Histone Deacetylase Inhibitors Enhance Candida albicans Sensitivity ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2002, p. 3532–3539 0066-4804/02/$04.00⫹0 DOI: 10.1128/AAC.46.11.3532–3539.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 46, No. 11

Histone Deacetylase Inhibitors Enhance Candida albicans Sensitivity to Azoles and Related Antifungals: Correlation with Reduction in CDR and ERG Upregulation W. Lamar Smith and Thomas D. Edlind* Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129 Received 22 April 2002/Returned for modification 21 June 2002/Accepted 14 August 2002

Histone acetylation and deacetylation play important roles in eukaryotic gene regulation. Several histone deacetylase (HDA) inhibitors have been characterized, including trichostatin A (TSA), apicidin, and sodium butyrate. We tested their effects on Candida albicans in vitro growth, heat sensitivity, and germ tube formation; minimal effects were observed. However, there was a dramatic effect of TSA on C. albicans sensitivity to the azoles fluconazole, itraconazole, and miconazole. Similar effects were observed with other HDA inhibitors and with the antifungals terbinafine and fenpropimorph, which target, as do azoles, enzymes in the ergosterol biosynthetic pathway. In contrast, HDA inhibitors had minimal effect on the activities of amphotericin B, flucytosine, and echinocandin, which have unrelated targets. Specifically, addition of 3 ␮g of TSA/ml lowered the itraconazole MIC for five susceptible C. albicans isolates an average of 2.7-fold at 24 h, but this increased to >200-fold at 48 h. Thus, the primary effect of TSA was a reduction in azole trailing. TSA also enhanced itraconazole activity against Candida parapsilosis and Candida tropicalis but had no effect with four less related yeast species. To examine the molecular basis for these effects, we studied expression of ERG genes (encoding azole and terbinafine targets) and CDR/MDR1 genes (encoding multidrug transporters) in C. albicans cells treated with fluconazole or terbinafine with or without TSA. Both antifungals induced to various levels the expression of ERG1, ERG11, CDR1, and CDR2; addition of TSA reduced this upregulation 50 to 100%. This most likely explains the inhibition of azole and terbinafine trailing by TSA and, more generally, provides evidence that trailing is mediated by upregulation of target enzymes and multidrug transporters. Candida species are the most common opportunistic fungal pathogens, in particular Candida albicans, which is among the normal mucosal flora in many humans. They typically cause mucosal (oropharyngeal, vaginal, urinary tract) infections and less commonly skin, nail, and systemic infections. Predisposing factors for candidiasis include immunodeficiency, broad-spectrum antibacterial therapy, and endocrine abnormalities. Candidiasis is generally treated with antifungal azoles such as topical miconazole and oral or intravenous fluconazole and itraconazole. Azoles inhibit lanosterol demethylase, one of about 20 enzymes involved in the biosynthesis of the major fungal membrane component ergosterol (for reviews, see references 19 and 35). The major limitation of antifungal azoles is their lack of fungicidal activity, which may contribute to treatment failures common with the severely compromised. Furthermore, surviving yeasts provide a reservoir for the development of azole resistance. In vitro, it is apparent that azoles not only fail to kill but also fail to truly suppress growth, resulting in trailing growth in broth microdilution assays even at concentrations well above the MIC (21, 24, 25, 27). C. albicans also exhibits trailing with other sterol biosynthesis inhibitors (SBIs) such as the squalene epoxidase inhibitor terbinafine (29). We and others are exploring the molecular basis for SBI trailing (T. D. Edlind, W. L. Smith, K. W. Henry, S. K. Katiyar, and J. T.

Nickels, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 241, 2000; T. D. Edlind, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. J-1844, 2001; D. Sanglard, F. Ischer, O. Marchetti, and J. Bille, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. J-1845, 2001). A likely possibility is that SBI trailing derives at least in part from the ability of C. albicans to upregulate, in response to drug exposure, the transcription of genes encoding lanosterol demethylase (ERG11), squalene epoxidase (ERG1), or azole and terbinafine efflux transporters (CDR1, CDR2, or MDR1) (1, 5, 6, 9–11, 17, 18). However, direct evidence in support of this has not been reported. It has been recognized in recent years that histone acetylation and deacetylation play important roles in eukaryotic gene regulation (for reviews, see references 8 and 33). The ε-amino groups of lysine residues within the flexible amino-terminal tails of the core histones are the primary targets for acetylation. These modifications reduce the electrostatic interaction between histones and DNA and therefore typically activate transcription by increasing the exposure of a promoter region to RNA polymerase and associated factors. In the yeast Saccharomyces cerevisiae, histone acetyltransferase activity is found in two multisubunit complexes, SAGA and ADA. These complexes share subunits, several of which are highly conserved from yeast to humans. Histone deacetylases (HDAs) are similarly conserved. S. cerevisiae encodes at least six HDAs, including RPD3 and HDA1 (28, 40). HDA1 and HDA6 are examples of closely related human homologs (7). Important tools in the experimental study of histone acetylation and deacetylation are HDA

