ABSTRACT
Although the echinocandin caspofungin primarily inhibits the synthesis of cell wall 1,3-β-d-glucan, its fungicidal activity could also potentially perturb the expression of virulence factors involved in the ability of Candida albicans to cause infection. Expression of the C. albicans secretory aspartyl proteinase (SAP) and phospholipase B (PLB) virulence genes was determined by reverse transcription-PCR after the addition of caspofungin to cells grown for 15 h in Sabouraud dextrose broth. In cells that remained viable, expression of SAP1 to SAP3, SAP7 to SAP9, and PLB1 was unaltered after exposure to fungicidal concentrations (4 to 16 μg/ml) of caspofungin over a period of 7 h. However, expression of SAP5 increased steadily beginning 1 h after exposure to caspofungin. These results indicate that caspofungin is rapidly fungicidal against C. albicans, before any suppression of SAP or PLB1 gene expression can occur.
The cyclic lipopeptide pneumocandins and echinocandins and the glycolipid papulacandins are members of a new class of antifungal agents which exert their activity by noncompetitive inhibition of fungal 1,3-β-d-glucan synthase (5, 17, 22, 30, 37). This enzyme is essential for the synthesis of cell wall glucan which provides structural integrity and osmotic stability for fungi but is not found in cells from higher eukaryotes including humans. Disruption of cell wall structure by inhibition of glucan synthesis results in osmotic instability and lysis of the fungal cell (12, 37). Caspofungin (MK-0991) is a water-soluble semisynthetic amine derivative of the natural product pneumocandin Bo (17, 26, 37), which in turn is a fermentation product derived from the fungus Glarea lozoyensis (9). Caspofungin was developed as a potential antifungal and anti-Pneumocystis agent (17, 26, 37). In vitro, caspofungin is fungicidal against Candida species, including azole-resistant species, and is fungistatic against Aspergillus species (6, 8, 15, 28, 33, 39, 45). However, it is inactive against Fusarium, Rhizopus, Trichosporon, and Cryptococcus neoformans (8, 15, 28, 39). In addition to prolonging survival in mouse models of disseminated candidiasis and aspergillosis (1, 2, 20, 21), caspofungin has recently shown promising clinical activity for the treatment of life-threatening infections caused by Candida and Aspergillus species (46; J. Maertens, I. Raad, C. A. Sable, A. Ngai, R. Berman, T. F. Patterson, D. Denning, and T. Walsh, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1103, 2000; A. Villanueva, E. Gotuzzo, E. Arathoon, L. M. Noriega, N. Kartsonis, R. Lupinacci, J. Smietana, R. S. Berman, M. J. Dinubile, and C. A. Sable, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. J-675, 2001).
Because caspofungin is fungicidal, it could potentially affect a diversity of cellular processes which contribute to the ability of Candida albicans to cause infection. The primary mechanism of action of pneumocandins and echinocandins is inhibition of synthesis of cell wall 1,3-β-d-glucan, and these compounds do not inhibit other C. albicans membrane-bound enzymes, such as chitin synthase or serine palmitoyltransferase (29). However, echinocandin antimycotics inhibit the incorporation of glucan-associated enolase into the growing cell wall of C. albicans (3). In addition, although the synthesis of C. albicans RNA is only modestly inhibited at the MIC of the pneumocandin L-733,560, a close analogue of caspofungin, marked inhibition occurs at 250 times the MIC (29).
Fungal virulence factors are attracting attention as potential targets for drug development (18, 38). C. albicans secretory aspartyl proteinases (Saps), under the control of a multigene family (SAP1 to SAP9) expressing distinct isoenzymes which are regulated differentially at the mRNA level in vitro (24, 34) and in vivo (35, 41), are implicated in the breakdown of several host substrates (23). There is evidence that phospholipase B expressed by at least two genes (PLB1 and PLB2) (32, 44) also contributes to the pathogenesis of candidiasis by the degradation of host tissues (19, 25). The potential impact of antifungal therapies on expression of C. albicans virulence factors is exemplified by the enhanced production of Sap by azole-resistant C. albicans isolates from a patient infected with human immunodeficiency virus after growth in subinhibitory concentrations of fluconazole (49). In the present study, we sought to determine whether caspofungin could potentially exert added anticandidal activity by inhibiting the expression of C. albicans SAPs and PLB1, as a consequence of nonspecific effects on transcription.
