The Antifungal Echinocandin Caspofungin Acetate Kills Growing Cells of Aspergillus fumigatus In Vitro

MATERIALS AND METHODS

RESULTS

We sought to better understand the physiological effects of caspofungin treatment using CFDA and DiBAC, which are fluorescent indicators of cell viability (28). First, C. albicans strain MY1055 was grown in buffered RPMI 1640 medium and challenged with caspofungin to determine the appropriate conditions and timing for cell killing. Exposure of cells in the early logarithmic phase to 0.25 μg of caspofungin per ml reduced the number of CFU by 2 log₁₀ in 15 h (data not shown). Cultures grown under these conditions were incubated with 0.15 μg of AMB per ml, 0.2 μg of caspofungin per ml, or 2.5 μg of FLC per ml or without drug. The cells were stained with either CFDA or DiBAC and were examined microscopically (Fig. 1). Nearly all cells from drug-free cultures stained with CFDA but not with DiBAC. Conversely, very few cells from AMB- or caspofungin-treated cultures stained with CFDA, while nearly all cells stained with DiBAC. C. albicans cells incubated with FLC showed a unique staining pattern; CFDA fluorescence was uniform but less intense than that seen for untreated cells, and only a few FLC-treated cells stained faintly with DiBAC.

The effect of AMB, caspofungin, or FLC on dye staining of live or dead cells was quantified by fluorometry. Because fluorescence intensity is a function of both the physiological state of the cells and the culture density, we normalized each signal to the cell number determined by counting the cells with a hemocytometer. Titrations were performed with AMB, FLC, or caspofungin, and cells from drug-treated cultures were either stained with CFDA or DiBAC or spread onto agar plates to quantify the CFU (Fig. 2). At AMB concentrations ≥0.15 μg/ml, the number of CFU was reduced and there was a decrease in the amount of CFDA fluorescence per cell and a nearly 70-fold increase in the level of normalized DiBAC staining. Similarly, concentrations of caspofungin ≥0.04 μg/ml reduced the number of C. albicans CFU and caused a 4-fold decrease in the level of CFDA staining and a greater than 140-fold increase in the level of DiBAC staining compared to those for the controls. Concentrations of caspofungin or AMB that did not reduce the CFU counts also did not produce changes in staining with either dye. Exposure of C. albicans cells to FLC at concentrations ≥0.5 μg/ml caused a decrease in the level of CFDA staining, which we attribute to decreased fluorescence intensity in all cells rather than the all-or-none staining evident in caspofungin- and AMB-treated cultures (Fig. 1). There also was a <10-fold increase in the level of DiBAC staining of FLC-treated cells, but there was no reduction in the number of CFU at any concentration of FLC tested.

Having established that staining with CFDA and DiBAC reflects a loss of viability of caspofungin-treated C. albicans cells, we adapted this method to further investigate the activity of caspofungin against A. fumigatus. When A. fumigatus is grown in buffered RPMI 1640 medium, it forms tangled hyphal masses; aliquots removed by standard methods are not representative of the total culture. We modified the medium by adding the polyacrylate Junlon (5, 23) to a final concentration of 0.15% (wt/vol), which reduced mycelial masses and produced a culture of small, punctate clumps within a turbid suspension of individual germlings (Fig. 3A). The presence of Junlon did not have a deleterious effect on the amount of fungal growth, as evidenced by equivalent biomass (measured by determination of the total amount of protein) in cultures grown with or without the polyacrylate (data not shown). Samples of CFDA-stained cultures of A. fumigatus, grown in medium with or without Junlon, were serially diluted in the same medium, and the fluorescence was measured. The linear titration evident in the presence of Junlon (Fig. 3B) contrasted with the scattered, nonlinear fluorescence titration observed with cultures grown in RPMI 1640 medium alone. Individual hyphae of A. fumigatus had a similar appearance when grown in medium with or without Junlon (data not shown), and the addition of Junlon did not affect the susceptibility of A. fumigatus MF5668 to caspofungin, AMB, or ITC in standard liquid broth microdilution assays (Table 1).

