Oxidative and amphotericin B-mediated cell death in the opportunistic pathogen Aspergillus fumigatus is associated with an apoptotic-like phenotype

S. Amin A. Mousavi and Geoffrey D. Robson

School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Manchester M13 9PT, UK

Correspondence
Geoffrey D. Robson
geoff.robson{at}man.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
When protoplasts of the opportunistic fungal pathogen Aspergillus fumigatus were treated with low but toxic levels of hydrogen peroxide (0·1 mM) or amphotericin B (0·5 µg ml–1), loss of cell viability and death were associated with a number of phenotypic changes characteristic of apoptosis. The percentage of protoplasts staining positive with annexin V-FITC, an indicator of the externalization of phosphatidylserine and an early marker of apoptosis, rose to ~55 % within 1 h. This was followed by a similar increase in apoptotic DNA fragmentation detected by the TUNEL assay, and led to a loss of cell permeability and death in ~90 % of protoplasts, as indicated by the uptake of propidium iodide. The development of an apoptotic phenotype was blocked when protoplasts were pre-treated with the protein synthesis inhibitor cycloheximide, indicating active participation of the cell in the process. However, no significant activity against synthetic caspase substrates was detected, and the inclusion of the cell-permeant broad-spectrum caspase inhibitor Z-VAD-fmk did not block the development of the apoptotic-like phenotype. Higher concentrations of H2O2 (1·8 mM) and amphotericin B (1 µg ml–1) caused protoplasts to die without inducing an apoptotic phenotype. As predicted, the fungistatic antifungal agent itraconazole, which inhibits growth without causing immediate cell death, did not induce an apoptotic-like phenotype.


Abbreviations: PI, propidium iodide; PS, phosphatidylserine; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The incidence of life-threatening invasive aspergillosis in immunocompromised hosts has increased dramatically over the last two decades (Groll et al., 1996; Vogeser et al., 1997; Latgé, 1999; Denning et al., 2002). Aspergillus fumigatus is the most common aetiological agent of invasive aspergillosis, being responsible for approximately 90 % of human infections (Derouin, 1994). The incidence of invasive aspergillosis varies between 1 and 19 % for patients who have undergone solid organ transplantation (Patel & Paya, 1997; Verweij & Denning, 1997), and patients with leukaemia, AIDS and granulomatous disease are also at risk (Brown et al., 1998; Denning, 1998; Kaizer et al., 1998). In contrast, the disease is rarely found in immunocompetent hosts (Karim et al., 1997). A. fumigatus is a common, widespread saprophytic fungus, and environmental surveys indicate that humans inhale several hundred A. fumigatus conidia per day (Hospenthal et al., 1998). Ingestion and killing of spores by alveolar macrophages represent the first line of defence against infection, mainly by non-oxidative mechanisms, whilst neutrophils employ an oxidative respiratory burst to kill germinating conidia that have escaped macrophage engulfment (Schaffner et al., 1986; Levitz et al., 1986; Morgenstern et al., 1997; Roilides et al., 1998; Latgé, 2001). Invasive aspergillosis has a high mortality and morbidity rate, with only 34 % of patients showing a favourable response to antifungal therapy (Denning, 1996). The fungicidal agent amphotericin B is widely used to treat invasive aspergillosis, although it can have serious side-effects (Clements & Peacock, 1990; Pathak et al., 1998).

Previously, we reported that the viability of A. fumigatus decreases rapidly when the organism enters stationary phase in liquid culture, and that this is associated with the appearance of an apoptotic-like phenotype (Mousavi & Robson, 2003). Although programmed cell death and the underlying mechanisms are well documented in mammalian cells (Strasser et al., 2000; Hengartner, 2000; Kaufmann & Hengartner, 2001), there have been few studies on the mechanism of cell death in fungi, and in filamentous fungi in particular (Umar & Van Griensven, 1997; Raju & Perkins, 2000; Lu et al., 2003; Cheng et al., 2003). In the yeast Saccharomyces cerevisiae, expression of the mammalian pro-apoptotic protein Bax induces an apoptotic-like phenotype, which is suppressed by simultaneous overexpression of Bcl-XL, a member of the mammalian anti-apoptotic Bcl-2 family (Ligr et al., 1998). Moreover, death in S. cerevisiae during the starvation phase, and following treatment with hydrogen peroxide, has also been shown to be associated with an apoptotic-like phenotype and to involve a metacaspase, a protease related to the mammalian caspase family (Madeo et al., 2002). Treatment of S. cerevisiae with toxic levels of acetic acid is also associated with an apoptotic-like phenotype (Ludovico et al., 2001), and was subsequently shown to involve the mitochondrion and cytochrome c release (Ludovico et al., 2002), in a similar manner to that seen in mammalian cells (Hengartner, 2000). Recently, Cheng et al. (2003) reported that the antifungal sphingoid long-chain base phytosphingosine induces an apoptotic phenotype in Aspergillus nidulans in a pathway that did not appear to involve metacaspase activity (Cheng et al., 2003).

