Susceptibility of Blastomyces dermatitidis conidia to products of oxidative metabolism

EXPERIMENTAL

MYCOLOGY

12, &l-89(1988)

BRIEF NOTE Susceptibility of Blastomyces dermatitidis Conidia to Products of Oxidative Metabolism ALAN M. SUGAR AND KEITH G.FIELD Evans Memorial

Department

of Clinical Research and the Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118

Accepted for publication

November 1, 1987

SUGAR, A. M., AND FIELD, K. G. 1988. Susceptibility of Blastomyces dermatitidis conidia to products of oxidative metabolism. Experimental Mycology 12, 84-89. Conidia obtained from Blastomyces dermatitidis were exposed to H,O,, halide, and peroxidase, alone and in combination, and to hypochlorous acid, in order to determine fungal susceptibility to these compounds. B. dermatitidis strains V (ATCC 26199) and GA-l (ATCC 26197) were used in this study. When exposed to H,O,, conidia from both strains were killed in a dose dependent fashion. LD,,‘s for strains V and GA-l were 7 + 4 and 3 ? 4 mM H,O,, respectively. A study of the kinetics of killing with 2.5 mM H,O,revealed similarities in the rate of killing, except at 30 and 60 minutes, when the GA-l strain was killed more rapidly than the V strain (59% vs 26% and 90% vs 42% dead at 30 and 60 minutes, respectively). All of the GA-1 conidia were killed within 120 minutes and 92 ? 5% of strain V were dead by this time. Exposure of conidia to KI or KC1 demonstrated a lack of toxicity of either halide at concentrations up to 500 JLM. Similarly HZ02 and halide together did not kill more conidia of either strain than did either agent alone. When HZ02, KI, and either myeloperoxidase or horseradish peroxidase were incubated with strain V or GA-l, complete killing was seen at 60 minutes. Hypochlorous acid (IO PM) was markedly fungicidal, killing 100% of both strains in 15 minutes. We conclude that both strains of B. dermatitidis conidia are susceptible to products of the phagocytic cell respiratory burst, but the concentrations of H,O, required are beyond those produced in vivo. However, peroxidase, in concert with H,O, and halide, as well as hypochlorous acid, is fungicidal in physiologically r&VaIH COIW%ItI2fiOI'IS. 0 1988 Academic Pms,Inc. INDEX DESCRIPTORS: Blastomyces dermatitidis; conidia; hydrogen peroxide; peroxidase; oxidative metabolism.

dis conidia, usually first encounter bronchoalveolar macrophages in the mammalian lung (Murphy and Florman, 1983), which is often followed by recruitment of neutrophils into the area (Cohen and Rossi, 1983; Sugar and Field, 1985a). These phagocytic cells have well-defined biochemical mechanisms for the production of potent reactive oxygen intermediates such as O,-, H,O,, and hypohalous acids. We therefore investigated the susceptibility of B. dermatitidis conidia to these compounds in an in vitro cell free system. Two strains of B. dermatitidis were used in these experiments: ATCC 26199 (V), which has been shown to be virulent in the

The infectious particIe responsible for the initiation of blastomycosis is thought to be the conidium (Garrison and Boyd, 1978). However, despite recent advances in our understanding of the host response to infection with Blastomyces dermatitidis, the study of the interaction of host defenses and B. dermatitidis conidia has only recently begun (Drutz and Frey, 1985; Williams and Moser, 1987; Sugar and Field, 1987). The mechanisms to initiate infection and the resolution or progression of inhaled conidia remain an enigma, yet are crucial to our understanding of the early events associated with this infection. Foreign particles, including B. dermatiti84 0147-5975188 $3.00 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

