Virulence of Sporothrix schenckii conidia and yeast cells, and their susceptibility to nitric oxide

Abstract

The involvement of nitric oxide (NO) in macrophage (Mφ) fungicidal activity against Sporothrix schenckii, and the relationship between NO susceptibility and the differential virulence of conidia and yeast cells, were investigated. Confirming a previously reported correlation between the length of time in culture and virulence of S. schenckii, conidia isolated from 12-day mycelial cultures (Ss-12) were less virulent to mice than conidia from 7-day cultures (Ss-7) or yeast cells. Indicative of NO production, infected animals showed a significant increase in serum levels of nitrite that was lower in mice infected with Ss-12 than in mice infected with Ss-7 or yeast. Stimulation of murine Mφ with interferon-γ (IFN-γ) induced NO production and inhibition of fungal growth. The cytotoxic activity of Mφ against Ss-12 was significantly greater than against Ss-7 or yeast cells, the highly virulent fungal forms. The addition of NO synthase inhibitors abrogated Mφ cytotoxic activity against all fungal forms. The phagocytic activity of Mφ against Ss-7 was significantly lower than against Ss-12 or yeast cells. Although the ingestion of fungal cells triggered the oxidative burst in Mφ, the fungicidal activity was not altered in the presence of superoxide dismutase (SOD) and catalase. In addition, Ss-12 and yeast cells were more susceptible than Ss-7 to the direct fungicidal activity of the NO donors S-nitroso-N-acetyl-dl-penicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholinosydnonimine (SIN-1). The results of this study indicate that NO is a key cytotoxic mediator involved in the murine Mφ defence against S. schenckii, and that the virulence of Ss-7, Ss-12 and yeast cells may be related to a differential susceptibility to NO.

Introduction

Sporothrix schenckii, the aetiological agent of sporotrichosis, is a dimorphic fungus widely distributed in nature, presenting a saprophytic mycelial form on plant debris and soil.¹ The traumatic inoculation of conidia and hyphae of S. schenckii leads to this subcutaneous mycosis and within the infected tissue the fungus differentiates to the yeast form and may spread to other tissues.² The systemic form of sporotrichosis may evolve from an initial cutaneous lesion or be associated with inhalation of conidia.³ Recently, more severe clinical forms of this disease have been associated with immunocompromised patients, such as human immunodeficiency virus (HIV)-infected patients, suggesting that S. schenckii is an emerging opportunistic pathogen.^4–6

In a new murine model of sporotrichosis, using a single strain of S. schenckii, it was demonstrated that the virulence of the infective conidia was related to the length of time in culture, which correlates with a variation of the carbohydrate composition of the fungus cell wall. Thus, conidia harvested from mycelial cultures after 12 days (Ss-12) were less virulent to mice than conidia cultured for 7 days (Ss-7). The difference in the virulence of these conidia correlated with the rhamnose/mannose ratio in the cell wall of each cell type.⁷

Although the host defence mechanisms against S. schenckii are not fully understood, previous studies have suggested that cell-mediated immunity plays an important role in the protection of the host against this fungus.⁸ Tachibana et al. have recently demonstrated that acquired immunity against S. schenckii is mainly expressed by T-cell-activated macrophages (Mφ).⁹

It has been extensively reported that nitric oxide (NO) produced by activated Mφ is a primary defence mechanism against many pathogens¹⁰ For instance, Penicillium marneffei,¹¹ Histoplasma capsulatum,¹² Cryptococcus neoformans,¹³ and Candida albicans¹⁴ are susceptible to NO. In addition, differences in the degree of Mφ-mediated cytotoxicity between the yeast and the hyphal forms of dimorphic fungi, such as C. albicans¹⁴ and P. marneffei,¹¹ have also been described.

The present work had three main objectives: to investigate whether Ss-7, Ss-12 and yeast cells of S. schenckii displayed different sensitivities to Mφ-derived NO; to assess whether NO donors were able to kill the fungus; and to investigate the other oxidative mechanisms involved in the fungicidal activity of Mφ. In addition, we sought to correlate these data with the differences in the virulence of the conidia and yeast cells of S. schenckii.