In this study, we report that cell death in A. fumigatus, induced either by hydrogen peroxide (oxidative death) or by treatment with the widely used antifungal agent amphotericin B, is associated with the induction of an apoptotic-like phenotype, suggesting that these agents cause death in A. fumigatus by inducing a primitive form of apoptosis. Moreover, unlike entry into the stationary phase, the development of the apoptotic-like phenotype induced by these two fungicidal agents was not blocked by the broad-spectrum caspase inhibitor Z-VAD-fmk, suggesting the presence of two apoptotic-like pathways in A. fumigatus.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Organism and growth conditions.
A. fumigatus AF10, a clinical isolate (ATCC 90240), was stored in 20 % (v/v) glycerol at –80 °C. Cultures were grown on modified Vogel's medium (Vogel, 1956), with 1 % (w/v) glucose replacing 2 % (w/v) sucrose. For liquid cultures, A. fumigatus was grown in 50 ml modified Vogel's medium supplemented with 50 mM MES (pH 5·5) and 0·15 % (w/v) of the polyacrylate polymer Junlon PW 110 (Honeywell and Stein, UK) to induce filamentous growth (Trinci, 1983), in 250 ml conical flasks, and inoculated with 1 ml conidial suspension (~1x108 conidia ml–1). Flasks were incubated with shaking (250 r.p.m.) at 37 °C, and growth of the cultures was followed by measuring optical density (540–560 nm) in an EEL colorimeter (Trinci, 1972). Viability was measured by determining c.f.u. ml–1 on modified Vogel's medium solidified with 1·5 % (w/v) agar. Spore suspensions were prepared from 5-day-old cultures grown on Sabouraud's glucose agar in 250 ml tissue culture flasks at 37 °C, harvested by gentle agitation with 0·01 % (v/v) Tween 80 and filtered through two layers of lens tissue.

Effect of H2O2, amphotericin B and itraconazole on mycelial growth.
To study the effect of H2O2, amphotericin B and itraconazole on the growth and viability of A. fumigatus, various concentrations of H2O2 (from a 30 %, v/v, stock solution), amphotericin B (from a 1 mg ml–1 stock) or itraconazole (from a 1 mg ml–1 stock) were added to early exponential phase cultures (OD ~1·5 EEL units), and viability monitored by determining c.f.u. ml–1 at 30 min intervals. For H2O2-treated mycelia, 0·25 % (w/v) catalase (Sigma) was added after sampling to remove H2O2 from the sample, whilst for amphotericin B or itraconazole treatments, mycelia were washed briefly in sterile deionized water. To determine if DNA degradation had occurred, mycelium was frozen in liquid nitrogen, ground to a fine powder in a mortar and pestle, and genomic DNA extracted according to Reader & Brody (1985). DNA was run on a 1·5 % (w/v) agarose gel in TPE buffer (0·09 M Tris/phosphate, pH 8·0, 2 mM EDTA) and visualized following ethidium bromide staining (0·4 µg ml–1 in TPE buffer).

Analysis of apoptotic markers.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL; TdT-FragEL DNA fragmentation detection kit, Oncogene Research Products) and annexin V-FITC (Oncogene Research Products) were used as markers of apoptosis, and uptake of propidium iodide (PI) was used as a marker of cell-membrane integrity, as previously described (Mousavi & Robson, 2003). In order to detect the expression of these apoptotic markers, the cell wall was first removed by digesting mycelium from mid-exponential-phase growth with Novozyme, as previously described (Mousavi & Robson, 2003), and protoplasts were then treated with various concentrations of H2O2, amphotericin B or itraconazole for up to 6 h. Following treatment, protoplasts were washed twice by centrifugation (1500 g) for 10 min and resuspended in an equal volume of regeneration buffer (0·1 M phosphate buffer, pH 7·0, 0·9 M sorbitol). In the case of H2O2-treated protoplasts, 0·25 % (w/v) catalase was added prior to washing. To determine protoplast viability, protoplasts were regenerated by spreading gently over the surface of modified Vogel's medium solidified with 1·5 % (w/v) agar (supplemented with 0·9 M sorbitol), and plates incubated at 37 °C until colonies became visible. To inhibit metacaspase activity or protein synthesis, respectively, 25 µg ml–1 of the broad-spectrum caspase inhibitor Z-VAD-fmk (Calbiochem) or 50 µg ml–1 cycloheximide (Sigma) was added to the protoplast suspension, 1 h prior to treatment with H2O2 or amphotericin B. DAPI staining was used to determine the percentage of protoplasts containing nuclei, as previously described (Mousavi & Robson, 2003).

Caspase activity.
Intracellular caspase activity was determined using a colorimetric assay based on the cleavage of a p-nitroaniline dye from the C-terminal of specific peptide substrates (Caspase Colorimetric Substrate/Inhibitor Quantipak, Calbiochem). Mycelium was ground in liquid nitrogen and the biomass resuspended in ice-cold lysis buffer (50 mM HEPES, pH 7·4, 1 mM DTT, 0·5 mM EDTA and 0·1 % (v/v) CHAPS), centrifuged at 1500 g for 10 min, and the caspase activity of the supernatant against substrates for caspase-1, -3 and -8 determined according to the manufacturer's instructions. Protein concentration was determined according to Bradford (1976).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth of A. fumigatus treated with H2O2 and amphotericin B
Exponentially growing A. fumigatus cultures were treated with various concentrations of H2O2 (0–3·6 mM; Fig. 1) and amphotericin B (0–3 µg ml–1; Fig. 2) 16 h after inoculation. Growth was monitored by measuring the optical density of the cultures (Figs 1a and 2a), and viability by determining c.f.u. ml–1 (Fig. 2a, b). Growth was not affected by the addition of 0·1 mM H2O2, whereas 1·2 and 1·8 mM H2O2 caused a cessation of growth for 5 h, after which growth resumed. When cultures were treated with concentrations of 2·4 mM H2O2 and above, growth ceased immediately and did not recover. As shown in Fig. 1(b), the number of colonies was not affected by concentrations up to 0·1 mM H2O2, whereas 1·2 and 1·8 mM H2O2 initially caused a drop in cell viability for the first 4 h, from which there was subsequent recovery. Treatment for 2 h with 3·0 mM H2O2 and above was lethal. Cultures treated at amphotericin B concentrations less than 1 µg ml–1 had no effect on growth, whereas addition of 2 µg ml–1 or more inhibited growth within 1 h (Fig. 2a). Fig. 2(b) shows that exposure to low concentrations of amphotericin B (0·5 or 1 µg ml–1) had no effect on cell viability, whereas cell viability was abolished after treating with 2 µg amphotericin B ml–1.