SUSCEPTIBILITY

OF Blastomyces CONIDIA

yeast form in a mouse model of pulmonary blastomycosis, and ATCC 26197 (GA-l), which is avirulent in the yeast form in that mouse model. We have, found that conidia derived from V and GA-l are virulent and avirulent in mice, respectively (unpublished data). The strains were maintained in the yeast form on Sabouraud dextrose agar (Difco Laboratories, Detroit, MI) slants at 4°C under sterile mineral oil. Mycelial cultures were grown in 25cm2 tissue, culture flasks (Corning Glass Works, Corning, NY) on Sabouraud dextrose agarat 22°C in the dark. Subcultures were passaged every 14 days for a maximum of 10 passages. Conidia were harvested from9L.to 25day-old cultures using a modification of the spin bar method employed by Huppert et d.. (1972), as previously described (Sugar a&! Field, 1985b). The conidia were counted! i& a hemacytometer and resuspended; at 2 x lo4 cells/ml. Purity of the conidial suspension was >87%. Viability of-the inoa$&are%provided with each experiment and: general@ exceeded 70%. Unless otherwise noted, al& &e,nr&a&s. were purchased from Sigma Chemi&a& Co.. (St. Louis, MO). Catalase (from bovine liver, 13,000 IJ/mg) was suspended in phos; phate buffer at 2 x 104. U/ml and dialyzed against buffer for 24 h at 4X,. fil?&red, sterilized (0.2~pm Millex-G’V filter, Millipore, Redford, MA), and stored at 4°C until use. On the day of use, the stock solution was diluted 1: 2 in phosphate: buffer an& used at a final concentration of 500 Udml, Catalase was inactivated by boiling for 10 minutes.. Reagent grade H,O, was stored at 4°C i’n the dark and assayed weekly (by absorbance at 230 nm) to determine the exact concentration. Stock solutions of KI and KC1 (50 mM) in phosphate buff= were ste.rilized by filtration and stored at 4°C.. Horseradish peroxidase (HRPOl; Type II from ’ Abbreviations used: HRPO, horseradkh dase; MPO, myeloperoxidase.

pwwi-

85

horse heart, 190-200 U/mg) was dissolved in distilled water at a concentration of 3.8 mg/ml and stored at -20°C. Myeloperoxidase (MPO) was prepared by the method of Agner and obtained from Dr. Robert A. Clark (Iowa City, IA). MPO was stored at -70°C at a concentration of 200 nm as determined by absorbance of reduced M 472 nm (Metcalf et al., 1986). Reagents were added to 96-well, flat bottom, tissue culture plates (Corning) in the following sequence to a final volume of 200 ~1: buffer, peroxidase, catalase, I-I.@,, lides, and conidia. Each variable was ru duplicate. The plates were incubated for various times (as noted for each experiment) at 37°C in a humidified chamber. The reaction was terminated by adding 10 ~1 of catalase to each well. The plates were centrifuged for 15 minutes at lOOOg, 1 of the supernatant was removed, and the wells were washed twice with 150 p,l of Sabouraud dextrose broth. After the addit~Q~ of 150 ~1 of fresh Sabouraud dextrose broth (final volume 200 pl), the plates were incubated at room temperature in the dark up to 72 h in a humidified chamber. E well was examined with an inverted microscope and percentage germination of each microcolony was recorded by counting 100 colonies in each well. In preliminary experiments, cultures were observed for up to 10 days and no change in percentage germination occurred. Thus, we interpret this lack of germination as killing of the fungi, Results are expressed as the mean + standard deviation (SD). Differences between means were compared by Student’s f test using a commercially available statistics program (StatSoft, Tulsa, OK), Significance is defined as P < 0.05. L calculated by the method of Muench (1938). Ourinitial experiments were designed to evaluate the fungicidal activity of reagent H,Oz, since this compound is an integral proximal toxic product of the p~ag~cyt~ re-