Materials and methods

Culture medium and reagents

RPMI-1640 medium, Hanks' balanced salt solution (HBSS), HEPES, streptomycin, penicillin, lipopolysaccharide (LPS; Escherichia coli serotype 026:B6), N^w-mono-methyl-l-arginine (l-NMMA), l-arginine (l-arg), aminoguanidine (AMG), polyethylene glycol superoxide dismutase from bovine erythrocytes (PEG-SOD; 4000 U/mg of protein), catalase (from bovine liver, thymol-free, 10 000–25 000 U/mg of protein), dihydrorodhamine 123 (DHR) and poly (2-hydroxyethyl metacrylate) (Poly HEMA) were purchased from Sigma Chemical Co. (St Louis, MO). Brain heart infusion (BHI) broth and agar were purchased from Oxoid Reagents (Basingstoke, Hampshire, UK). Recombinant murine interferon gamma (IFN-γ) was obtained from Genzyme (San Diego, CA). S-nitroso-N-acetyl-dl-penicillamine (SNAP) and its non-nitrosylated parent molecule N-acetyl-dl-penicillamine (SAP), GSNO and 3-morpholinosydnonimine (SIN-1) were a kind gift of Dr Jamil Assreuy (Departamento de Farmacologia, Universidade Federal de Santa Catarina, Brazil).

Animals

Male C57Bl/6 mice (6–8 weeks old) were obtained from Oswaldo Cruz Foundation (Fiocruz, Rio de Janeiro, Brazil). Mice were housed in controlled temperature rooms at 23–25°, with ad libitum access to food and water during all experiments. All procedures were approved by our Institutional Ethics Committee and are in accordance with National Institutes of Health Animal Care Guidelines.

Fungus

S. schenckii strain 1099-18 (Mycology Section of the Department of Dermatology, Columbia University, New York, NY) was used throughout this study. The different forms of the fungus were obtained as described previously.⁷ Briefly, the mycelial phase of S. schenckii was grown in Sabouraud dextrose broth under mild agitation at 25°, for 7 or 12 days. Conidia were separated from hyphae by gauze filtration and designated as Ss-7 or Ss-12, corresponding to the length of time in culture, 7 or 12 days, respectively. Yeast cells were cultured in BHI broth for 7 days at 37° under orbital agitation (150 r.p.m.). The cells harvested from the culture medium were > 95% differentiated into the yeast-like form. For the experiments, each cell type was washed three times with HBSS, pH 7·4, followed by determination of cell number in a Neubauer chamber. The viability of each cell sample was ascertained by determining the number of colony-forming units (CFU) after 5 days of incubation on BHI agar plates at 37°.

Animal infection and mortality

For the mortality assay, C57Bl/6 mice were anaesthetized and inoculated intravenously (i.v.) with 5 × 10⁶ fungal cells suspended in 0·2 ml of sterile phosphate-buffered saline (PBS), as previously described.⁷ Control groups were injected with the same volume of vehicle. On day 14 postinfection, some animals were bled by cardiac puncture for serum nitrate measurement. Infected mice were observed for up to 30 days and survivors beyond this day were killed by cervical dislocation.

Mφ cultures

Peritoneal Mφ were obtained from C57Bl/6 mice injected intraperitoneally (i.p.) with 1 ml of sterile thioglycollate (3% w/v). After 4 days, peritoneal cells were harvested, centrifuged (500 g, 10 min) and resuspended in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and 10 mm HEPES. Total and differential counts were performed as described previously and the cell number was adjusted to 10⁶ Mφ/ml.¹⁵ Cell viability was ascertained by Trypan Blue exclusion and was routinely > 95%.

Mφ activation and fungicidal activity

Peritoneal Mφ (5 × 10⁵ cells) were plated on 24-well plates and incubated for 2 hr at 37° in an atmosphere of 5% CO₂. Non-adherent cells were removed by two washes with prewarmed medium. Adherent Mφ were activated with IFN-γ (20 U/ml) plus LPS (20 ng/ml) in the presence or absence of l-NMMA (200 µm), AMG (200 µm) and/or l-Arg (2 mm). Four hours later, cultures were infected with 5 × 10⁴ conidia (Ss-7 or Ss-12) or yeast cells. In some cultures, PEG-SOD (130 U/ml) and catalase (50 µg/ml) were added to the cultures 1 hr before infection. Routinely, some wells were filled with medium alone and inoculated with the fungus as a control for fungal growth. All treatments were performed in triplicate. After adding the fungi, cultures were incubated for 18 hr at 37° in an atmosphere of 5% CO₂. Plates were then centrifuged (500 g for 10 min) and cell-free supernatants were collected and stored at −20° for nitrite determination. Cells were then lysed with 1 ml of sterile distilled water. Each well was scraped and the cell lysate was serially diluted with sterile HBSS, pH 7·4, to determine fungal viability. For this, aliquots in duplicate were plated on BHI agar containing streptomycin (100 µg/ml) and penicillin (100 U/ml), and CFU counts were performed after 5 days of culture at 37°. Mφ fungicidal activity was evaluated as the percentage of killing, calculated as:

Assays in a cell-free system

The direct fungicidal activity of NO against S. schenckii was evaluated using two NO donors, SNAP and GSNO (0·1, 0·3 and 1 mm), and the peroxynitrite donor SIN-1 (0·1 mm), added directly to fungal cultures. Conidia or yeast cells (5 × 10⁴ cells/0·5 ml) were incubated in RPMI-1640 for 18 hr at 37° in the presence of the compounds. The fungal viability was evaluated as the percentage of killing, using the calculation:

Phagocytosis assay

Suspensions of Ss-7, Ss-12 or yeast cells were placed onto slides precoated with a monolayer of adherent peritoneal Mφ (activated or not activated with IFN-γ and LPS) at a ratio of 10 : 1 (target : effector). The cultures were incubated at 37° in an atmosphere of 5% CO₂ for 2 hr. Phagocytosis was stopped by cooling. Cultures were washed three times with cold RPMI-1640 to remove non-ingested fungi. Cells were then fixed with methanol : acetone (1 : 1; v/v) for 10 min, stained with May–Grunwald–Giemsa and examined using an optical microscope (× 100 objective). The percentage of phagocytic Mφ (PM) and the average number of ingested fungal cells per Mφ (F) were determined after counting a total of 200 cells. Phagocytic index (I) was calculated by using the formula:

Evaluation of intracellular oxidative burst by flow cytometry analysis

The ability of fungal cells to trigger an oxidative burst in Mφ was assessed by the intracellular oxidation of DHR.¹⁶ Freshly harvested peritoneal Mφ (1 × 10⁶ cells/ml) were plated onto 24-well plates precoated with poly HEMA in order to prevent Mφ adherence. Cells were incubated with DHR (10 µm) for 1 hr at 37° in the presence of medium alone, phorbol ester (phorbol 12-myristate 13-acetate [PMA]; 30 nm) or fungal cells, at a ratio of 10 : 1 (target : effector). Plates were cooled and flow cytometry analysis was performed for evaluating cell viability and the increase in DHR intracellular fluorescence.¹⁶ Using a fluorescence-activated cell sorter (FACScalibur; Becton- Dickinson, Franklin Lakes, NJ), cell gating was performed based on the forward and side (FSC/SSC) light scattering. Mφ were identified by their forward- and right-angle scattering properties. Viability was determined as the percent of propidium iodide (PI)-negative events. Viable cells were > 95% per group. The green fluorescence related to oxidized DHR of viable cells was collected through a 530-band pass filter. Data acquired in a mode of 10 000 gated events were analysed using the cellquest software (Becton-Dickinson) and were expressed as median of fluorescence.

Serum nitrate and nitrite measurement

The concentrations of nitrite (NO₂^–), a stable metabolite of NO, was determined in the culture supernatants by using the Griess method, as previously described¹⁷ and results are expressed as µm of . In serum, levels of nitrate + nitrite (NO_x) were determined as previously described.¹⁸ Briefly, serum was deproteinized by zinc sulphate and diluted 1 : 1 with water. Nitrate was converted to nitrite using E. coli nitrate reductase for 2 hr at 37°. Samples were centrifuged and 100 µl of each supernatant was mixed with Griess reagent in a 96-well plate and read at 540 nm in a plate reader. Standard curves of nitrite and nitrate (0–150 µm) were run simultaneously. As under these conditions nitrate conversion was always greater than 90%, no corrections were made. Values are expressed as µm NO_x (nitrate + nitrite).

Statistical analysis

The Wilcoxon rank sum statistical test was used for comparing differences in the mortality rates between groups; P < 0·05 was considered significant. For the remaining data, statistical analysis was assessed by analysis of variance (anova) followed by the Student's t-test; P < 0·05 was considered as statistically significant.