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Fig. 1. Effect of the addition of various concentrations of H2O2 on the growth (a) and viability (b) of A. fumigatus 16 h after inoculation (arrow) in shake flask cultures at 37 °C. {lozenge}, 0 mM; {square}, 0·1 mM; {triangleup}, 1·2 mM; {circ}, 1·8 mM; {blacksquare}, 2·4 mM; {blacktriangleup}, 3 mM; {bullet}, 3·6 mM H2O2. Data represent the mean of five replicates±SEM.

 


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Fig. 2. Effect of the addition of various concentrations of amphotericin B on the growth (a) and viability (b) of A. fumigatus 16 h after inoculation (arrow) in shake flask cultures at 37 °C. {lozenge}, 0 µg ml–1; {square}, 0·5 µg ml–1; {triangleup}, 1 µg ml–1; {circ}, 2 µg ml–1; {blacksquare}, 3 µg amphotericin B ml–1. Data represent the mean of five replicates±SEM.

 
In many mammalian cells, apoptosis is often associated with the action of specific endonucleases that attack nuclear DNA in the internucleosomal linker regions, resulting in double-stranded, low-molecular-mass oligonucleosomal DNA fragments in multiples of about 180 to 200 bp, which are visible after electrophoresis as a DNA ‘ladder’ (De Mario et al., 1997). Fig. 3(a) shows that incubation of mycelium with 3 mM H2O2 did not cause DNA fragmentation after 2 h; however, exposure for 4 h and above resulted in complete loss of intact DNA and the appearance of fragmented DNA as a smear (Fig. 3a). A DNA ladder was not visible. Exposure of mycelium to 3 µg amphotericin B ml–1 for up to 4 h had no visible effect on DNA integrity; however, complete loss of intact DNA occurred after 8 h exposure, and fragmented DNA was visible as a smear. As with H2O2 treatment, a DNA ladder was not visible (Fig. 3b).



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Fig. 3. Agarose gel electrophoresis of DNA extracted from A. fumigatus mycelium exposed to 3 mM H2O2 (a) or 3 µg amphotericin B ml–1 (b) for up to 24 h. Lanes in Fig. 3a: 1, molecular mass markers (1 kb ladder); 2, DNA extracted after 16 h incubation; lanes 3–6, DNA extracted after treating for 2 h with 3 mM H2O2 (lane 3), 4 h (lane 4), 8 h (lane 5) and 24 h (lane 6). Lanes in Fig. 3b: 1, molecular mass markers (1 kb ladder); 2, DNA extracted after 16 h incubation; lanes 3–6, DNA extracted after treating for 2 h with 3 µg amphotericin B ml–1 (lane 3), 4 h (lane 4), 8 h (lane 5) and 24 h (lane 6).

 
Effect of H2O2 and amphotericin B on protoplast viability
The effect of various concentrations of H2O2 and amphotericin B on protoplast viability is shown in Figs 4(a) and 4(b), respectively. Protoplast viability was unaffected by 0·01 mM H2O2, whereas, following the addition of 0·1 mM H2O2, less than 50 % of viable protoplasts survived after 1 h, and less than 20 % after 2 h exposure. No protoplasts were viable 2 h after treatment with 1·8 mM H2O2 (Fig. 4a). When exposed to 0·25 µg amphotericin B ml–1, more than 70 % of protoplasts retained viability after 2 h, whereas less than 20 % of protoplasts were viable 1 h after the addition of 0·5 µg ml–1. No protoplasts remained viable 2 h after the addition of 1 or 2 µg amphotericin B ml–1 (Fig. 4b and data not shown).



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Fig. 4. Effect of various concentrations of H2O2 (a) and amphotericin B (b) on protoplast viability after exposure for 1 h (white bars) or 2 h (grey bars), before being diluted on osmoticum and spread onto Vogel's medium containing 50 mM sorbitol. The viability (67 % total count) of untreated protoplasts at t0 was considered as 100 %. Data represent the mean of five replicates±SEM.