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen La., Philadelphia, PA 19129. Phone: (215) 991-8377. Fax: (215) 848-2271. E-mail: [email protected]. 3532

HDA INHIBITORS ENHANCE C. ALBICANS AZOLE SENSITIVITY

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inhibitors, which include trichostatin A (TSA), sodium butyrate, apicidin, and trapoxin (31, 41). These and related compounds were initially studied for their effects on mammalian cells, and in particular for their ability to reverse the transformed phenotype of many tumor cells (26). Their common effect on HDA activity, mediated by related structural elements that mimic the lysine side chain, was only subsequently appreciated. Comparable studies with fungi have been limited, although effects of TSA on S. cerevisiae HDAs (4) and global gene expression (3) have been reported. We tested the effects of HDA inhibitors on C. albicans in vitro growth, heat sensitivity, and germ tube formation; only minimal effects were observed. However, there was a dramatic effect of TSA and, to variable extent, of other HDA inhibitors on SBI activity against C. albicans, which correlated with TSA effects on SBI-dependent ERG and CDR upregulation. MATERIALS AND METHODS HDA inhibitors and antifungals. HDA inhibitors were obtained as follows: TSA (Cayman Chemical, Ann Arbor, Mich.), apicidin (Calbiochem, San Diego, Calif.), sodium butyrate (Sigma-Aldrich, St. Louis, Mo.), and trapoxin (generous gift of M. Yoshida and K. Sugita). TSA was provided as a 1-mg/ml solution in ethanol; all others were dissolved in dimethyl sulfoxide. Antifungal agents were obtained as follows: fenpropimorph (Crescent Chemical, Hauppauge, N.Y.), fluconazole (Pfizer, New York, N.Y.), itraconazole (Janssen, Titusville, N.J.), terbinafine (Novartis, East Hanover, N.J.), echinocandin L-774967 (Merck, Rahway, N.J.), miconazole, amphotericin B, and flucytosine (Sigma). Fluconazole and flucytosine were dissolved in saline or water; all others were dissolved in dimethyl sulfoxide. Strains and culture conditions. C. albicans strains were obtained from T. White (strains Ca2-76 and Ca12-99 [38]), J. Rex (strains CaLL, CaLH, and CaHH, corresponding to isolates 630-15.3, 707-15, and UTR-14, respectively [25]), the American Type Culture Collection (Manassas, Va.) (strains Ca66027 and Ca90028) or were recent oral isolates from healthy volunteers (strains CaTE2 and CaTE8). Candida tropicalis 750 and 66029, Candida parapsilosis 22019 and 90018, Candida glabrata 2001 and 66032, Candida krusei 6258 and 14243, and Cryptococcus neoformans 6352 and 28958 were all obtained from the ATCC. S. cerevisiae diploid strain BY4743 and derivatives with homozygous deletions of HDA genes were obtained from ResGen (Huntsville, Ala.). The medium employed was yeast extract-peptone-dextrose (YPD; 1% yeast extract, 2% peptone, and 2% dextrose, pH approximately 6.3) or, where indicated, RPMI (RPMI-1640 minus glutamine, with 2% dextrose and 0.165 M MOPS [morpholinepropanesulfonic acid], pH 7.0). Candida and C. neoformans strains were incubated at 35°C; S. cerevisiae strains were incubated at 30°C. Broth microdilution assays. Fresh overnight cultures were diluted 1:100 in YPD (or, where indicated, RPMI), incubated 4 h with aeration, and then counted in a hemocytometer and diluted again to 104 cells/ml. HDA inhibitor was added as indicated, and cells were aliquoted to wells of a flat-bottomed 96-well plate (100 ␮l per well, except for row A wells, which received 200 ␮l). Antifungal (or in initial experiments HDA inhibitor) was added to row A (final dimethyl sulfoxide vehicle concentration, ⬍0.5%) and twofold serially diluted to rows B through G (by transferring 100 ␮l); row H served as antifungal-free control. In some experiments, row A wells received 150 ␮l and serial threefold dilutions were done by transferring 50 ␮l. Plates were incubated (in bags, to minimize evaporation) at 35°C, except where indicated. Growth was measured by reading absorbance at 630 nm in a microplate reader; background due to the medium was subtracted from all samples. MIC was defined as the concentration inhibiting growth ⬎80% for assays using RPMI or ⬎60% for assays using YPD (in which growth rate and trailing were greater). Germ tube assay. Fresh overnight cultures of strains CaLL and Ca2-76 were diluted to 106 cells/ml in YPD plus 20% heat-inactivated fetal bovine serum and treated with the indicated concentrations of TSA; controls received an equivalent volume of ethanol vehicle (final concentration, 0.1 to 2.7%). Cultures were incubated at 37°C for 2 h, germ tubes and total cells were counted microscopically, and the results were expressed as percent germ tubes. Hybridization analysis of antifungal and TSA-treated cultures. A log-phase culture of strain CaLL in YPD medium (3 ⫻ 106 cells/ml) was divided into six 25-ml portions and treated with or without 3 ␮g of TSA/ml and with or without