We determined the effect of drug treatment on A. fumigatus hyphal morphology, cell staining, and CFU counts. Spores of A. fumigatus strain MF5668 inoculated into RPMI-Junlon and incubated with shaking at 37°C germinated and grew into small germlings within 14 h. Compound (either 0.15 μg of AMB per ml, 0.3 μg of caspofungin per ml, or 2.5 μg of ITC per ml) or vehicle was added to the actively growing cultures at this point. After 6 h of incubation with drug or vehicle, aliquots of the cultures were stained with either CFDA or DiBAC and examined microscopically. As with untreated C. albicans cultures, the vast majority of untreated A. fumigatus cells stained with CFDA but not with DiBAC (Fig. 4). In contrast, none of the AMB-treated A. fumigatus cells was stained by CFDA, but all AMB-treated A. fumigatus cells were stained by DiBAC. ITC treatment produced a forked or club-like morphology among a small number of apical cells, some of which failed to stain with CFDA or stained with DiBAC. Of the CFDA-positive ITC-treated cells, there was a trend toward decreased fluorescence of all cells, similar to the findings for FLC-treated C. albicans. Caspofungin-treated germlings had a high proportion of cells at hyphal tips and branch sites that did not stain with CFDA or that did stain with DiBAC (Fig. 4). Inspection of caspofungin-treated, DiBAC-stained hyphae at a higher magnification revealed plumes of stained material apparently outside of transparent cells at the tips (Fig. 5). This is most likely cellular phospholipid debris which was trapped in the viscous Junlon-containing medium after individual cells lysed and released their contents. The high prevalence of damaged cells at hyphal tips and the branch points of caspofungin-treated germlings was not reflected by a change in the number of CFU (Fig. 6). A. fumigatus germlings in RPMI-Junlon were exposed to 0.3 μg of caspofungin, AMB, or ITC per ml or vehicle and were monitored for the number of CFU during a 15-h incubation. AMB treatment produced an ∼2 log₁₀ reduction in the number of A. fumigatus CFU within 3 h, and ITC or caspofungin treatment did not affect the number of CFU. Cultures grown in the absence of drug displayed obvious increases in hyphal mass without a commensurate increase in the number of CFU.

The dose dependence of drug-induced changes in A. fumigatus staining with CFDA or DiBAC was evaluated by titrating each compound and measuring the fluorescence quantitatively. To relate any changes in staining to cell growth and viability, parallel cultures were monitored for the reduction of Alamar Blue in the presence or the absence of drug. A change in the color of the Alamar Blue dye results from the production of reducing equivalents during fungal growth; there is little to no change in dye color if cells die or stop growing. AMB-treated A. fumigatus cells exhibited a threshold for increased DiBAC staining and decreased CFDA staining at AMB concentrations ≥0.15 μg/ml (Fig. 7). This threshold correlated with the reduction of Alamar Blue and was consistent with the staining patterns observed in photomicrographs (Fig. 4). Caspofungin-treated A. fumigatus cultures exhibited a continuum of normalized fluorescence changes and Alamar Blue reduction across a broad range of concentrations, with no clear threshold. A. fumigatus treated with 0.012 or 0.06 μg of caspofungin per ml displayed moderate DiBAC staining; the maximal response was at caspofungin concentrations ≥0.3 μg/ml. This correlated with a decreased level of Alamar Blue reduction. ITC at concentrations ≥0.5 μg/ml prevented Alamar Blue reduction, with little quantitative change in fluorescent dye staining. At these concentrations, ITC appears to stop the production of reducing equivalents by A. fumigatus while minimally inducing the physiological changes reflected by changes in CFDA or DiBAC staining.

The preponderance of staining changes among cells at the tips and branch points of caspofungin-treated A. fumigatus germlings prompted an analysis of the effects of caspofungin as a function of cell position. A. fumigatus cells in photomicrographs were categorized as either apical, subapical, or subapical branched (Fig. 8) and were scored as fluorescent or nonfluorescent. As seen in Fig. 4 and Table 2, AMB-treated A. fumigatus cells failed to stain with CFDA and stained with DiBAC, regardless of their position in the germ tube. Among caspofungin-treated germlings, only 12% of apical cells stained with CFDA, and 72% stained with DiBAC, while 39% of subapical branching cells stained with CFDA and 56% stained with DiBAC (Table 2). For subapical cells within caspofungin-treated germlings, 76% stained with CFDA and 10% stained with DiBAC. ITC treatment led to a staining pattern that was intermediate in its cell specificity; there was a modest preference for apical and subapical branching cells that was not as pronounced as the selective staining pattern seen with caspofungin-treated germlings.

