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Antimicrobial Agents and Chemotherapy, October 2007, p. 3747-3751, Vol. 51, No. 10
0066-4804/07/$08.00+0     doi:10.1128/AAC.00929-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Ssk1p Response Regulator and Chk1p Histidine Kinase Mutants of Candida albicans Are Hypersensitive to Fluconazole and Voriconazole{triangledown}

Neeraj Chauhan, Michael Kruppa, and Richard Calderone*

Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC 20057

Received 18 July 2007/ Accepted 20 July 2007


   ABSTRACT
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Hypersensitivity to the triazoles fluconazole and voriconazole associated with two-component signal transduction proteins has not been reported in Candida albicans. Herein, we show that strains of C. albicans lacking the response regulator Ssk1p or the Chk1p histidine kinase signal transduction proteins are hypersensitive to fluconazole and voriconazole compared to wild-type (wt) as well as gene-reconstituted strains, reflecting an increased hypersensitivity to these drugs of about 16- to 500-fold. In comparison to wt cells, both mutants had elevated levels of fluconazole accumulation and reduced viability upon incubation with either drug, suggesting that in the absence of Ssk1p or Chk1p, fluconazole and voriconazole have significantly increased fungicidal effects on C. albicans.


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The incidence of fungal infections has increased throughout the world. Patient management of these infections begins with an early diagnosis and the use of antifungal drugs. In some cases, diagnosis of blood-borne diseases is problematic since current assays for antigen detection are not sufficiently sensitive, especially in the case of candidiasis. Management of patients with invasive disease has increasingly utilized the newer triazoles and the echinocandins. For example, voriconazole is quite effective against most forms of primary aspergillosis, and the echinocandins appear to be fungicidal against most Candida species (3, 16). Triazoles appear to be mostly fungistatic and, therefore, resistance to these drugs is commonly reported, especially among several of the non-C. albicans species of Candida such as C. glabrata (5, 16, 25). In this regard, mechanisms of resistance in Candida species include overproduction of efflux pumps, mutations in the ERG11 whose gene product is the target of triazoles, as well as overproduction of Erg11p. Less understood is the hypersensitivity of Candida species to antifungals, which has been reported previously (4, 11, 14, 15, 24, 28). In these reports, hypersensitivity was associated with a gene mutation and/or iron deprivation. For example, an erg1 mutant of C. glabrata had increased susceptibility to fluconazole, itraconazole, and terbinafine that was associated with decreased levels of ergosterol in the mutant and low oxygen tension during growth (28). In C. glabrata, deletion of the calcium channel proteins (Cch1-Mid1) conferred only a minimum hypersensitivity to drugs that target the cell membrane, such as amphotericin B, triazoles, and amorolfine, but augmented killing of the same mutants occurred during long-term incubation with fluconazole (15). A recent report demonstrated that iron deprivation of C. albicans resulted in hypersensitivity to fluconazole as well as a variety of other inhibitors such as cycloheximide (24). Iron deprivation was established as a cause of the drug hypersensitivity by several approaches, including removal of iron from the growth medium and the use of iron mutants (such as ftr1 and ftr2).

Two-component signal transduction has been described in prokaryotes, lower eukaryotes such as fungi, and plants but not in mammalian cells (9, 17). This type of signal pathway includes histidine kinase (HK) and response regulator (RR) proteins that are associated with functions such as adaptation to stress and the regulation of virulence. In prokaryotes, two-component signal transduction is associated with resistance to ß-lactam drugs in Streptococcus pneumoniae and in Mycobacterium tuberculosis, where the response regulator protein EmbR is associated with increased expression of the EmbAB operon genes which in turn confer altered resistance to ethambutol, one of the antituberculosis drugs (18, 26). In human-pathogenic fungi such as C. albicans, two-component signal proteins are required for virulence, cell wall biosynthesis, and stress adaptation, but their role in responses to antifungal drugs is unknown (6-10, 17). Based upon the data from the bacteria mentioned above, we determined the sensitivity/resistance of several two-component HKs and RRs from C. albicans. Of these, below we describe the heightened susceptibilities of the ssk1 RR and the chk1 HK mutants to the triazole antifungals fluconazole and voriconazole.