FIG. 1. Broth microdilution assays of HDA inhibitor effects on growth of C. albicans strain CaLL. Incubation was for 24 h at 35°C (solid lines) or 42°C (dashed lines) in the presence of the indicated concentrations of TSA (triangles; in micrograms per milliliter), apicidin (circles; in micrograms per milliliter), or sodium butyrate (squares; in millimolar concentrations).

8 ␮g of fluconazole/ml or terbinafine/ml, as indicated. Cultures were incubated at 35°C with aeration. At 0, 1, 2, 4, and 8 h the control culture cells (without TSA and antifungal) were counted in a hemocytometer and volumes corresponding to 3 ⫻ 107 cells were removed to centrifuge tubes; equivalent volumes of the treated cultures were similarly removed. Cells were pelleted, resuspended in 50 mM sodium acetate–10 mM EDTA (pH 5.0) buffer, and frozen immediately at ⫺70°C. RNA was extracted and analyzed by slot blot hybridization as described previously (12). 32P-labeled probes were prepared from the indicated PCRamplified gene fragments as described previously (10).

RESULTS HDA inhibitors had minimal effects on C. albicans growth, heat sensitivity, and germ tube formation. Broth microdilution assays were used to test the effects of HDA inhibitors TSA, apicidin, and sodium butyrate on the growth of C. albicans strain CaLL under optimal conditions (YPD medium at 35°C) (Fig. 1). There were no effects of TSA or apicidin over the range of 1 to 32 ␮g/ml and only minor effects of sodium butyrate at high concentration (32 mM). In contrast, tumor cells are affected by these compounds at concentrations about 10-fold lower (14, 41). The assay above was repeated at 42°C to determine if HDAregulated genes played a role in growth under conditions of temperature stress (Fig. 1, dashed lines). Again, apicidin had no effect at concentrations up to 32 ␮g/ml, while sodium butyrate inhibition at a high concentration was slightly enhanced. TSA at its highest concentration reduced growth by 80%, but this effect was transient and full growth was observed by 48 h (not shown). Two strains were tested for effects of HDA inhibitor TSA on germ tube formation induced by 2 h of incubation at 37°C in 20% serum. Minimal effects were observed over the range of 1 to 9 ␮g of TSA/ml (Table 1). At the highest concentration tested (27 ␮g/ml), germ tube formation was reduced ⱖ3-fold,

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ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Effects of HDA inhibitor TSA on C. albicans germ tube formationa % of ethanol vehicle

TSA concn (␮g/ml)

0.1

0 1 0 3 0 9 0 27

0.3 0.9 2.7 a

% Germ tube formation in strain: CaLL

Ca2-76

59 62 61 57 54 30 7 13

50 59 52 54 51 50 10 18

Data represent the averages of two independent experiments.