Results

Differential virulence of S. schenckii conidia and yeast cells to C57Bl/6 mice and serum nitrate levels in infected animals

Similarly to what has been observed previously for BALB/c and Swiss mice,⁷ the severity of infection induced by S. schenckii in C57Bl/6 mice depends upon the type of fungal form inoculated. Figure 1 shows that animals inoculated with Ss-7 conidia or yeast cells succumbed to the infection and began to die 14 days after infection. The profile of the mortality rate for both groups was similar, with only 25% still alive on day 30 postinfection. In contrast, 100% of mice infected with Ss-12 conidia survived for up to 30 days postinfection (Fig. 1). It is noteworthy that differences in the virulence were not related to a possible loss in the viability of fungal cells, as the CFU counts showed 95–100% viability. Table 1 shows that infected animals exhibited a significant increase in serum NO_x levels when compared with uninfected animals. In addition, mice infected with Ss-12 conidia displayed lower NO_x levels than those infected with Ss-7 conidia or yeast cells.

Figure 1.

Survival of C57Bl/6 mice infected with Sporothrix schenckii. Animals were injected intravenously (i.v.) with 5 × 10⁶ conidia isolated from 7-day (○) or 12-day (▵) mycelial cultures, or with yeast cells (□). Control animals were injected with phosphate-buffered saline (PBS) (•). The mortality rate was followed-up for 30 days after infection (n = 30/group). *Significant difference (P < 0·05) compared with uninfected mice.

Table 1.

Serum nitrate + nitrite (NO_x) levels^* in mice infected with conidia isolated from 7-day (Ss-7) or 12-day (Ss-12) mycelial cultures or with yeast cells of S. schenckii

Uninfected	Ss-7	Ss-12	Yeast cells
26·3 ± 2·7	95·4 ± 6†‡	80 ± 4†	106·1 ± 3†‡

^*Data are presented as [NO_x] (µm). NO_x levels were assayed on day 14 postinfection, n = 5.

^†P < 0·05 compared with uninfected mice.

^‡P < 0·05 compared with Ss-12-infected mice.

Fungicidal activity of IFN-γ/LPS-activated Mφ

The ability of activated Mφ to kill the different cell phases of S. schenckii is shown in Fig. 2. In general, accumulation of nitrite correlated with Mφ fungicidal activity. However, the Mφ fungicidal activity varied with the fungal cell form. Killing activity towards Ss-12 conidia (60%) was significantly greater than that towards Ss-7 conidia (37%) or the yeast form (44%) (Fig. 2). The killing activity of Mφ can be attributed to NO as its production by Mφ was strongly inhibited by the NOS inhibitor, l-NMMA, where a complete inhibition of the killing activity against the three fungal cell forms was observed (Fig. 2). Addition of l-Arg to the medium reversed the inhibitory effect of l-NMMA on nitrite production and restored the fungicidal activity (data not shown). Non-activated Mφ produced virtually no nitrite and did not kill any type of fungal cell. Importantly, addition of fungal cells to non-activated Mφ did not induce the production of NO and, in fact, these cells were themselves a good substrate for S. schenckii growth, as observed by the fact that all fungal forms grew 50% more effectively in the presence of non-activated Mφ when compared to growth observed with medium alone (data not shown).

Figure 2.

Production of nitric oxide (NO) and fungicidal activity of activated macrophages (Mφ). Mice peritoneal Mφ were activated with interferon-γ (IFN-γ) (20 U/ml) and lipopolysaccharide (LPS) (20 ng/ml) for 4 hr before addition of conidia isolated from 7-day (Ss-7) or 12-day (Ss-12) mycelial cultures, or yeast cells of Sporothrix scheneckii. In some experiments N^w-mono-methyl-l-arginine (l-NMMA) (0·2 mm) was added to the cultures. The percentage of killing (left panels), as evaluated by CFU counts, and nitrite concentration in the supernatants (right panels) were determined after 18 hr. The values are expressed as mean ± SD from triplicate determinations of a representative experiment. *Significant difference (P < 0·05) compared with Ss-12 conidia. †Significant difference compared with the addition of l-NMMA.