We explored the effect of extended incubation in the presence of each antifungal drug on dye staining. Aliquots removed at 6, 24, 48, and 72 h after drug addition from cultures grown in RPMI-Junlon were stained and evaluated by microscopy, and parallel cultures were grown in the presence of Alamar Blue. There was substantial growth in the untreated culture during the course of the experiment (data not shown), and the CFDA-stained hyphae fluoresced strongly at all time points. There was a slight increase in DiBAC staining of untreated germlings with incubation for ≥24 h, although this was primarily due to staining of conidia and conidium-bearing structures (Fig. 9A). AMB at concentrations ≥0.27 μg/ml caused changes in staining by CFDA and DiBAC, regardless of the cell position within the germlings, over the entire 72-h time course (data not shown). Of interest, 0.09 μg of AMB per ml had antifungal activity at 6 and 24 h, but there was significant growth by 48 h. This was confirmed both by staining with CFDA and DiBAC and by Alamar Blue reduction. At all time points, caspofungin preferentially affected actively growing cells at the tips and branch points of A. fumigatus hyphae, producing staining changes in roughly 75% of apical cells (Fig. 9 and data not shown). The extent of hyphal branching became more pronounced with prolonged incubation, but the proportion of fluorescent apical and subapical branching cells was consistent at all time points. With as much as 72 h of incubation with caspofungin at concentrations ≥0.06 μg/ml, there was no evidence of hyphae with the slender, tapered morphology of untreated A. fumigatus germlings. Exposure to ITC caused only subtle changes in the CFDA and DiBAC staining patterns at all concentrations tested, although there was growth inhibition and no detectable Alamar Blue reduction at ITC concentrations ≥0.5 μg/ml (data not shown).

Morphological changes associated with sporulation were observed with prolonged incubation in both drug-free and caspofungin-treated cultures. This phenomenon is distinct from microcycle conidiation (direct production of conidiophores without prior mycelial formation), but like microcycle conidiation, it was most likely a consequence of growth at 37°C in shake flasks (3, 10). Without drug, abundant conidiophores formed from submerged hyphae within 24 h, and their morphology resembled that of structures produced by aerial mycelia. In contrast, aberrant conidiophores formed by caspofungin-treated A. fumigatus appeared only after 72 h, they were much less prevalent, and they appeared transparent and flattened (Fig. 9). At least one-half failed to stain with CFDA or stained with DiBAC, and none appeared to produce any mature conidia.

DISCUSSION

The effects of antifungal compounds on fungal pathogens are frequently tested by spreading samples from either treated cultures or organs from infected animals on agar plates and enumerating the CFU. However, for A. fumigatus, the filamentous nature of growth in both liquid culture and infected tissues makes it difficult to assess activity based on the number of CFU. Increases in cell mass are not usually reflected by increases in the number of CFU (29), and a CFU by definition reflects the viability only of the entire germling, not of individual cells within the germling. Because of the limitations of CFU measurements for A. fumigatus, alternative methods have been developed which utilize dye staining to assess various aspects of cell viability, most notably, membrane potential and intracellular enzymatic activity. Examples of these stains include tetrazolium dyes (14, 15) and the halogenated dye FUN-1 (27). Another dye, propidium iodide, has been used in flow cytometric analysis of C. albicans and C. neoformans (21), but its use with A. fumigatus to date has been limited to conidia (30).

A previous report (28) described the use of CFDA and DiBAC to characterize the response of C. albicans to AMB. Among other things, these dyes have been used to study the membrane potentials of mammalian cells (7, 12) and the viabilities of both bacteria (24) and S. cerevisiae (16). By using at least two different dyes, which provide information about independent aspects of cell physiology, drugs with distinct mechanisms of action can be compared. It is important to relate preliminary dye-staining results to other viability measurements, including CFU quantitation or Alamar Blue reduction, to ensure that the staining changes correspond to true changes in cell vitality.