but this was due to the 2.7% ethanol vehicle as demonstrated by parallel controls with vehicle alone. HDA inhibitors enhanced azole activity against C. albicans. As has been previously reported, C. albicans strains exhibit variable degrees of trailing growth at azole concentrations well above the MIC (21, 24, 25, 27). The results of broth microdilution assays of a strain (CaLL) exhibiting typical azole trailing

are shown in Fig. 2A and B (YPD and RPMI media, respectively). After 22 h of incubation, trailing growth (beginning above the MICs at 1 or 0.25 ␮g/ml in YPD and RPMI, respectively) was about 12% of the fluconazole-free control growth. By 48 h, this increased to 65% in YPD and 35% in RPMI. Consequently, the 48-h MICs (defined as the concentration inhibiting growth ⬎80% in RPMI or ⬎60% in YPD) increased to ⬎8 ␮g/ml. The addition of HDA inhibitor TSA (3 ␮g/ml) had no effect on growth by itself (as noted above) and had only a minor effect on fluconazole activity in terms of MIC or 50% inhibitory concentration (Fig. 2A and B); specifically, the MIC decreased twofold at the 22 h time point in YPD medium but was otherwise unchanged. In contrast, TSA had a dramatic effect on fluconazole trailing at 48 h, reducing CaLL growth to undetectable levels above 1 or 0.5 ␮g of fluconazole/ml in YPD or RPMI, respectively. Similar experiments demonstrated comparable effects with 2 ␮g of TSA/ml, but 0.5 ␮g of TSA/ml had little effect (not shown). YPD was employed in further studies (except where noted), since trailing and effects of HDA inhibitors were somewhat more pronounced in this medium. More-extensive studies were done with itraconazole, which,

FIG. 2. Broth microdilution assays of HDA inhibitor effects on fluconazole activity against strain CaLL. (A) YPD medium with (circles) or without (triangles) 3 ␮g of TSA/ml, incubated for 22 h (open symbols) or 48 h (closed symbols). (B) Same as for panel A, except that RPMI medium was used. (C) YPD medium with (circles) or without (triangles) 16 ␮g of apicidin/ml, incubated for 18 h (open symbols) or 42 h (closed symbols). (D) Same as for panel C, except with 4 mM sodium butyrate in place of apicidin.


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TABLE 2. Effects of HDA inhibitor TSA (3 ␮g/ml) on itraconazole trailinga Avg MICb (␮g/ml) at: C. albicans strain

CaLL Ca66027 Ca2-76 CaTE2 CaLH Ca90028 CaTE8 CaHH Ca12-99

24 h

Trailing

48 h

Itra

Itra plus TSA

Itra

Itra plus TSA

0.010 0.005 0.009 0.009 0.031 0.009 0.005 0.11 ⬎3

0.003 0.005 0.003 0.003 0.005 0.005 0.005 0.11 0.17

⬎3 ⬎3 ⬎3 ⬎3 ⬎3 0.020 0.013 0.27 ⬎3

0.013 0.009 0.009 0.005 0.058 0.013 0.009 0.17 ⬎3

Without TSA

With TSA

⬎300 ⬎500 ⬎300 ⬎300 ⬎100 2 3 2 —c

4 2 3 2 12 3 2 2 ⬎18

a

Trailing is the ratio of the 48-h MIC to the 24-h MIC. Itra, itraconazole. MIC values were determined in two independent experiments using serial twofold or threefold dilutions. c —, cannot be determined. b

like fluconazole, is an azole administered both orally and parenterally. Nine C. albicans strains were tested with itraconazole with or without TSA (3 ␮g/ml). Seven strains were susceptible, and five of these demonstrated clear itraconazole trailing, i.e., the ratio of 48-h MICs to 24-h MICs was ⬎100-fold (Table 2). For four of these five, TSA reduced itraconazole trailing dramatically so that the ratio was only 2- to 4-fold; for the hightrailing strain CaLH it was reduced to 12-fold. TSA had relatively little effect on two strains that had intrinsically low itraconazole trailing (Ca90028 and CaTE8) and on fluconazole-resistant strain CaHH. With fluconazole-resistant strain Ca12-99, TSA effects were apparent at 24 h only. This strain is known to have an ERG11 mutation that confers fluconazole resistance (38), which could explain the minimal effect of TSA. Similar effects on azole trailing of strain CaLL were observed with other HDA inhibitors. Apicidin at 16 ␮g/ml was not inhibitory by itself and lowered the fluconazole MIC 2-fold at 18 h, but this increased to a ⬎16-fold reduction at 42 h (Fig. 2C). Comparable effects were observed with 32 ␮g of apicidin/ ml, while a partial effect on trailing was observed with 8 ␮g of apicidin/ml (not shown). Sodium butyrate at 4 mM actually increased the fluconazole MIC twofold at 18 h, but by 42 h there was a ⬎4-fold decrease in MIC (Fig. 2D). Comparable effects were observed with 8 mM sodium butyrate, but at 2 mM it was inactive (not shown). Trapoxin was not available in sufficient quantities for full analysis, but preliminary studies suggested that it too blocked fluconazole trailing. Since pH has been shown to affect trailing (21), the effect of addition of TSA (3 ␮g/ml), apicidin (16 ␮g/ml), or sodium butyrate (4 mM) on the pH of the medium was tested: there was no change. HDA inhibitor effects were limited to antifungals that inhibit sterol biosynthesis. TSA was tested for its effects on the activity of other widely used antifungals. The topical imidazole miconazole has the same lanosterol demethylase target as the triazoles fluconazole and itraconazole, and as expected, HDA inhibitors had the same effect, reducing miconazole trailing in strain CaLL (not shown). Terbinafine and related allylamines also inhibit ergosterol biosynthesis, except that their specific target is squalene epoxidase rather than lanosterol demethylase. C. albicans is susceptible to terbinafine but exhibits high