The strains used in this study are described in Table 1. We compared the parental strain CAF2-1 to mutants with the SSK1 and CHK1 genes deleted (strains SSK21 and CHK21) as well as strains reconstituted with a single gene of SSK1 or CHK1 (strains SSK23 and CHK23) (6-10). For our experiments, broth microdilution assays were performed in accordance with the guidelines in CLSI (formerly NCCLS) document M27-A2 (19, 20, 22, 23). The total volume of cells and drug was 200 µl per microtiter well, and each drug was diluted in RPMI to achieve final concentrations of 0.004 to 128 µg/ml. Uninoculated cultures were used as a reference standard. Stock inoculum suspensions were prepared from 24-h cultures on Sabouraud dextrose agar at 37°C. For MIC determinations, an inoculum of 1.0 x 103 cells per well was used, and cells were prepared in RPMI 1640 medium; all incubations were at 37°C without or with voriconazole or fluconazole. MICs were determined both visually at 24 and 48 h and spectrophotometrically at 550 nm using a microplate reader (TECAN). The lowest concentration of drug that caused a significant growth inhibition (≥90%) below those of the growth control strains (no drug) was noted as the MIC for that drug.


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  		TABLE 1. C. albicans strains used in this study

 
The MICs we obtained for voriconazole and fluconazole against the parental strain CAF2-1 fall within (or are close to) the published sensitivities for clinical isolates (2, 12). In regard to the mutants, our data indicate that both the SSK21 and CHK21 mutants are hypersensitive to the triazoles, fluconazole and voriconazole, compared to the sensitivities of the wild-type (wt) strain CAF2-1 and the gene-reconstituted strains, SSK23 and CHK23 (Table 2). We also tested mutants of the other RR from C. albicans (SKN7) and the SLN1 HK but found them to be either similar in their sensitivities (skn7) or to have slightly increased sensitivities (sln1) to these drugs in comparison to the sensitivity of the parental strain CAF2-1. Interestingly, both CHK21 and SSK21 strains are as sensitive as wt cells (CAF2-1) to amphotericin B, caspofungin, and two imidazoles, miconazole and ketoconazole (data not shown). These results suggest that the signal proteins Ssk1p and Chk1p are critical to the response of cells to the triazoles tested. Also, our data imply that these proteins or their downstream effectors may be exploited in the development of combined therapy with fluconazole or voriconazole or even utilized as new drug targets.


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  		TABLE 2. Susceptibility of C. albicans strains to the triazoles, voriconazole and fluconazole

 
One possibility for the hypersensitivity to the triazoles is an increased or altered uptake of the compounds. To test this, [3H]fluconazole (Amersham Biosciences) uptake was measured according to the procedure of Tsai et al. (28). All C. albicans strains were grown at 30°C overnight in yeast extract-peptone-dextrose (YPD) medium. Cells were diluted with fresh culture medium to 108 cells/ml, as determined by optical density at 600 nm. Fluconazole accumulation was measured by using drug that had been tritiated by gas exchange to a specific activity of 629 GBq/mM. [3H]fluconazole was added to 8-ml cell suspensions at a final concentration of 7.4 kBq/ml (0.2 µCi/ml, 3.6 ng/ml). After 60 min of incubation with rotation at 225 rpm (30°C), triplicate 2.5-ml samples were filtered on a vacuum manifold (Millipore) with 24-mm-diameter GF/C glass fiber filters (Whatman, United Kingdom) which had been presoaked in 100 µM unlabeled fluconazole in phosphate-buffered saline (PBS) (pH 7.0). Zero-time samples were chilled in ice for 30 min before the addition of tritiated fluconazole and kept on ice until they were filtered. Cells were washed on the filters twice with 8 ml of PBS containing 100 µM unlabeled fluconazole. The filters were dried at 37°C for 1 h, placed in Hydrofluor scintillation fluid (National Diagnostics, Atlanta, GA), and allowed to stand overnight, and the radioactivity of the cells was then counted with a LS6500 liquid scintillation counter (Beckman Coulter). Results are expressed as differences in counts per minute per 106 cells at 0 and 60 min; data were evaluated using the analysis of variance two-factor test. We observed that compared to wt cells (strain CAF2-1), uptake of [3H]fluconazole over a 60-min incubation time was increased twofold in the SSK21 mutant, while uptake in the CHK21 mutant (Fig. 1) was slightly increased. In the case of gene-reconstituted strains (CHK23 and SSK23), uptake was comparable to that of CAF2-1. Since net accumulation of fluconazole is a function of both transport and efflux, in all strains we also used RT-PCR to measure the transcripts of several efflux pump genes described for C. albicans, including CDR1, CDR2, CDR4, CDR11, and MDR1.