 
H2O2- and amphotericin B-induced death of A. fumigatus is associated with an apoptotic phenotype
To assess whether H2O2- or amphotericin B-induced cell death in A. fumigatus displayed characteristics associated with apoptosis, we determined the percentage of protoplasts (containing nuclei) that stained positive with annexin V-FITC, TUNEL and PI, following addition of H2O2 and amphotericin B (Fig. 5a, b, respectively). Annexin V binds specifically to phosphatidylserine (PS), and is widely used to detect the exposure of PS on the outside of the plasma membrane, which occurs early in the apoptotic process (Champagne et al., 1999; Martinet et al., 1999). Protoplasts prior to treatment showed a low level of staining (<7 %) with annexin V-FITC, TUNEL and PI. When protoplasts were treated for 2 h with 0·01 mM H2O2, annexin V-FITC-positive protoplasts increased to ~20 %, whilst there was a small but insignificant (P>0·05) increase in TUNEL- and PI-positive protoplasts (Fig. 5a). When incubated with 0·1 mM or 1·8 mM H2O2, ~55 % of protoplasts stained positive for annexin V-FITC. H2O2 at 0·1 mM induced apoptotic-like nuclear degradation in ~55 % of protoplasts (TUNEL-positive), but at the higher concentration of 1·8 mM, this number fell to ~20 %. Treatment with 0·1 mM H2O2 increased PI staining to ~17 %; however, 1·8 mM led to >80 % of the protoplasts staining positive with PI.



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Fig. 5. Appearance of apoptotic markers in protoplasts treated for 2 h with (a) 0 mM (black bar), 0·01 mM (white), 0·1 mM (diagonal hatching) or 1·8 mM (grey) H2O2, or (b) 0 µg ml–1 (black bar), 0·25 µg ml–1 (white), 0·5 µg ml–1 (diagonal hatching) or 1 µg ml–1 (grey) amphotericin B. Results are shown as the percentage of DAPI-positive protoplasts staining positive with annexin V-FITC, TUNEL or PI. Results represent the mean of five replicate samples±SEM.

 
A similar pattern was observed when protoplasts were treated with amphotericin B (Fig. 5b). When treated with 0·25 µg ml–1 amphotericin B, ~15 % stained positive with annexin V-FITC. This rose to ~50 % when treated with 0·5 or 1 µg amphotericin B ml–1. When treated with 0·5 µg amphotericin B ml–1, the proportion of TUNEL-positive protoplasts increased to ~65 %; however, the level of staining was less than 20 % when treated with 1 µg ml–1. The proportion of PI-positive protoplasts was less than 20 % when treated with 0·25 or 0·5 µg amphotericin B ml–1, but rose to ~85 % when treated with 1 µg amphotericin B ml–1.

To test if the apoptotic-like phenotype was induced by agents that inhibited growth, without being fungicidal, we used the fungistatic azole itraconazole, which in the short term inhibits growth, without killing the cell (Lamb et al., 1999). Treatment of protoplasts with 10 µg itraconazole ml–1, a concentration that inhibited protoplast regeneration without causing death, did not cause a significant increase in annexin V-FITC, TUNEL or PI-positive staining (data not shown).

Influence of H2O2 on the appearance of apoptotic markers over time
To study the development of the apoptotic phenotype over time, we treated protoplasts with 0·1 mM H2O2 and monitored the proportion of protoplasts staining positive for annexin V-FITC, TUNEL and PI over 6 h (Fig. 6). Initially, prior to treatment, the proportion of protoplasts staining positive for any of the markers was <10 %. Within 1 h of treatment with 0·1 mM H2O2, the proportion of protoplasts staining positive with annexin V-FITC rose to ~45 %, and remained approximately constant thereafter. The proportion of TUNEL-positive protoplasts rose slightly to ~15 % after 1 h exposure to H2O2, but had risen to 50 % after 2 h and continued to increase to a maximum of ~65 % after 4 h, before decreasing to ~40 % after 6 h. PI-positive protoplasts increased slightly to ~10 % after 1 h, increased to ~25 % after 2 h, and thereafter rose steadily to reach a maximum of ~90 % after 5 h.



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Fig. 6. Appearance of apoptotic markers over time in protoplasts treated with 0·1 mM H2O2 for up to 6 h. Results are shown as the percentage of DAPI-positive protoplasts staining positive with annexin V-FITC (black bars), TUNEL (white bars) or PI (grey bars). Results represent the mean of five replicate samples±SEM.

 
Blocking protein synthesis prevents the development of an apoptotic-like phenotype
Programmed cell death and the development of an apoptotic phenotype is dependent on the active participation of the cell and the synthesis of novel proteins. To determine whether the development of the apoptotic-like phenotype, following treatment with H2O2 or amphotericin B, was dependent on novel protein synthesis, protoplasts were treated with the protein synthesis inhibitor cycloheximide for 1 h, prior to the addition of H2O2 or amphotericin B (Fig. 7a, b). Pre-incubation with cycloheximide completely blocked the increase in TUNEL-positive protoplasts following treatment with either H2O2 or amphotericin B, but had no significant effect on the proportion of PI-positive protoplasts induced by 1·8 mM H2O2 or 1 µg amphotericin B ml–1.



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Fig. 7. Effect of cycloheximide on the appearance of apoptotic markers in protoplasts treated with (a) H2O2 or (b) amphotericin B. Protoplasts were incubated for 1 h in the absence (black bars, white bars) or presence (hatched bars, grey bars) of 50 mM cycloheximide, before treating for 2 h with (a) 0·1 mM H2O2 (black bars, white bars), or 1·8 mM H2O2 (hatched bars, grey bars) or (b) 0·5 µg ml–1 amphotericin B (black bars, white bars) or 1 µg ml–1 amphotericin B (hatched bars, grey bars). Results are shown as the percentage of DAPI-positive protoplasts staining positive with TUNEL or PI, and represent the mean of five replicate samples±SEM.