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trailing, as reflected in the shallow dose-response curves (Fig. 3A). Addition of TSA (3 ␮g/ml) clearly reduced both MIC and trailing, although distinguishing these is more difficult with terbinafine than with azoles. Similar results were obtained with the morpholine fenpropimorph (not shown), an agricultural fungicide whose enzyme targets in the ergosterol biosynthetic pathway follow those of allylamines and azoles (19). In contrast, amphotericin B, echinocandin L-774967, and flucytosine generated steep dose-response curves (Fig. 3B, C, and D). The MIC increased two- to fourfold with longer incubation (18 versus 66 h), but trailing was not apparent. TSA (3 ␮g/ml) had no effect on the activities of these three antifungals, all of which have mechanisms of action unrelated to ergosterol biosynthesis. Specifically, amphotericin B disrupts membranes by binding to ergosterol, echinocandins inhibit glucan synthase and hence cell wall synthesis, and flucytosine disrupts RNA synthesis by acting as a nucleotide analog. Apicidin (16 and 32 ␮g/ml) and sodium butyrate (4 and 8 mM) were similarly tested for effects on terbinafine and amphotericin B activity. Both HDA inhibitors at both concentrations tested had modest (two- to fourfold) effects on terbinafine MIC and trailing but had no effect on amphotericin B activity (data not shown). TSA also enhanced azole activity against C. tropicalis and C. parapsilosis but not against four additional yeast species. Two isolates each of six additional yeast species were tested for effects of TSA on itraconazole activity. TSA (3 ␮g/ml) enhanced the activity of itraconazole against both isolates of C. parapsilosis and C. tropicalis; representative results for each are shown (Fig. 4). C. parapsilosis demonstrated relatively little azole trailing, and the effects of TSA were primarily on the MIC; the converse was observed for C. tropicalis. Minimal or no effects of TSA on itraconazole activity were observed with the C. glabrata, C. krusei, Cryptococcus neoformans, and S. cerevisiae strains tested (data not shown). Since the lack of effect of TSA could have a trivial basis (e.g., poor uptake or rapid efflux), we examined antifungal sensitivities of S. cerevisiae strains in which each of the HDA gene homologs had been specifically deleted (3, 40). There were only very minor differences in fluconazole, itraconazole, miconazole, and terbinafine sensitivities of the parent BY4743 and its hda1, rpd3, hos1, hos2, and hos3 homozygous deletion derivatives (data not shown), consistent with the lack of effect of TSA. TSA reduced azole- and terbinafine-dependent upregulation of CDR and ERG genes. To examine the molecular basis for the HDA inhibitor effects, we studied expression of ERG genes encoding azole and terbinafine target enzymes and CDR/ MDR1 genes encoding multidrug transporters in C. albicans cells treated with fluconazole or terbinafine with or without TSA. Log-phase cultures of strain CaLL were treated for 1 to 8 h with fluconazole (8 ␮g/ml), terbinafine (8 ␮g/ml), TSA (3 ␮g/ml), or specific combinations of these agents. RNA was purified and analyzed by slot blot hybridization with specific gene probes (Fig. 5). Consistent with the broth microdilution results (above), combinations of fluconazole or terbinafine with TSA had the most apparent effects on C. albicans growth, as revealed by the reduction at 8 h in ACT1 RNA (equal culture volumes were analyzed at each time point). Consistent with a previous report (10), treatment with flu-

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FIG. 3. TSA effects on the activities of the indicated nonazole antifungals against strain CaLL. YPD was used in all assays except for the flucytosine assay (D), which used RPMI because flucytosine was inactive in YPD. TSA (3 ␮g/ml) was present (circles) or absent (triangles); incubation was for 18 h (open symbols) or 66 h (closed symbols).