Candida strains.
Sap- and phospholipase-producing (11, 13, 41) C. albicans LAM-1 (serotype A) was originally isolated from the blood of a patient with systemic candidiasis (31). The isolate was stored at −80°C in a solution containing 65% (vol/vol) glycerol, 10 mM Tris (pH 7.5), and 10 mM MgCl2. Prior to use, Candida was cultured at 37°C on Sabouraud dextrose agar (SDA) (Becton Dickinson and Company, Cockeysville, Md.).
Antifungal susceptibility testing.
The susceptibility of C. albicans strain LAM-1 to antifungal agents was determined by the broth microdilution version (16) of the M27-A method defined by the National Committee for Clinical Laboratory Standards (36). The method was performed in RPMI 1640 medium (Gibco BRL, Gaithersburg, Md.) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (MOPS-buffered RPMI; ICN Biochemicals, Cleveland, Ohio) with readings at 48 h, except for amphotericin B, which was tested in antibiotic medium 3 and read after 24 h (47). The MICs of the azoles and flucytosine were defined as the lowest concentrations giving prominent growth reduction. For the other drugs, the MIC was the lowest drug concentration giving an optically clear well. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 (40) were included as quality control isolates (7). After 48 h, 20 μl of each clear well was plated on SDA, and the minimum fungicidal concentration (MFC) was the lowest concentration giving no growth (>98% killing).
Time-kill curve procedures.
Caspofungin (MK-0991) was provided as a pure white powder by the Merck Research Laboratories (Rahway, N.J.) and stored at −80°C. A stock solution at a concentration of 400 μg/ml was prepared in sterile distilled water for each experiment.
Three to five colonies of C. albicans grown for 24 to 48 h on SDA were suspended in 5 ml of 0.01 M phosphate-buffered saline (pH 7.2), and the cells were counted using a hemacytometer. An appropriate volume of fungal suspension was then added to Erlenmeyer flasks containing 70 ml of either Sabouraud dextrose broth (SDB; Difco Laboratories, Detroit, Mich.) or MOPS-buffered RPMI to yield a starting inoculum of approximately 103 CFU/ml. Caspofungin was added to individual cultures with resulting concentrations ranging from 0.125 to 16 μg/ml, either at the start of culture or after 15 h of incubation with rotary agitation at 37°C. Culture medium without drug served as a growth control. At predetermined time points, duplicate 100-μl volumes were withdrawn from each culture and serially diluted (10-fold), and a 100-μl aliquot from each dilution was plated on SDA for determination of CFU. The results were expressed as the averages of the duplicate CFU determinations. When colony counts were anticipated to be less than 103 CFU/ml, duplicate 0.1-ml aliquots of the cultures were plated directly on SDA without dilution. A 1-ml aliquot of each culture was also removed at the same time points and centrifuged, and the pellet was immediately frozen at −80°C for analysis of C. albicans SAP and PLB1 gene expression.
RT-PCR.
Isolation of RNA from the pelleted cells and reverse transcription-PCR (RT-PCR) were performed as previously described (41). In order to assess gene expression by the portion of C. albicans which remained viable during the time-kill experiments, RT-PCR for each gene and time point was conducted using an amount of RNA originating from 1,400 CFU of C. albicans. For the detection of gene expression, individual digoxigenin-labeled probes were used to specifically detect each of the RT-PCR products by Southern blotting (41). Positive controls for expression of each of the individual SAP and PLB1 genes were included in the RT-PCR assay, using RNA from C. albicans LAM-1 grown in vitro under conditions known to induce their expression. The EFB1 gene was amplified as a positive mRNA control and to detect any contaminating genomic DNA by the presence of a 365-bp intron (41). A negative control for expression of each gene was obtained by omitting the RNA in the RT-PCR assay (41).