 
Caspase activity
To determine whether caspases contribute to cell death, following treatment with H2O2 or amphotericin B, a colorimetric assay based on the cleavage of p-nitroaniline dye from the C-terminal of specific peptide substrates was performed (Thornberry, 1998). Total protein was extracted from the mycelium at various time points up to 6 h, following the addition of 3 mM H2O2 or 3 µg amphotericin B ml–1 to the culture, and assayed for activity against substrates specific for caspases-1, -3 and -8. No significant increase in activity against the caspase substrates was observed (data not shown). Moreover, addition of the broad-spectrum caspase inhibitor Z-VAD-fmk to protoplasts, prior to treatment with 0·1 mM H2O2 or 0·5 µg amphotericin B ml–1, did not block the subsequent increase in the percentage of TUNEL-positive or PI-positive protoplasts (data not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Apoptosis is controlled by a complex regulatory network, which can be activated by external signals (e.g. reactive oxygen species, ROS; Madeo et al., 1999; Bustamante et al., 2000) and internal processes (e.g. replication failures or developmental programmed cell death; Okuno et al., 1998). During the course of this work, H2O2 was used to generate oxidative stress to mimic killing of cells by the respiratory burst, whilst cell death caused by amphotericin B was investigated, as it is still the most widely prescribed antifungal agent for the treatment of aspergillosis, despite its nephrotoxicity (Wingard et al., 1999; Patterson et al., 2000). Analysis of chromosomal DNA from H2O2- or amphotericin B-treated mycelia by agarose electrophoresis showed smearing of the DNA. However, evidence of a DNA ladder pattern, which is found in many apoptotic systems as the result of internucleosomal DNA cleavage (Hofmann et al., 1999; Bustamante et al., 2000), was not found. DNA laddering was also absent during cell death in the stationary phase in A. fumigatus (Mousavi & Robson, 2003), and was also absent during cell death in S. cerevisiae and in Pichia pastoris (Martinet et al., 1999; Madeo et al., 1999). Moreover, DNA smearing was also reported as a late response in A. nidulans, following treatment with phytosphingosine (Cheng et al., 2003). Apoptotic cell death in certain mammalian cells has also been shown to occur in the absence of a DNA ladder (Oberhammer et al., 1993; Knapp et al., 1999).

A more sensitive test of apoptotic DNA fragmentation is based on labelling the free 3'-OH termini, which are exposed during apoptotic DNA cleavage. The TUNEL assay, which relies on the incorporation of biotinylated or fluorescein-labelled dUTP, catalysed by terminal deoxynucleotidyl transferase (TdT), enables DNA breakage to be visualized in individual cells undergoing apoptosis and has become one of the most widely used indicators of apoptosis (Gavrieli et al., 1992; Frohlich & Madeo, 2000). Previously, we demonstrated that cell death during the stationary phase in A. fumigatus was associated with a marked increase in the proportion of TUNEL-positive nuclei indicating apoptotic-like cleavage of the DNA (Mousavi & Robson, 2003), and TUNEL staining was also reported during cell death in A. nidulans treated with phytosphingosine (Cheng et al., 2003). In this study, lower concentrations of H2O2 and amphotericin B (0·1 mM and 0·5 µg ml–1, respectively) increased the proportion of TUNEL-positive protoplasts indicating apoptotic-like DNA fragmentation, whereas higher concentrations (1·8 mM and 1 µg ml–1, respectively) caused a lower increase in TUNEL-positive protoplasts (Fig. 5). This decrease in TUNEL-staining at higher concentrations correlates with a large increase in PI-staining, indicating loss of membrane permeability and necrotic death. The induction of necrosis by high concentrations of numerous cytotoxic substances, and apoptosis at lower concentrations, is a well known phenomenon (Lieberthal & Levine, 1996), and has also been reported in S. cerevisiae treated with H2O2 and acetic acid (Madeo et al., 1999; Ludovico et al., 2001).

In mammalian cells, an earlier indicator of apoptosis is the translocation of PS from the inner to the outer leaflet of the cytoplasmic membrane (Champagne et al., 1999), and can be detected with FITC-labelled annexin V, which specifically binds to PS (Martinet et al., 1999). As with TUNEL staining, treatment with low concentrations of H2O2 or amphotericin B led to a large increase in annexin V-FITC staining. Moreover, this increase occurred prior to the increase in TUNEL staining (Fig. 6), and this early increase in PS translocation has also been reported in A. nidulans (Cheng et al., 2003).

Apoptosis requires the active participation of the cell in the synthesis of new proteins that contribute to cell death. Consequently, protein synthesis inhibitors can actively block apoptosis in cells, and this is widely used to demonstrate the participation of the cell during death (Hiraoka et al., 1997; Sanchez et al., 1997). In S. cerevisiae, cycloheximide has been shown to block the development of an apoptotic phenotype in response to various stimuli (Madeo et al., 1999), to block TUNEL staining in A. fumigatus following entry into the stationary phase, and to block TUNEL staining in A. nidulans following treatment with phytosphingosine (Cheng et al., 2003; Mousavi & Robson, 2003). When added prior to treatment with low concentrations of H2O2 or amphotericin B, cycloheximide blocked the development of a TUNEL-positive phenotype, whilst having no effect on the later increase in PI-positive staining that indicates loss of membrane integrity and cell death (Fig. 7). Thus, the development of an apoptotic-like phenotype requires protein synthesis and active participation of the cell. However, although entry into an apoptotic pathway can be prevented by blocking protein synthesis, subsequent cell death through a necrotic process is not prevented, as reported previously when A. fumigatus enters stationary phase (Mousavi & Robson, 2003).