Figure 1
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  		FIG. 1. [3H]fluconazole uptake in C. albicans. Strains CAF2-1, SSK21 (ssk1/ssk1), SSK23 (ssk1/SSK1), CHK21 (chk1/chk1), and CHK23 (chk1/CHK1). All strains were grown as described in the text and then pulsed with labeled fluconazole for 1 h. Cells were also labeled with fluconazole and immediately transferred to an ice bath (zero time). For CAF2-1 versus SSK21, P equals 0.006 for the 60-min treatment; and for CAF2-1 versus CHK21, P equals 0.06 for the 60-min treatment (analysis of variance two-factor test).

 
Cells were grown in YPD broth at 30°C to log phase without fluconazole and then were treated with fluconazole (1x MIC) or were not treated. Samples were taken at different times (0, 30, 60, and 120 min), total RNA was extracted by the hot phenol method for each strain, and RT-PCR was performed by using a QIAGEN one-step PCR kit; 1 µg of total RNA was used for each PCR. All PCR products were resolved on 1% agarose gels, which were scanned, using an Alpha Imager 2000 (Alpha Innotech Corp.), imported as TIFF files, and evaluated for differential expression (10). Actin expression served as an internal control. We found that transcript levels from all strains were comparable, suggesting that the net increase in accumulation of fluconazole in CHK21 and SSK21 is due to transport and not a reduction in drug efflux (data not shown). These data suggest that the hypersensitivity of the mutants was at least partially associated with greater drug net accumulation, although accumulation was slightly lower in strain CHK21.

Because of the increased sensitivity of each mutant to fluconazole and voriconazole, we also measured the fungicidal effects of each drug on all strains. Cells were grown in YPD medium at 30°C overnight and inoculated to an optical density at 600 nm of 0.01 in minimal medium (0.67% yeast nitrogen base, 2% glucose) containing a 4x MIC concentration of each drug. After 24 h of incubation, 100 µl of cell suspension was collected, cell counts were determined with a hemocytometer, and appropriate cell dilutions in PBS were plated on YPD agar plates to assess the number of viable cells. CFU on YPD plates were counted after 2 days of incubation at 30°C. The results of these experiments using 4x MIC concentrations of each drug are shown in Fig. 2 (fluconazole) and Fig. 3 (voriconazole). For the fluconazole experiment, at an MIC of 4x that of strain SSK21, we observed increased killing versus wt cells that was statistically significant, while strain SSK23 was killed slightly less than CAF2-1, and that difference was statistically insignificant. The CHK21 mutant was also killed more than wt and CHK23 cells but less than strain SSK21. With voriconazole, the killing rates observed in the wt strain (CAF2-1) and gene-reconstituted strains (SSK23 and CHK23) were greater than with fluconazole, but both null mutants (SSK21 and CHK21) were killed significantly more than the wt and gene-reconstituted strains (Fig. 3). When we also used 2x and 1x MIC concentrations of each drug, a reduction in killing was observed at the 1x MIC concentration (data not shown). Thus, the fungicidal activities of these triazoles increased dramatically for both mutants compared to the activity with CAF2-1.


Figure 2
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  		FIG. 2. Killing assays of C. albicans strains in the presence or absence of fluconazole. All strains described in Fig. 1 were incubated in medium containing a 4x (MIC of SSK21 or CHK21) concentration of fluconazole and incubated for 24 h in shake culture at 30°C. At that time, dilutions of untreated and treated cultures were plated on YPD agar and CFU were determined. The percent viability is expressed for all strains compared to CAF2-1. P equals 0.0007 for CAF2-1 versus SSK21 in drug-treated cultures; and for SSK21 versus CHK21, P equals 0.04.

 

Figure 3
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  		FIG. 3. Killing assays of C. albicans strains in the presence or absence of voriconazole. All strains were similarly prepared as in Fig. 2, except that a 4x (MIC) concentration of voriconazole was used for strains SSK21 and CHK21. Viability is also shown for the wt and gene-reconstituted strains. P equals 0.006 for drug-treated CAF2-1 versus SSK21 and CHK21.