Phagocytosis of S. schenckii by Mφ

Table 2 shows that activation of mice peritoneal Mφ with IFN-γ/LPS did not significantly alter their ability to phagocytose any morphological phase of the fungus when compared with non-stimulated Mφ. However, the phagocytic index of both activated and non-activated Mφ towards Ss-7 conidia was significantly lower than to Ss-12 conidia and yeast cells.

Table 2.

Phagocytic index^* of mice peritoneal macrophages (Mφ) against Sporothrix schenckii

Fungal form	Non-stimulated Mφ	Activated† Mφ
Ss-7	15 ± 3‡	16 ± 3‡
Ss-12	40 ± 2	45 ± 5
Ss-Y	35 ± 3	43 ± 4

^*The phagocytic index was calculated as described in the Materials and methods.

^†Mφ were activated with interferon-γ (IFN-γ) (20 U/ml) plus lipopolysaccharide (LPS) (20 ng/ml).

^‡P < 0·05 in relation to the phagocytic index of Ss-12.

Ss-7, conidia isolated from 7-day mycelial cultures of S. schencki; Ss-12, conidia isolated from 12-day mycelial cultures of S. schencki; Ss-Y, yeast cells of S. schencki.

Mφ oxidative burst

The ability of S. schenckii to trigger an oxidative burst following ingestion by Mφ was investigated by flow cytometry using DHR as a fluorescent probe.¹⁶ Fig. 3(a) shows representative histograms from one experiment, illustrating the variety of Mφ intracellular fluorescence after incubation with fungal cells. A significant increase in DHR fluorescence was clearly observed in the presence of the conidia and yeast cells of S. schenckii, indicating that they triggered the oxidative burst in Mφ with an intensity comparable to that induced by PMA (Fig. 3b).

Figure 3.

Oxidative burst in macrophages (Mφ) induced by Sporothrix schenckii. Intracellular oxidation of the dihydrorodhamine 123 (DHR) fluorescent probe was assayed in Mφ by flow cytometry 60 min after interaction with 7-day (Ss-7) or 12-day (Ss-12) conidia, or with yeast cells. Control: Mφ in the absence of fungal cells. Mφ stimulated with phorbol 12-myristate 13-acetate (PMA) alone were used as positive controls. (a) Shows, in histograms, the logarithmic displays of increasing fluorescence (x-axis) versus cell number (y-axis) from one typical experiment. From the frequency curves, the median fluorescence was calculated for each group and is shown as bars in (b). The layout of each bar/group in (b) was shared in the histograms for each group in (a).

Effects of SOD, catalase and AMG on the Mφ fungicidal activity

The role of superoxide and hydrogen peroxide in the fungicidal activity of IFN-γ/LPS-activated Mφ was investigated. Table 3 shows that treatment with SOD (a superoxide scavenger) or catalase (a hydrogen peroxide scavenger) did not alter the fungicidal activity of activated Mφ. Moreover, neither SOD nor catalase modified the differential cytotoxicity towards Ss-7, Ss-12 or yeast cells. On the other hand, AMG, a selective inhibitor of inducible nitric oxide synthase (iNOS)¹⁹ abolished the Mφ killing effect, confirming the important role of NO in the fungicidal activity.

Table 3.

Effect of treatment with superoxide dismutase (SOD), catalase or aminoguanidine (AMG) on the nitrite release and fungicidal activity of activated macrophages (Mφ)

	Fungal form
	Ss-7		Ss-12		Ss-Y
Treatment	Nitrite (µm)	% Killing	Nitrite (µm)	% Killing	Nitrite (µm)	% Killing
Activated Mφ*	44·5 ± 1	15 ± 4†	53·4 ± 2	30·8 ± 5	50·5 ± 6	17 ± 3†
+ SOD	42·2 ± 1	16 ± 3†	51·5 ± 0	27·7 ± 4	38·3 ± 10	17 ± 4†
+ Catalase	41·6 ± 1	18 ± 2†	45·8 ± 1	30·5 ± 3	42·9 ± 3	18 ± 3†
+ AMG	7·3 ± 0‡	0 ± 2‡	6·4 ± 1‡	0 ± 2‡	6·0 ± 0‡	0 ± 1‡

^*Mφ were activated with interferon-γ (IFN-γ) (20 U/ml) plus lipopolysaccharide (LPS) (20 ng/ml).

^†P < 0·05 compared with Ss-12.

^‡P < 0·05 compared with values in the absence of AMG.