86

SUGAR

AND

spiratory burst (Babior, 1978). When incubated in the presence of 250 rnM H202, conidia of both strains were killed completely after 60 minutes (Fig. 1). At a lo-fold lower concentration of H,Oz, however, differential susceptibility to H,O, was evident; strain GA-l was markedly more susceptible than the V strain, 89 -+ 6% vs 66 zt 6% (P < 0.01, GA-l vs V). At lower concentrations of H,O*, no significant differences between strains were found and baseline viability values were obtained at ~0.25 mM &02. Compared to control cultures, significant killing of V and GA-1 strains occurred down to 2.5 mM. Confirmation that the lethal effect was entirely due to H202 was obtained by completely abrogating killing by the addition of catalase to the reaction mixtures (data not shown). LDsO’s of strains V and GA-1 were 7 -+ 4 and 3 _+ 4 n-&f H202. Differences in the rate of lethality of 2.5 mM H20, on the V and GA-l strains were observed (Fig. 2). The GA-l strain was killed more quickly than strain V (P < 0.004 at 30 and 60 minutes), but at 90 and 120 minutes, killing of V (65 -t 23% and 92 -+ 5%) and GA-l (94 -+ 5% and 100 + 0%) were similar. We chose to conduct these

0

0

O+cd 250 [Hydrogen

7.5

23

Peroxide

.25 .025 .0025 mM]

FIG. 1. Susceptibility of Blastomyces dermatitidis conidia to hydrogen peroxide. Conidia were incubated with the indicated concentrations of reagent H,Oz for 60 min and viability was determined by counting germinating conidia following incubations of up to 72 h as detailed in the text. Compared to control, significant killing was observed at 250 and 25 m&I H,O,; P < 0.01. Points represent means 2 SD of three or four individual experiments, with each variable run in duplicate.

FIELD

0

30

60

60

120

Minutes FIG. 2. Kinetics of killing conidia with H,Oz. Conidia were incubated for the indicated times in H,OZ (2.5 m44). Differences between killing of V and GA-1 were significant at 30 and 60 min; P < 0.004. Data represent means 2 SD of three individual experiments, with each variable run in duplicate.

killing kinetic experiments with an approximate LD,, of H,O, (in 1 h), so that some killing could be observed during the course of the experiment and an increase in dead fungi could be demonstrated with a prolongation of the incubation time. Incubation of both V and GA-l conidia in 5000 mM KI resulted in 399% killing of the fungus. At concentrations of 500 to 5 mM, no lethal effects could be observed. Similarly, KC1 at concentrations of up to 5000 PM for 60 minutes did not kill either strain. Addition of KI or KC1 to 2.5 mM H20, did not augment the ability of that concentration of H,O, to kill either strain (data not shown). Experiments were performed with purified human myeloperoxidase and commercially available horseradish peroxidase, to evaluate the role of peroxidase in enhancing the lethal effects of H,Oz and halide. In simultaneous experiments, both sources of peroxidase activity gave equivalent results (Table 1). Therefore, additional experiments used the more readily available horseradish peroxidase. From these experiments, it is apparent that peroxidase enhances the effects of otherwise nontoxic concentrations of H,O, and KI when incubated with conidia. Conidia from both strains of B. dermati-

SUSCEPTIBILITY

OF

Effect of Peroxidase-H,O,KI

Blasfomyces

CONIDIA

7

TABLE 1 on Killing of B. dermatifidis

Conidia

Percentage killed Strain

H,O, + KI c PO” IATCC 26199 (V) I r 0.4 421 4+-o 100 I 4) 22 2 12 100 I 0 ATCC 26197 (GA-l) 22 2 8 21 i 12 -~ a Conidia were incubated with 25 )LM H,O,, 50 pJ4 KI, and MPO (1 U/ml) or HRPO (1 U/ml) for 60 min. Data are means t SD of two individual experiments. Data for MPO and HRPO were combined. Control