A number of studies in yeast and mammalian cells have demonstrated that accumulation of ROS within the cytoplasm plays a central role in apoptotic-like cell death (Greenlund et al., 1995; Slater et al., 1995; Ligr et al., 1998; Madeo et al., 1999). It is possible that treatment with low concentrations of H2O2 or amphotericin B may trigger an apoptotic-like phenotype through the accumulation of ROS, and that continued accumulation in the cytoplasm ultimately causes physical damage and loss of cell integrity, as indicated by the increase in PI-positive staining. Treatment of protoplasts with low but toxic concentrations of the strong oxidizing agent sodium hypochlorite also induced an apoptotic phenotype similar to that observed with H2O2, suggesting that this is a general response to oxidative stress (results not shown). Previously, we reported an increase in intracellular activity toward caspase substrates as cultures entered the stationary phase. Moreover, the cell-permeant broad-spectrum caspase inhibitor z-VAD-fmk was able to block the increase in TUNEL and annexin V-FITC staining, suggesting a role for an upstream caspase-like activity in nuclear degradation and PS externalization (Mousavi & Robson, 2003). In S. cerevisiae, a metacaspase with caspase-like activity, YCA1, has been cloned and shown to mediate apoptosis induced by H2O2 and in chronologically aged cells (Madeo et al., 2002). However, in this study, no significant activity against caspase substrates was induced following H2O2 or amphotericin treatment. Moreover, inclusion of z-VAD-fmk prior to treatment did not block the development of an apoptotic-like phenotype, suggesting an alternative pathway to that involved in stationary-phase-induced cell death. Caspase-independent death has been described in other systems under specific conditions (De Mario et al., 1997; Brunet et al., 1998; Kroemer et al., 1998; Okuno et al., 1998), and the caspase inhibitor z-VAD-fmk found not to block the development of an apoptotic-like phenotype (Villa et al., 1997). In A. nidulans, a metacaspase homologue, casA, has been identified, and a {Delta}casA disrupted strain shown still to undergo apoptosis in response to phytosphingosine, suggesting death by a caspase-independent pathway (Cheng et al., 2003). As previously reported, A. fumigatus appears to contain two metacaspase homologues (Mousavi & Robson, 2003), both with a high level of identity to A. nidulans casA. Interrogation of the A. nidulans genome sequence at the Whitehead Institute also revealed a second metacaspase homologue in A. nidulans (data not shown). It is possible, therefore, that one metacaspase homologue is activated through starvation, upon entry into the stationary phase, and is active against caspase substrates, whereas the second may be activated by H2O2 and amphotericin B, but is inactive against the synthetic substrates tested in this study.

In this study, we have demonstrated that cell death induced by low but toxic concentrations of H2O2 or amphotericin B triggers the development of a protein-synthesis-dependent apoptotic-like phenotype. Thus, cell death in A. fumigatus in infected humans, as a consequence of phagocyte action or of treatment with the antifungal agent amphotericin B, may actively involve the participation of the fungal cells in their own death, through triggering an apoptotic-like pathway. Further work on the role of ROS, metacaspase and the mitochondrion will be needed to understand better the mechanisms underlying the apoptotic-like pathway(s) in A. fumigatus.


   ACKNOWLEDGEMENTS
 
The authors thank the Iranian Ministry of Health for financial support to S. A. A. M.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein–dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]

Brown, M. J., Worthy, S. A., Flint, J. D. A. & Muller, N. L. (1998). Invasive aspergillosis in the immunocompromised host: utility of computed tomography and bronchoalveolar lavage. Clin Radiol 53, 255–257.[Medline]

Brunet, C. L., Gunby, R. H., Benson, R. S. P., Hickman, J. A., Watson, A. J. M. & Bardy, G. (1998). Commitment to cell death measured by clonegenicity is separable from the appearance of apoptotic markers. Cell Death Differ 5, 107–115.[CrossRef][Medline]

Bustamante, J., Bersier, G., Romero, M., Badin, R. A. & Boveris, A. (2000). Nitric oxide production and mitochondrial dysfunction during rat thymocyte apoptosis. Arch Biochem Biophys 376, 239–247.[CrossRef][Medline]

Champagne, M., Pierre, D., Sergei, O., Martin, B., Pavel, H. & Johanne, T. (1999). Protection against necrosis but not apoptosis by heat stress proteins in vascular smooth muscle cells: evidence for distinct modes of cell death. Hypertension 33, 906–913.[Abstract/Free Full Text]

Cheng, J., Park, T.-S., Chio, L.-C., Fischl, S. & Ye, X. S. (2003). Induction of apoptosis by sphingoid long-chain bases. Mol Cell Biol 23, 163–177.[Abstract/Free Full Text]

Clements, J. S. & Peacock, J. E. (1990). Amphotericin B revisited: reassessment of toxicity. Am J Med 88, 22N–27N.[Medline]