Ss-7, conidia isolated from 7-day mycelial cultures of Sporothrix schencki; Ss-12, conidia isolated from 12-day mycelial cultures of S. schencki; Ss-Y, yeast cells of S. schencki.

Inhibition of S. schenckii growth by NO donors

In order to provide evidence of a direct deleterious effect of NO on S. schenckii cultures, the three different fungal forms were incubated with either of SNAP or GSNO, two NO donors. Table 4 shows that both compounds decreased the survival of the fungus in a concentration-dependent manner. Ss-12 conidia and yeast cells were more susceptible than Ss-7 to the fungicidal activity of SNAP or GSNO. The non-nitrosylated parent molecule of SNAP (SAP) did not affect fungal growth (data not shown). We also investigated whether peroxynitrite could kill fungal cells. For this, cells were incubated with SIN-1, a compound that releases NO and superoxide simultaneously and is thought to be a peroxynitrite donor.²⁰ SIN-1 strongly inhibited the growth of Ss-12 conidia but showed a lesser inhibitory effect on the growth of either Ss-7 or yeast cells. Addition of SOD together with SIN-1 greatly augmented the fungicidal activity against all fungal forms (Table 4).

Table 4.

Effect of nitric oxide (NO) donors on the growth of Sporothrix schenckii

	% Of killing
Treatment	Ss-7	Ss-12	Ss-Y
SNAP
0·1 mm	3·5 ± 3	10 ± 7	15 ± 4
0·3 mm	8·8 ± 3*	46 ± 5‡	40 ± 11‡
1·0 mm	41·0 ± 15*‡	68 ± 7‡	62 ± 12‡
GSNO
0·1 mm	13 ± 1	2 ± 2	9 ± 3
0·3 mm	15 ± 2*	32 ± 2‡	30 ± 0‡
1·0 mm	20 ± 8*‡	75 ± 6‡	74 ± 7‡
SIN-1 (0·1 mm)	14 ± 8*	68 ± 13‡	20 ± 4*‡
+ SOD	60 ± 16†‡	100 ± 0†‡	68 ± 12†‡

^*P < 0·05 compared with Ss-12.

^†P < 0·05 compared to cells treated with SIN-1 in the absence of superoxide dismutase (SOD).

^‡P < 0·05 compared with untreated fungal cells.

Ss-7, conidia isolated from 7-day mycelial cultures of S. schencki; Ss-12, conidia isolated from 12-day mycelial cultures of S. schencki; Ss-Y, yeast cells of S. schencki.

GSNO, S-nitrosoglutathione; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acetyl-dl-penicillamine.

Discussion

It was recently reported that BALB/c and Swiss mice inoculated with Ss-7 conidia developed an acute systemic disease with a high mortality.⁷ On the other hand, animals inoculated with Ss-12 conidia developed a less severe disease with a high survival rate. This difference in the virulence between the two types of conidia is also seen in the present study using C57Bl/6 mice. Moreover, it was also found that the high mortality rate of mice inoculated with yeast cells, the main infective fungal form in vivo, was similar to that obtained with Ss-7 conidia. In previous work it was suggested that the differences in the virulence correlate to the rhamnose : mannose ratio in the cell wall of both types of conidia.⁷ Interestingly, the cell wall of the more virulent fungal forms (Ss-7 and yeast cells), present a high rhamnose and a low mannose content when compared with Ss-12, the less virulent conidia.²¹ Hence, data shown here support the suggestion that the differential virulence of S. schenckii morphological phases may be associated with differences in the cell wall composition.

A significant increase in NO_x serum levels was evident on day 14 postinfection for all infected groups, suggesting that NO is indeed produced by the infection in vivo. Interestingly, there appeared to be a relationship between virulence and serum NO_x levels, as demonstrated by the fact that NO_x levels were 20% lower in the less virulent conidia than in the more virulent forms.