The MICs for C.albicans LAM-1 were typical of those for other C.albicans isolates (flucytosine, 0.125 μg/ml; amphotericin B, 1 μg/ml; fluconazole, 0.25 μg/ml; itraconazole, 0.125 μg/ml; posaconazole, 0.031 μg/ml; voriconazole, 0.031 μg/ml; caspofungin, 1 μg/ml; micafungin [FK-463], 0.031 μg/ml; and anidulafungin [LY-303366], 0.031 μg/ml). This strain was thus susceptible to all classes of antifungal agents tested, including the echinocandins. The MFC for caspofungin (1 μg/ml) was equivalent to the MIC, demonstrating fungicidal activity against C. albicans and in agreement with reported in vitro susceptibility data of C. albicans to this antifungal (6, 8, 15, 28, 33, 45).
When caspofungin was added at concentrations (0.125 to 1 μg/ml) at or below the MIC to late-log-phase C. albicans grown in SDB for 15 h (Fig. 1A), the apparent rate of reduction of the number of CFU per milliliter was unexpectedly inversely proportional to the drug concentration. This effect was correlated with increasing macroscopic and microscopic clumping of C. albicans cells as the concentration of caspofungin decreased. Interestingly, aggregates of C. albicans blastoconidia have been previously observed in an enriched synthetic liquid medium after 4 h of treatment with caspofungin, L-733,560, as well as acidic terpenoid (1, 3)-β-d-glucan synthase inhibitors, at concentrations at or below the MIC or MFC (37; M. Kurtz, personal communication). In another report, however, caspofungin concentrations below that required to inhibit 80% of the strains resulted in growth curves similar to that for the control in the absence of caspofungin for all C. albicans isolates tested and no report of aggregation (14).
Plots of the number of C. albicans treated with different concentrations of caspofungin over time. The mean values for log10 of the number of CFU per milliliter versus time for C. albicans strain LAM-1 tested against different concentrations of caspofungin. The following concentrations (in micrograms per milliliter) of caspofungin were used: 0 (control) (□), 0.0625 (▾), 0.125 (♦), 0.25 (•), 1 (▴), 4 (○), 8 (▵), and 16 (▪). Caspofungin was added to cultures in SDB after 15 h of incubation (A) or at the start of culture (B). Representative results are shown from four (A) and two (B) independent experiments.
The apparent enhanced killing of C. albicans at caspofungin concentrations below the MIC may have resulted from the formation of increasingly large aggregates, each giving rise to a single colony and consequently being counted as 1 CFU. Aggregation was not prevented by adding 0.01% (wt/vol) Tween 20 or 0.0025% Tween 80 (Fisher Scientific, Fair Lawn, N.J.) to the SDB culture medium (data not shown). In addition, time-kill curves constructed by a proposed standardized method (27) in RPM1 1640 buffered with MOPS also resulted in aggregation at values at or below the MIC and in a rate of killing comparable to that in SDB at concentrations (4 to 16 μg/ml) above the MIC or MFC (data not shown). As expected, these higher concentrations of caspofungin (4 to 16 μg/ml) all produced approximately 90% killing of C. albicans within 7 h in SDB medium (Fig. 1A) (8, 14) with no visible aggregation but abundant cellular debris microscopically (14). The absence of additional killing effect against C. albicans at increasing concentrations of caspofungin (4 to 16 μg/ml) has been observed by other investigators (14). Taken together, the results of the time-kill studies suggest that inhibition of synthesis of cell wall glucan by caspofungin primarily alters the surface properties of C. albicans at concentrations below the MIC, causing the cells to aggregate, while killing at concentrations above the MIC or MFC disrupts the cells and aggregation is thus not observed. Interestingly, caspofungin at concentrations below the MIC induces a high-affinity fibronectin receptor that may promote the adhesive properties of C. albicans (M. L. Pendrak, T. J. Walsh, and D. D. Roberts, Prog. Abstr. 6th ASM Conference on Candida and Candidiasis, abstr. 124, 2002).