De Mario, R., Lenti, L., Malisan, F., d'Agostino, F., Tomassini, B., Zeuner, A., Rippo, M. R. & Testi, R. (1997). Requirement for GD3 ganglioside in CD95 and ceramide-induced apoptosis. Science 277, 1652–1655.[Abstract/Free Full Text]

Denning, D. W. (1996). Therapeutic outcome in invasive aspergillosis. Clin Infect Dis 23, 608–615.[Medline]

Denning, D. W. (1998). Invasive Aspergillosis. Clin Infect Dis 26, 781–805.[Medline]

Denning, D. W., Anderson, M. J., Turner, G., Latgé, J.-P. & Bennett, J. W. (2002). Sequencing the Aspergillus fumigatus genome. Lancet Infect Dis 2, 251–253.[CrossRef][Medline]

Derouin, F. (1994). Special issue on Aspergillosis. Pathol Biol 42, 625–736.

Frohlich, K. U. & Madeo, F. (2000). Apoptosis in yeast, a monocellular organism exhibits altruistic behaviour. FEBS Lett 473, 6–9.[CrossRef][Medline]

Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. A. (1992). Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 119, 493–501.[Abstract]

Greenlund, L. J., Deckwerth, T. L. & Johnson, E. M. (1995). Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron 14, 303–315.[Medline]

Groll, A. H., Shah, P. M., Mentzel, C., Schneider, M., Just-Nuebling, G. & Huebner, K. (1996). Trends in post-mortem epidemiology of invasive fungal infections at a university hospital. J Infect 33, 23–32.[Medline]

Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature 407, 770–776.[CrossRef][Medline]

Hiraoka, W., Fuma, K. & Kuwabara, M. (1997). Concentration-dependent modes of cell death in Chinese hamster V-79 cells after treatments with H2O2. J Radiat Res 38, 95–102.[Medline]

Hofmann, F., Ohnimus, H., Strupp, W., Zimmerman, U. & Jassoy, G. (1999). Electric field pulses can induce apoptosis. J Membr Biol 169, 103–109.[CrossRef][Medline]

Hospenthal, D. R., Kwon-Chung, K. J. & Bennet, J. E. (1998). Concentrations of airborne Aspergillus compared to the incidence of invasive aspergillosis: lack of correlation. Med Mycol 36, 165–168.[CrossRef][Medline]

Kaizer, L., Huguenint, T., Lew, P. D., Chapuis, B. & Pittet, D. (1998). Invasive aspergillosis. Clinical features of 35 proven cases at a single institution. Medicine 77, 188–194.[CrossRef][Medline]

Karim, M., Alam, M., Shah, A. A., Ahmed, R. & Sheikh, H. (1997). Chronic invasive aspergillosis in apparently immunocompetent hosts. Clin Infect Dis 24, 723–733.[Medline]

Kaufmann, S. H. & Hengartner, M. O. (2001). Programmed cell death: alive and well in the new millennium. Trends Cell Biol 11, 526–534.[CrossRef][Medline]

Knapp, P. E., Bartlett, W. P., Williams, L. A., Yamada, M., Ikenaka, K. & Scoff, R. P. (1999). Programmed cell death without DNA fragmentation in the jimpy mouse: secreted factors can enhance survival. Cell Death Differ 6, 136–145.[CrossRef][Medline]

Kroemer, G., Dallaporter, B. & Rosche-Rigon, M. (1998). The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60, 619–647.[CrossRef][Medline]

Lamb, D., Kelly, D. & Kelly, S. (1999). Molecular aspects of azole antifungal action and resistance. Drug Resist Updat 2, 390–402.[CrossRef][Medline]

Latgé, J.-P. (1999). Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12, 310–350.[Abstract/Free Full Text]

Latge, J.-P. (2001). The pathobiology of Aspergillus fumigatus. Trends Microbiol 9, 382–389.[CrossRef][Medline]

Levitz, S. M., Selsted, M. E., Ganz, T., Lehrer, R. I. & Diamond, R. D. (1986). In vitro killing of spores and hyphae of Aspergillus fumigatus and Rhizopus oryzae by rabbit neutrophil cationic peptides and bronchoalveolar macrophages. J Infect Dis 154, 483–489.[Medline]

Lieberthal, W. & Levine, J. S. (1996). Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury. Am J Physiol 271, F477–F488.[Medline]

Ligr, M., Madeo, F., Frohlich, E., Hilt, W., Frohlich, K. & Wolf, D. H. (1998). Mammalian Bax triggers apoptotic changes in yeast. FEBS Lett 438, 61–65.[CrossRef][Medline]

Lu, B. C., Gallo, N. & Kijes, U. (2003). White-cap mutants and meiotic apoptosis in the basidiomycete Coprinus cinereus. Fungal Genet Biol 39, 82–93.[CrossRef][Medline]

Ludovico, P., Sousa, M. J., Silva, M. T., Leao, C. & Côrte-Real, M. (2001). Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid. Microbiology 147, 2409–2415.[Abstract/Free Full Text]

Ludovico, P., Rodrigues, F., Almeida, A., Silva, M. T., Barrientos, A., Leao, C. & Côrte-Real, M. (2002). Cytochrome C release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae. Mol Biol Cell 13, 2598–2606.[Abstract/Free Full Text]