The mycelial masses which form as A. fumigatus grows in liquid culture confounded our ability to withdraw representative samples or to focus on a field of cells under the microscope. We found that the addition of the polyacrylate Junlon promoted a more homogeneous culture of A. fumigatus in shaking flasks. After drug treatment and dye staining, a linear titration was obtained when germlings grown in RPMI-Junlon were diluted serially and assayed for fluorescence. Junlon has been used to force dispersed growth of other organisms, including Streptomyces spp. (23) and Aspergillus niger (5, 39). Additionally, by selecting germlings trapped in the Junlon layer, we focused on a form of A. fumigatus which not only fit entirely within a single microscope field but which was also representative of the total culture. The ability to photograph entire germlings and discriminate individual cells within these germlings was especially useful for assessing the preferential lysis of growing A. fumigatus cells by caspofungin.

The abundance of 1,3-β-d-glucan in cell walls formed during different stages of the A. fumigatus life cycle is not well characterized. During vegetative growth, the focus of new cell wall synthesis is the hyphal apex (4, 9, 37), and inhibition of 1,3-β-d-glucan synthesis has profound effects on cell wall structure in A. fumigatus (26). As predicted from work with A. nidulans mutants, whose altered cell wall composition led to hyphal “balloons” which lysed without osmotic protection (40), we found that cells at the hyphal tips and branch points were more susceptible to lysis than subapical cells with mature cell walls. Even with prolonged incubation (up to 72 h), the focus of caspofungin killing was on apical and subapical branching cells. As aberrant, conidiophore-like structures formed in the presence of caspofungin, their staining pattern with CFDA or DiBAC was also consistent with cell death. Therefore, our results demonstrate that inhibition of 1,3-β-d-glucan synthesis during either germ tube or conidiophore formation can be a lethal event. Understanding the effect of glucan synthesis inhibition as spores germinate, which is another key developmental step for A. fumigatus, will require further study.

Because filamentous fungi grow by apical extension, continued killing of cells at the hyphal tips would be expected to have a profound impact on the growth of the organism in vivo. Petraitiene et al. (33) reported that caspofungin treatment improved survival compared to that for the untreated controls in a rabbit model of pulmonary aspergillosis. This improvement in survival was at least as good as that seen with AMB therapy. Caspofungin treatment reduced several clinical measures of disease, including total lung weight, pulmonary infarcts, and pulmonary injury (determined by computed tomography), despite an increase in the number of CFU. In a recent report, caspofungin treatment yielded a reduced fungal burden (assessed by quantitative PCR) compared to that in untreated animals, and the reduction in the fungal burden was comparable to that determined for an AMB-treated group (11).

The drugs used in these studies (AMB, FLC, ITC, and caspofungin) have been categorized as either fungicidal or fungistatic. AMB is accepted as fungicidal against both C. albicans and A. fumigatus, based on the reductions in the number of CFU. Conversely, azoles such as FLC and ITC are considered fungistatic against C. albicans and A. fumigatus, respectively. It is interesting that both FLC- and ITC-treated cells exhibited weak but widespread staining with CFDA in our study. Perhaps azole-mediated ergosterol inhibition causes increased permeability of the cell membranes, such that azole-treated cells only partially retain free carboxyfluorescein following esterase cleavage of CFDA. Caspofungin has been shown to be fungicidal against C. albicans (2, 6), and the fluorescence patterns observed in cells stained with CFDA and DiBAC correlate with decreases in the number of CFU in a manner comparable to that seen with AMB-treated cells. The activity of caspofungin against A. fumigatus is more difficult to categorize. While there was no reduction in the number of CFU from standard killing curve measurements, we did see changes in the patterns of staining by dyes that stain viable and nonviable cells analogous to those observed with caspofungin-treated C. albicans. Plumes of DiBAC-stained debris at hyphal tips suggest caspofungin caused lysis of the actively growing A. fumigatus cells. Whether lysis occurs in vivo remains to be determined. Work with persistently neutropenic mice illustrated that caspofungin prevents significant mortality in A. fumigatus-infected animals, even for up to 21 days after therapy is withdrawn (2). If caspofungin is strictly fungistatic against A. fumigatus, a reemergence of the infection should lead to mortality in the face of continued immunosuppression. Perhaps significant injury to cells at the hyphal tips renders the fungus unable to invade tissue beyond a focus of infection. As subapical cells within the mycelium form branches, their requirement for de novo synthesis of 1,3-β-d-glucan may render them susceptible to lysis by caspofungin. The inability of A. fumigatus to sustain polarized growth in the presence of multiple doses of caspofungin could lead to significant fungal cell death in tissues.