Time-kill curves were also constructed by adding caspofungin to a smaller inoculum of C. albicans (103 CFU) at the start of culture in SDB and quantitating CFU at determined time points over 20 h of incubation. The apparent reduction in CFU was again inversely proportional to the concentration of caspofungin because of aggregation, but at least 99% killing was achieved within 9 h at higher concentrations (Fig. 1B). However, a persistent fraction of C. albicans was not killed, and growth of the fungus quickly resumed until the end of incubation. It is hypothesized that this rebound in CFU may be the result of decreased drug stability under the extended study conditions (14). Monitoring of the pH at each time point revealed pH values of 5 to 5.5, making it unlikely that caspofungin was inactivated by deviating from the recommended pH range of 5 to 7 (Merck Research Laboratories).
Expression of the SAP1 to SAP9 and PLB1 genes of C. albicans was determined by RT-PCR after the addition of caspofungin (4 to 16 μg/ml) to cells grown for 15 h in SDB. In comparison to a control culture without caspofungin, expression of SAP1 to SAP3, SAP7 to SAP9, PLB1, and the EFB1 positive-control gene was unaltered at these drug concentrations over a period of 7 h (Fig. 2). Interestingly, however, expression of SAP5 increased steadily beginning 1 h after exposure to these concentrations of caspofungin (Fig. 2). Although SAP5 is expressed in vitro by the hyphal form of C. albicans at neutral pH (24, 48), expression has been found using in vivo expression technology in a murine model of esophageal candidiasis when only blastoconidia are observed (42). Although regulation of expression of the SAP genes has not been completely elucidated at the molecular level, it could be hypothesized that induction of expression of SAP5 may have resulted from signaling events occurring in response to cell wall damage by caspofungin. The C. albicans transcriptional regulators CPH1 and EFG1 mediate the activation of the SAP5 gene (43) and may have been up-regulated after exposure to caspofungin. Alternatively, peptide hydrolysis products of the cyclic hexapeptide caspofungin (4) may have induced expression of SAP5, because peptides up-regulate expression of the SAP genes in vitro (24). Because the RT-PCR analysis was conducted on the portion of C. albicans cells exposed to but not yet killed by the antifungal agent, inhibition of 1,3-β-d-glucan synthesis by caspofungin was rapidly fungicidal against C. albicans before the expression of virulence genes could be perturbed. The absence of suppression of expression of SAP genes, PLB1, and the control EFB1 gene by caspofungin concurs with the highly specific inhibition of glucan synthase by the close analogue L-733,560, which begins to show nonspecific effects at 25× MIC (29, 37). In conclusion, it is unlikely that suppression of SAP and PLB1 gene expression by caspofungin occurs at the caspofungin concentrations in plasma found in clinical treatment of candidiasis (10), and such an indirect mechanism would not be expected to contribute to clearance of C. albicans during treatment with caspofungin.
Expression of the SAP2, SAP5, and EFB1 genes of C. albicans over a period of 7 h after the addition of caspofungin (4 to 16 μg/ml) to cells grown for 15 h in SDB. Representative results are shown from two independent experiments. EFB1 was used as a positive control. Expression of the remaining SAP genes and PLB1 was similar to that of SAP2.
ACKNOWLEDGMENTS
This study was supported in part by grants from Merck Frosst Canada & Co. and the Canadian Institutes of Health Research/Rx & D Research Program.
We thank Claire St-Onge for preparation of the manuscript.
FOOTNOTES
- Received 15 March 2002.
- Returned for modification 27 March 2002.
- Accepted 11 June 2002.
- Copyright © 2002 American Society for Microbiology