Madeo, F., Fröhlich, E., Ligr, M., Grey, M., Sigrist, S. J. & Wolf, D. H. (1999). Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 145, 757–767.[Abstract/Free Full Text]

Madeo, F., Herker, E., Maldener, C. & 8 other authors (2002). A caspase-related protease regulates apoptosis in yeast. Mol Cell 9, 911–917.[Medline]

Martinet, W., van den Plas, D., Raes, H., Reekmans, R. & Contreras, R. (1999). Bax-induced cell death in Pichia pastoris. Biotechnol Lett 21, 821–829.[CrossRef]

Morgenstern, D. E., Gifford, M. A. C., Li, L. L., Doerschuk, C. M. & Dinauer, M. C. (1997). Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response to Aspergillus fumigatus. J Exp Med 185, 207–218.[Abstract/Free Full Text]

Mousavi, S. A. A. & Robson, G. D. (2003). Entry into the stationary phase is associated with a rapid loss of viability and an apoptotic-like phenotype in the opportunistic pathogen Aspergillus fumigatus. Fungal Genet Biol 39, 221–229.[CrossRef][Medline]

Oberhammer, F., Wilson, J. W., Dive, C., Morris, I. D., Hickman, J. A., Wakeling, A. E., Walker, P. R. & Sikorska, M. (1993). Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J 12, 3679–3684.[Abstract]

Okuno, S., Shigeomi, S., Ito, T., Nomura, M., Hamada, E., Ysujimoto, Y. & Matsuda, H. (1998). Bcl-2 prevents caspase-independent cell death. J Biol Chem 273, 34272–34277.[Abstract/Free Full Text]

Patel, R. & Paya, C. V. (1997). Infections in solid-organ transplant recipients. Clin Microbiol Rev 10, 86–124.[Abstract]

Pathak, A., Pien, F. D. & Carvalho, L. (1998). Amphotericin B use in a community hospital, with special emphasis on side effects. Clin Infect Dis 26, 334–338.[Medline]

Patterson, T. F., Kirkpatrick, W. R., White, M., Hiemenz, J. W., Wingard, J. R., Dupont, B., Rinaldi, M. G., Stevens, D. A. & Graybill, J. R. (2000). Invasive aspergillosis. Disease spectrum, treatment practices, and outcomes. Medicine 79, 250–260.[CrossRef][Medline]

Raju, N. B. & Perkins, D. D. (2000). Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora. Fungal Genet Biol 30, 213–221.[CrossRef][Medline]

Reader, U. & Brody, P. (1985). Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1, 17–20.

Roilides, E., Katsifa, H. & Walsh, T. J. (1998). Pulmonary host defences against Aspergillus fumigatus. Res Immunol 149, 454–465.[CrossRef][Medline]

Sanchez, A., Álvarez, A. M., Benito, M. & Fabregat, I. (1997). Cycloheximide prevents apoptosis, reactive oxygen species production, and glutathione depletion induced by transforming growth factor {beta} in fetal rat hepatocytes in primary culture. Hepatology 26, 935–943.[CrossRef][Medline]

Schaffner, A., Davis, C. E., Schaffner, T., Markert, M., Douglas, H. & Braude, A. I. (1986). In vitro susceptibility of fungi to killing by neutrophil granulocytes discriminates between primary pathogenicity and opportunism. J Clin Invest 78, 511–524.[Medline]

Slater, A. F. C., Stefan, C., Nobel, I., Van den Dobbelsteen, D. J. & Orrenius, S. (1995). Signalling mechanisms and oxidative stress in apoptosis. Toxicol Lett 83, 149–153.[CrossRef]

Strasser, A. O'Connor L. & Dixit, V. M. (2000). Apoptosis signalling. Annu Rev Biochem 69, 217–245.[CrossRef][Medline]

Thornberry, N. A. (1998). Caspases: key mediators of apoptosis. Chem Biol 5, 97–103.

Trinci, A. P. J. (1972). Culture turbidity as a measure of mould growth. Trans Br Mycol Soc 58, 467–473.

Trinci, A. P. J. (1983). The effect of Junlon on the morphology of Aspergillus niger and its use in making turbidity measurements of fungal growth. Trans Br Mycol Soc 81, 408–412.

Umar, M. H. & Van Griensven, L. J. L. D. (1997). Morphologic cell death in developing primordia of Agaricus bisporus. Mycologia 89, 274–277.

Verweij, P. E. & Denning, D. W. (1997). Diagnostic and therapeutic strategies for invasive aspergillosis. Respir Crit Care Med 18, 203–215.

Villa, P., Kaufmann, S. H. & Earnshaw, W. C. (1997). Caspases and caspase inhibitors. Trends Biochem Sci 22, 388–393.[CrossRef][Medline]

Vogel, H. J. (1956). A convenient growth medium for Neurospora (medium N). Microb Genet Bull 13, 42–44.

Vogeser, M., Haas, A., Aust, D. & Ruckdeschel, G. (1997). Post-mortem analysis of invasive aspergillosis in a tertiary care hospital. Eur J Clin Microbiol Infect Dis 16, 1–6.[Medline]

Wingard, J. R., Kubilis, P., Lee, L. & 7 other authors (1999). Clinical significance of nephrotoxicity in patients treated with amphotericin B for suspected or proven aspergillosis. Clin Infect Dis 29, 1402–1407.[CrossRef][Medline]

Received 14 October 2003; revised 6 February 2004; accepted 25 February 2004.



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