 
Two-component phosphorelay systems include a membrane-bound, sensor HK protein, which in bacteria is autophosphorylated in response to an environmental stimulus. The phosphate is then transferred to a cytoplasmic RR protein which in turn directly regulates transcriptional events in response to the stimulus (27). In fungi, phosphotransfer via a membrane-bound HK (Sln1p) to a phosphotransfer intermediate protein (Ypd1p) precedes phosphorylation of the cytoplasmic RR protein Ssk1p. In the absence of stress, phosphorylated Ssk1p is unable to activate the downstream HOG1 mitogen-activated protein kinase pathway. During stress, Ssk1p is not phosphorylated and therefore activates Hog1p, resulting in the adaptation of the cells to several kinds of stress conditions (9, 10). As for other two-component proteins that regulated HOG1 MAPK, no Ypd1 mutant has been constructed, and the sln1 HK kinase and skn7 RR mutants are either slightly more sensitive than or equal in sensitivity to CAF2-1 with both triazoles (data not shown). In C. albicans, Ssk1p activation of Hog1p is required for oxidant adaptation in vitro, optimum survival in human neutrophils, disease in a murine model of invasive candidiasis, and adherence to human esophageal tissue in vitro (8-10). The Chk1p HK of C. albicans regulates cell wall biosynthesis, and its absence results in avirulence and decreased adherence to mammalian cells (17). Since both mutants have similar phenotypes in their sensitivity to voriconazole, it is possible that they may reside in the same signal pathway. However, two lines of evidence argue against this idea. First, as stated above, the mutants differ in regard to their sensitivity to fluconazole, as strain SSK21 is over 30-fold more sensitive to fluconazole than strain CHK21. Second, as stated above, while both genes are required for virulence, CHK1 is required for cell wall biosynthesis while SSK1 is not; SSK1 regulates adaptation to a variety of oxidants and CHK1 is functionally less important in this regard (compared to control strains), suggesting that each participates in a dissimilar pathway. Nevertheless, genetic data to resolve the issue of common or different pathways for these proteins are needed. We also observed that in some assays gene-reconstituted strains were equal phenotypically to the parental strain. We attribute this observation to differences in assays. For example, CHK23 was equal to CAF2-1 in sensitivity to the drugs (Table 1) but was about intermediate in killing to CAF2-1 and CHK21 (null) in regard to the extent of its killing by voriconazole (Fig. 3).

In the current study, we have investigated the phenotypes of mutants in the two-component proteins Ssk1p and Chk1p when incubated with the triazoles fluconazole and voriconazole. Of the two RRs of C. albicans, Ssk1p and Skn7p, the skn7 mutant exhibits a sensitivity to both triazoles which is equal to the sensitivity of CAF2-1 and, as mentioned above, the sln1 HK mutant is only mildly sensitive to each drug (data not shown). We have also shown that the doubling times for each mutant and CAF2-1 in YPD medium are statistically similar, so the sensitivities of both mutants to triazoles compared to that of CAF2-1 are seen only in drug-treated cultures. Thus, deletion of either SSK1 or CHK1 results in a hypersensitivity to both drugs, and this phenotype is apparently associated with an increased net accumulation of at least fluconazole in mutant strains compared to those in wt and gene-reconstituted strains. There is a single report on triazole uptake by C. albicans that suggests transport occurs by facilitated diffusion (1). Also, in C. albicans, facilitated transport was associated with caspofungin uptake, but in each of these studies a transporter was not identified (1, 21). Of interest, compared to CAF2-1, both mutants are more readily killed by fluconazole and voriconazole, which are normally fungistatic towards this pathogen. An undesirable trait of triazole therapy in general is that resistance can develop, especially in the case of non-C. albicans species of Candida. We hypothesize, therefore, that compounds which target Ssk1p or Chk1p may be useful in cotherapy with triazoles to potentiate the activity of the latter compounds.


   ACKNOWLEDGMENTS
 
This study was supported by Public Health Service grants NIH-NIAID R01 AI47047 and NIH-NIAID AI43465 to R.C. N.C. is a recipient of an American Heart Association (AHA) National Scientist Development grant, 0635108N.

We thank John Bennett for providing [3H]fluconazole.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology & Immunology, Georgetown University Medical Center, 3900 Reservoir Rd. NW, Washington, DC 20057. Phone: (202) 687-1513. Fax: (202) 687-1800. E-mail: calderor{at}georgetown.edu Back

{triangledown} Published ahead of print on 30 July 2007. Back


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Antimicrobial Agents and Chemotherapy, October 2007, p. 3747-3751, Vol. 51, No. 10
0066-4804/07/$08.00+0     doi:10.1128/AAC.00929-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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