanning, Hamaker, and Mitchell, unpublished data). Those protein kinases are additional candidate SKO1 regulators. We found that SKO1 expression was similar to wt in eight mutants, reduced about twofold in four mutants, and increased about twofold in four mutants. We note that SKO1 expression was increased in all mutants of the PKC-signaling pathway (). SKO1 expression was most severely reduced in the psk1−/− mutant (). Indeed, several independent psk1Δ/Δ deletion strains were hypersensitive to caspofungin ( and data not shown), a phenotype that was complemented by a wt PSK1 allele (). SKO1 was expressed at its uninduced level in three independent psk1Δ/Δ mutant deletion mutants, regardless of caspofungin treatment (A and data not shown). Therefore, Psk1 is a positive regulator of SKO1 expression in caspofungin-treated cells.
Figure 6. SKO1 Expression in caspofungin-hypersensitive protein kinase mutants. SKO1 expression was monitored by RT-PCR in reference strain DAY286 and in the 17 protein kinase insertion homozygotes indicated. All strains were treated with caspofungin for 60 min. (more ...)
Figure 7. Psk1 Requirement for SKO1 expression. (A) RT-PCR analysis of SKO1 expression in reference strain DAY185, the prototrophic psk1Δ/Δ mutant strain (JMR192), and the psk1Δ/Δ/+-complemented strain (JMR188) with or without caspofungin (more ...)
Our observations predict that a psk1Δ mutation will affect expression of Sko1 target genes. RT-PCR assays showed reduced expression of PGA13 and MNN2 and the increased expression of HGT6 in psk1Δ/Δ cells, compared with wt or complemented strains (, A and B). Interestingly, HGT6 was overexpressed in the psk1Δ/Δ mutant only after caspofungin treatment (C), the circumstance in which the mutant has reduced expression of SKO1 (). These results support the model that Psk1 is required for functional expression of SKO1 in response to caspofungin.
Figure 8. Expression of SKO1 target genes in psk1Δ/Δ mutants. RT-PCR expression analysis of SKO1 target genes PGA13 (A), MNN2 (B), and HGT6 (C) with or without caspofungin treatment in reference strain DAY185, the prototrophic psk1Δ/Δ (more ...)