conazole or terbinafine alone resulted in coordinate transcriptional upregulation of ERG1 and ERG11 (Fig. 5). This was apparent as early as 1 h but was more pronounced by 2 to 4 h. Fluconazole treatment also resulted in a gradual upregulation of CDR1, and after 8 h CDR2 expression was just detectable. Terbinafine treatment, in contrast, strongly and rapidly up-

regulated both CDR1 and CDR2; indeed, their levels began to decline by 2 h. Neither antifungal detectably upregulated MDR1. TSA alone had no detectable effects on expression of any of the RNAs examined (Fig. 5). However, TSA addition almost completely blocked the fluconazole- or terbinafine-dependent

FIG. 4. TSA effects on itraconazole activity against C. parapsilosis strain 22019 (A) and C. tropicalis strain 750 (B). TSA (3 ␮g/ml) was present (circles) or absent (triangles); incubation was for 24 h (open symbols) or 48 h (closed symbols).


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FIG. 5. Hybridization analysis of ERG and CDR/MDR expression in the presence or absence of fluconazole, terbinafine, and TSA. RNA was prepared from equivalent CaLL cultures treated for the indicated times (1 to 8 h) with vehicle only (C), 8 ␮g of fluconazole/ml (Flu), fluconazole plus 3 ␮g of TSA/ml (Flu/Ts), TSA alone (Ts), 8 ␮g of terbinafine/ml (Ter), or terbinafine plus TSA (Ter/Ts). Replicate slot blots were prepared and hybridized to the indicated 32P-labeled gene probes as described in Materials and Methods.

upregulation of ERG1 and ERG11. It also blocked the fluconazole-dependent upregulation of CDR1 (and the barely detectable upregulation of CDR2). With respect to the potent terbinafine-dependent upregulation of CDR1 and CDR2, TSA was partially effective, reducing RNA levels approximately 50%. Finally, the combination of fluconazole and TSA actually upregulated MDR1 to a modest extent. DISCUSSION The nearly complete C. albicans genome sequence (http: //www-sequence.stanford.edu/group/candida) encodes three proteins with ⬎50% identity over much of their lengths to TSA-sensitive human and S. cerevisiae HDAs (data not shown). It was therefore anticipated that TSA and other HDA inhibitors would affect C. albicans gene expression in some manner. Nonetheless, the three inhibitors tested had no effect on C. albicans growth under optimal conditions and relatively minor effects at elevated temperature; also, TSA had no effect on serum-induced germ tube formation. However, HDA inhibitors had clear effects on the C. albicans trailing growth commonly observed with azoles, the allylamine terbinafine, and the morpholine fenpropimorph. These SBIs are structurally distinct and have different enzyme targets but share the property of inhibiting sterol biosynthesis. TSA also enhanced azole activity against C. tropicalis and C. parapsilosis (reducing trailing, MIC, or both) but not against four other yeast species tested; this result correlates with the evolutionary relatedness of these species (2). Reduction in HDA activity by TSA treatment or gene deletion was recently shown to also affect colonytype switching in C. albicans strain WO-1 (15, 32), although the relationship (if any exists) between this finding and SBI trailing is not clear. Genetic studies have demonstrated that many of the 20 or so enzymes involved in sterol biosynthesis are essential for growth (for a review, see reference 19). Nevertheless, none of the SBIs that target these essential enzymes are fungicidal, except at concentrations greatly above their MICs. Moreover, these antifungals are not truly fungistatic since growth continues at slower rates at concentrations well above the MIC (21, 24, 25,