Several authors have shown that plasma nitrate levels reflect the production detected in different tissues (such as spleen, liver and heart) and leucocytes,²² and NO production is induced by different cytokines.^18,22

It was recently reported that the expression of acquired protective immunity against S. schenckii in mice is dependent on both Mφ and CD4⁺ T cells. In addition, an essential role was demonstrated for IFN-γ produced by T cells from infected animals in the fungicidal activity of Mφ.⁹

In the present report we show that differences in the virulence of these two types of conidia and yeast cells from a single strain of S. schenckii could be ascribed to a differential susceptibility to NO produced by activated Mφ. Peritoneal Mφ activated in vitro with IFN-γ/LPS and producing high levels of NO were able to efficiently kill S. schenckii. However, the susceptibility to the microbicidal effect of activated Mφ varied among the three types of fungal cells. Whereas the growth of Ss-12 (the less virulent form) was strongly inhibited, the growth of Ss-7 and yeast cells (the more virulent forms) was only modestly affected. The role played by NO in the fungicidal activity of activated Mφ was confirmed by showing that AMG or l-NMMA inhibited NO production and completely abrogated the killing of all fungal forms.

Differences in the susceptibility to a NO-dependent microbicidal effect have already been described for other fungi. Indeed, the relationship between the resistance to NO and virulence have been frequently described for different strains of the same pathogen.^23,24 More recently, it was demonstrated that yeast form of P. marneffei was more sensitive to fungicidal activity of activated Mφ than conidia.^11,25 The authors attributed these results to the fact that P. marneffei yeast cells were able to enhance NO production by activated Mφ. This explanation does not support our data, as conidia or yeast cells of S. schenckii did not seem to induce iNOS or increase NO production by peritoneal Mφ.

Another important finding of this report is that there was a significant difference in the phagocytic index between the low and highly virulent S. schenckii conidia. The ability of Mφ to ingest Ss-12 conidia was significantly greater than their ability to ingest Ss-7 cells. Therefore, it is conceivable that the differential virulence of both conidia could be ascribed to this differential ability of Mφ to phagocytize them. In spite of the different phagocytosis index, all three fungal cell types were able to trigger the oxidative burst of Mφ. However, the cytotoxicity of activated Mφ against S. schenckii was not altered in the presence of SOD or catalase, scavengers of superoxide anion and hydrogen peroxide_, respectively. These findings suggest that the production of reactive oxygen species is probably not one of the main determinants of differences in virulence among these fungal forms. The mechanism of evasion for S. schenckii from oxygen reactive intermediates generated by phagocytes has been described previously. Sgarbi et al.²⁶ showed that ergosterol molecules from the surface of yeast cells are able to react with hydrogen peroxide, producing ergosterol peroxide which acts as a protective mechanism.

Our results show that NO kills S. schenckii. When NO donors were added to S. schenckii culture in the absence of Mφ, it was observed that Ss-12 (the less virulent conidia) were more susceptible than Ss-7 (the more virulent conidia). Therefore, Ss-7 are phagocytosed to a lesser extent and are more resistant to NO (either from Mφ or from NO donors). Taken together, these two characteristics of Ss-7 could explain why they cause severe disease in comparison to Ss-12 cells. Curiously, we could not find a comparable relationship among virulence in vivo, phagocytosis and resistance to the NO cytotoxic effect for yeast cells. Yeast cells were less susceptible than Ss-12 conidia to NO produced by activated Mφ but, similarly to Ss-12, they were efficiently phagocytosed and were highly sensitive to NO derived from NO donors. These data suggest that yeast cells may also have intracellular evasion mechanisms for protection against the deleterious effect of NO produced by activated Mφ.

Several reports have suggested that the cytotoxic effects of NO can be ascribed to the production of peroxynitrite following the reaction of NO with superoxide anion.²⁷ Our results show that SIN-1, a peroxynitrite donor, killed S. schenckii in a Mφ-free system. However, the addition of SOD increased the fungicidal effect of SIN-1, suggesting that peroxynitrite does not contribute to the NO-induced killing of S. schenckii. Similar findings have been described for other pathogens.^28,29

In summary, our data show that NO is a key cytotoxic mediator involved in the murine Mφ defence against S. schenckii. The differential susceptibility of Ss-7 and Ss-12 conidia to the cytotoxic effect of Mφ seems to depend not only on a differential phagocytosis but also on their differential resistance to the effects of NO. In addition, yeast cells, which are the infective form of S. schenckii, seem to have intracellular evasion mechanisms that are able to protect them against the toxic effects of both oxygen and nitrogen free radicals generated by activated Mφ. Taken together, our data suggest that the resistance of conidia and yeast cells of S. schenckii to the cytotoxic mechanisms of Mφ can explain, at least in part, their differential in vivo virulence.