H,O, + KI

HA

tidis were killed after 1.5 minutes of incubation at concentrations of 0.1 to 1000 pJ4 NaOCl (Table 2). No significant differences in the degree of killing between the two strains were observed. Fungicidal activity of OCH- was completely abrogated by adding sodium thiosulfate, a scavenger of hypochlorite ions, to the reaction mixture (data not shown). The kinetics of killing of conidia by OClwere then examined (Table 3). When conidia were incubated with 1 pM NaOCl, killing of both strains was observed as early as 5 minutes, although statistical significance was not achieved until after 1.5 minutes of incubation. By 30 minutes >90% of conidia of both strains were dead (P < 0.005, both strains compared to control). No differences in susceptibility to this oxidant were seen between the two fungal strains at any of the times studied. The susceptibility of B. dermatitidis conidia to products of the respiratory burst of mammalian phagocytes is of interest because of the demonstrated importance of oxidative metabolites in the ability of host effector cells to kill invading microorgan-

isms (Babior, 1978, 1984; Klebanoff, ~98~). Demonstration of lethal effects of components of the peroxidase-H,O,-halide system in in vitro systems, thus, is an initial step in defining potential avenues of research into the subject of host defense mechanisms operating in blastomycosis. The data presented in this paper support the potential efficacy of oxidative metabolites derived from macrophages or neutrophils in killing B. dermatitidis conidia, The susceptibility of the conidia to reagent H,Oz can be compared to that previously reported for yeast forms derived from the same strains of B. dermatitidis used in this study (Sugar et al., 1983). The yeast forms exhibited dose dependent H,O, susceptibility that correlated with vir~le~~~ of the fungus in a mouse pneumonia model (V < GA-l), with LD50’~ >50 and 3 m spectively. This compares to the de susceptibility of the conidia to < GA-I. Corresponding LD,,‘s GA-l conidia were determined i to be 7 and 3 m&I H,O,, respectively. Interestingly, the yeast form of the strain was the most resistant to H

TABLE 2 Killing of Conidia By NaOCl [NaOCl] Strain

0

1000

10

I

0.1

ATCC 26199 (V) ATCC 26197 (GA-l)

20 +- 8 32 -1-5

100 T 0.4 100 i 0

1ooi 1 100 t 6)

68 2 12 19 f 5

23 + 13 40

Q.QE 21 x9 31

a Concentration in micromolars; data are means -+ SD of two to four individual experiments except for 0.1 and 0.01 p,M NaOCl with GA-l. Incubations were for 15 min.

88

SUGAR

AND

FIELD

TABLE 3 Kinetics of Killing Blastomyces dermatitidis

Conidia by NaOCl”

Strain

Control

1 min

5 min

15 min

30 min

ATCC 26199 (V) ATCC 26197 (GA-l)

22 + 1 21 r 1

24 + 15 492 19

42 + 13 65 2 18

78 + 27 77 t 7

95 k 7 97 I- 5

a Conidia were incubated in 1 &4 NaOCl for the designated intervals. Data are expressed as means + SD of two or three individual experiments.

there were only slight differences noted between conidia. The relationship, if any, between susceptibility to H202 and virulence of the organism remains to be established. Furthermore, physiologically relevant concentrations of H,O, are on the order of <0.025 mM (Nathan et al., 1979), thus raising the question of whether H,02 is an important fungicidal agent by itself in viva. However, concentrations of H,O, present in the small microsite spaces between effector cells and their targets have not been measured and the precise concentration may be, in fact, higher than calculations using cell populations would suggest. Similar to the situation we previously described with yeast forms of these same strains of B. dermatitidis (Sugar et al., 1983), addition of peroxidase to nonlethal concentrations of H,O, and iodide resulted in rapid and significant killing of both strains of B. dermatitidis conidia, with no relationship to the virulence of the strain. In summary, we have studied the susceptibility of two strains of B. dermatitidis conidia to products of the phagocytic cell respiratory burst. The results indicate that conidia are killed by supraphysiologic concentrations of H,O,, but that a peroxidasemediated reaction is required for killing of this morphologic form of B. dermatitidis at physiologically relevant concentrations of H,O,. Since it is currently unknown whether bronchoalveolar macrophages or neutrophils are the first cells encountered by the organism once it is inhaled into the lungs, final interpretation of the significance of the data presented in this report awaits completion of the analysis of cell

types present nidia.

following

inhalation

of co-

ACKNOWLEDGMENT

This work AI22051-OlAl.

was supported

by NIH

Grant

No.

REFERENCES

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SUSCEPTIBILITY

OF Blnstomyces CONLDIA

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