27). It is therefore apparent that yeast cells have mechanisms that allow them to respond and adapt to the inhibition of sterol biosynthesis (or perhaps more generally the inhibition of membrane biosynthesis). Two potential mechanisms for this adaptation would be SBIinduced transcriptional upregulation of ERG genes or of multidrug transporter genes such as CDR1. Constitutive upregulation of one or more of these genes in many azole-resistant C. albicans isolates has been reported (for an example, see reference 22; for reviews, see references 20, 37, and 39). ERG or multidrug transporter gene overexpression from multicopy plasmids in S. cerevisiae can also result in azole or terbinafine resistance (16, 23, 30; T. Edlind, unpublished data). Therefore, a similar effect on SBI activity against susceptible C. albicans isolates should result from the SBI-induced ERG or CDR upregulation that has been described previously (5, 10, 11, 18). It was unclear, however, whether this ERG/CDR upregulation was responsible for trailing growth, a shift in MIC, or both. This was unclear because no mutants specifically defective in SBI-induced ERG/CDR upregulation have been characterized. In lieu of mutants, specific inhibitors can provide useful information. HDA inhibitor TSA on a molecular level reduced azole- or terbinafine-induced ERG and CDR upregulation and on a cellular level reduced azole or terbinafine trailing with generally minor effects on initial (18- to 24-h) MICs. In retrospect this may be the only logical result, because at SBI concentrations below the MIC the cellular signal that induces ERG and CDR upregulation is presumably lacking. A potential concern is that our studies of ERG and CDR expression were limited to 8 h and effects were most apparent at 1 to 4 h, while trailing was most apparent only after 24 h of incubation. If expression were to be examined at later time points, then cell density would become a major variable, since ERG expression decreases while CDR1 expression increases in late-log and stationary phases (10). Conversely, differences in cell growth are not significant after 1 to 4 h of incubation. Future studies will examine these early time points for cellular markers of trailing, such as changes in sterol composition. With S. cerevisiae, the lack of effect of TSA was consistent with a similar lack of effect of HDA gene deletions on SBI

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activity in that organism. Surprisingly, however, strains with disruptions of S. cerevisiae HAT complex genes (e.g., ada3 and spt7) have markedly enhanced azole sensitivity (16; our unpublished data). Thus, inhibition of histone acetylation in S. cerevisiae appears to have an effect on SBI sensitivity comparable to the inhibition of histone deacetylation in C. albicans, C. tropicalis, and C. parapsilosis. The basis for this intriguing difference remains to be elucidated. It was also somewhat surprising that TSA inhibited the azole- or terbinafine-induced upregulation of both CDR and ERG genes, since there are no previous reports describing a common regulatory mechanism for these two classes of genes in either C. albicans or S. cerevisiae. It is not likely that TSA and related HDA inhibitors are acting directly on CDR and ERG gene promoters, since decreased deacetylation (i.e., increased acetylation) should enhance transcriptional upregulation, which is the opposite of what was observed (but which was in fact observed to a modest degree with MDR1 and fluconazole treatment). Thus, we speculate that SBI treatment is associated with histone deacetylation of the promoter region of a transcriptional repressor (or repressors); the resulting downregulation of this repressor leads to CDR and ERG upregulation. According to this model, HDA inhibition by TSA and related compounds would result in constitutive expression of this repressor, blocking CDR/ERG upregulation. There is ample precedent for transcriptional repressor involvement in both CDR and ERG expression. In S. cerevisiae, expression of the CDR homolog and azole transporter PDR5 involves the transcriptional activators encoded by PDR1 and PDR3, and a C. albicans homolog of these genes, FCR1, complements the fluconazole hypersensitivity of a pdr1 pdr3 mutant and upregulates PDR5 expression (34). In C. albicans, however, FCR1 acts as a repressor of multidrug resistance: an fcr1 mutant was resistant to azoles and several unrelated drugs known to be CDR substrates (34). Less is known about ERG regulation in C. albicans, but in S. cerevisiae repressor sequences have been identified within ERG promoters (13, 36), and deletion of the repressor gene ROX1 has the predicted effect of decreasing SBI sensitivity (K. W. Henry, J. T. Nickels, and T. D. Edlind, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 239, 2000). Future studies will directly examine these hypothetical roles of transcriptional repressors and histone acetylation and deacetylation in antifungal trailing. Is there a potential clinical use for combinations of HDA inhibitor and SBIs such as azoles? Based on the in vitro data presented here, these combinations could provide a more rapid cure and also reduce the incidence of azole-resistant mutants which arise from the reservoir of surviving (and presumably slowly growing) cells during long-term treatment. Certainly, issues of toxicity would need to be addressed, as illustrated by the fact that several HDA inhibitors are currently in development as antitumor agents (26). A more general issue, however, is the relevance of SBI trailing to treatment efficacy. Using an immunocompetent mouse model of systemic infection and comparing two isolates with low (i.e., normal) and high trailing, Rex et al. (25) did not detect significant differences in fluconazole responses. Ideally these studies could be extended to include isogenic pairs of trailing and nontrailing isolates and to immunocompromised models of systemic and


mucosal infection. In a study of human immunodeficiency virus-positive patients with recurrent oropharyngeal candidiasis (3 to 12 episodes over 3- to 15-month periods), Revankar et al. (24) detected trailing isolates mixed with nontrailers; the patients in all cases responded to 7-day courses of fluconazole. Thus, trailing isolates, unlike resistant isolates, are not associated with treatment failure but could be associated with recurrent infection, i.e., by surviving treatment and seeding the next infection. If this proves to be the case, then trailing-blocking agents such as HDA inhibitors would indeed be beneficial additions to SBI therapy for immunocompromised patients. ACKNOWLEDGMENTS We thank K. Henry, S. Katiyar, and J. Nickels for useful comments, T. White and J. Rex for strains, and H. Profous-Juchelka for echinocandin L-774967. This work was supported by Public Health Service grant AI46768. REFERENCES 1. Bammert, G. F., and J. M. Fostel. 2000. Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol. Antimicrob. Agents Chemother. 44:1255–1265. 2. Barns, S. M., D. J. Lane, M. L. Sogin, C. Bibeau, and W. G. Weisburg. 1991. Evolutionary relationships among pathogenic Candida species and relatives. J. Bacteriol. 173:2250–2255. 3. Bernstein, B. E., J. K. Tong, and S. L. Schreiber. 2000. Genomewide studies of histone deacetylase function in yeast. Proc. Natl. Acad. Sci. USA 97: 13708–13713. 4. Carmen, A. A., S. E. Rundlett, and M. Grunstein. 1996. HDA1 and HDA3 are components of a yeast histone deacetylase (HDA) complex. J. Biol. Chem. 271:15837–15844. 5. De Backer, M., T. Ilyina, X.-J. Ma, S. Vandoninck, W. Luyten, and J. Vanden Bossche. 2001. Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray. Antimicrob. Agents Chemother. 45:1660–1670. 6. Dimster-Denk, D., J. Rine, J. Phillips, S. Scherer, P. Cundiff, K. DeBord, D. Gilliland, S. Hickman, A. Jarvis, L. Tong, and M. Ashby. 1999. Comprehensive evaluation of isoprenoid biosynthesis regulation in Saccharomyces cerevisiae utilizing the genome reporter matrix. J. Lipid Res. 40:850–860. 7. Gray, S. G., and T. J. Ekstrom. 2001. The human histone deacetylase family. Exp. Cell Res. 262:75–83. 8. Grunstein, M. 1997. Histone acetylation in chromatin structure and transcription. Nature 389:349–352. 9. Henry, K. W., M. C. Cruz, S. K. Katiyar, and T. D. Edlind. 1999. Antagonism of azole activity against Candida albicans following induction of multidrug resistance genes by selected antimicrobial agents. Antimicrob. Agents Chemother. 43:1968–1974. 10. Henry, K. W., J. T. Nickels, and T. D. Edlind. 2000. Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis inhibitors. Antimicrob. Agents Chemother. 44:2693–2700. 11. Hernaez, M. L., C. Gil, J. Pla, and C. Nombela. 1998. Induced expression of the Candida albicans multidrug resistance gene CDR1 in response to fluconazole and other antifungals. Yeast 14:517–526. 12. Katiyar, S. K., and T. D. Edlind. 2001. Identification and expression of multidrug resistance-related ABC transporter genes in Candida krusei. Med. Mycol. 39:109–116. 13. Kennedy, M. A., and M. Bard. 2001. Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae. Biochim. Biophys. Acta 1517:177–189. 14. Kim, M. S., M. W. Son, W. B. Kim, Y. In Park, and A. Moon. 2000. Apicidin, an inhibitor of histone deacetylase, prevents H-ras-induced invasive phenotype. Cancer Lett. 157:23–30. 15. Klar, A. J. S., T. Srikantha, and D. R. Soll. 2001. A histone deacetylation inhibitor and mutant promote colony-type switching of the human pathogen Candida albicans. Genetics 158:919–924. 16. Kontoyiannis, D. P. 1999. Genetic analysis of azole resistance by transposon mutagenesis in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 43:2731–2735. 17. Kontoyiannis, D. P., and G. S. May. 2001. Identification of azole-responsive genes by microarray technology: why are we missing the efflux transporter genes? Antimicrob. Agents Chemother. 45:3674–3676. 18. Krishnamurthy, S., V. Gupta, R. Prasad, S. L. Panwar, and R. Prasad. 1998. Expression of CDR1, a multidrug resistance gene of Candida albicans: transcriptional activation by heat shock, drugs, and human steroid hormones. FEMS Microbiol. Lett. 160:191–197.

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