Vol. 61, No. 10

INFECTION AND IMMUNITY, Oct. 1993, p. 4099-4104

0019-9567/93/104099-06$02.00/0 Copyright © 1993, American Society for Microbiology

Isolation and Characterization of a Secreted Metalloprotease of Aspergillus fumigatus MONOD,l* S. PARIS,2 D. SANGLARD,3 K. JATON-OGAY,4 J. BILLE,4 AND J. P. LATGE'2 Service de Dermatologiel and Institut de Microbiologie, 4 Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, and Institut fir Biotechnologie ETH-Honggerberg, 8093 Zurich,3 Switzerland, and Unite de MFycologie, Institut Pasteur, 70015 Paris, France2 M.

Received 26 March 1993/Returned for modification 23 May 1993/Accepted 7 July 1993

A metalloprotease (MEP) secreted by Aspergilusfumigatus was isolated from an alkaline protease-deficient

Several species of Aspergillus, in particular, Aspergillus

fiumigatus and A. flavus, have been implicated as the caus-

ative agents of a number of human and animal diseases (1, 8, 27). They are considered opportunistic organisms which can establish and develop inside their host only under a variety of underlying conditions, including impaired immune status. After inhalation of spores, clinical aspergillosis can develop in three major presentations. The first presentation is allergic bronchopulmonary aspergillosis, which occurs when Aspergillus sp. colonizes the bronchial tree and releases antigenic substances that cause a hypersensitivity pneumonitis. The second is aspergilloma (fungus ball), which generally develops in pulmonary cavities secondary to other lung diseases such as tuberculosis and anatomic defects. The last form is invasive pulmonary or disseminated aspergillosis, which is a life-threatening infection in severely immunocompromised patients such as neutropenic patients and transplant recipients. Proteolytic degradation of the lung tissue has been suggested as one of the key events involved in the physiopathology of A. fumigatus (7, 23). An extracellular serine protease of the subtilisin family (ALP) has been isolated from clinical isolates of A. fumigatus (5, 12, 16). Its high collagenolytic activity and ability to digest elastin favored a possible role in the invasion of the tissues by the fungus. Furthermore, this enzyme could be detected in hyphae of A. fumigatus in infected murine and human lungs (13, 16). Recently, we cloned the gene encoding ALP (6), and in order to determine whether the secreted protease plays a key role in the virulence of A. fumigatus, we disrupted the protease gene and assayed ALP-nonproducing strains in mice (11). Surprisingly, in the immunocompromised mouse model we studied, no difference in pathogenicity was observed between ALP-producing and ALP-nonproducing isogenic strains. The rate of mortality and invasion of lung tissues by mycelia of the two strains were similar. It was suggested that the presence of a significant residual proteolytic activity, not *

due to ALP, was sufficient to allow mycelial development on protein substrates in vitro and in vivo (11). In this report, we describe the isolation and characterization of a major metalloprotease (MEP) secreted byA. fiumigatus responsible for the residual proteolytic activity of the ALP-deficient mutants.

MATERIALS AND METHODS Materials. Malt agar was purchased from Oxoid (Basingstoke, Bucks, United Kingdom), and soluble collagen was from Serva (Heidelberg, Germany). Azocollagen and elastinCongo red were from Sigma (St. Louis, Mo.). Hydroxylapatite (Bio-Gel HTP), polyacrylamide P60 gel, and lowmolecular-weight protein standards were from Bio-Rad. Carboxymethyl (CM)-Sephadex, ampholytes of the pH range 3.5 to 9.5, and isoelectric focusing standards were from Pharmacia (Uppsala, Sweden). The protein inhibitors (4-amidinophenyl)-methanesulfonyl fluoride, antipain dihydrochloride, aprotinin, chymostatin, E64, EDTA, leupeptin, a2-macroglobulin, pepstatin, phenylmethylsulfonyl fluoride (PMSF), phosphoramidon, and soybean trypsin inhibitor were obtained from Boehringer (Mannheim, Germany). The MEP inhibitor 1,10-phenanthroline and the cysteine proteinase inhibitor iodoacetamide were from Sigma.

Strains and growth conditions. A. fiunigatus CBS 144-89

and A18, a CBS 144-89 mutant in which the gene encoding the secreted alkaline protease ALP was inactivated by gene disruption (12), were used in this work. The strains were maintained on malt agar slants at 4°C. For enzyme production, the strains were grown in liquid medium containing 0.2% (wt/vol) soluble collagen as sole nitrogen and carbon source (11). Flasks containing 500 ml of medium were inoculated with 107 conidia from a 5- to 10-day malt agar culture and incubated at 30°C on an orbital shaker at 200 rpm for 48 h. Enzyme purification. Two-liter batches of culture filtrate were used as the source of enzyme. Step 1. Dry hydroxylapatite was added to the filtrate at a concentration of 4 g/liter. After the mixture was shaken for

Corresponding author. 4099

Downloaded from http://iai.asm.org/ on June 7, 2018 by guest

mutant after the fungus was cultivated in the presence of collagen as the sole nitrogen and carbon source. The enzyme was purified 50-fold from the culture supernatant after adsorption to hydroxylapatite and carboxymethyl-Sephadex and after gel filtration. The molecular mass was determined to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was estimated at pH 5.5 by isoelectric focusing. Reducing agents and divalent cations strongly inhibited enzyme activity, whereas nonionic detergents had no effect. A. fimigatus MEP was totally inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. MEP is not able to cleave elastin and is thermosensitive. Sera from patients suffering from aspergilloma reacted with MEP in Western blotting (immunoblotting) analyses, suggesting that MEP promotes an antigenic response in these patients.

4100

INFECT. IMMUN.

MONOD ET AL.

c

80

40

420.

ii PMSF

+

EDTA

+

-

_

+ +

_

+ +

+

+

+

+

elastinolytic (right) activities were measured and/or EDTA (1 mM).

as

30 min, the gel was transferred to a glass column. The column was first washed with 10 mM sodium phosphate buffer (pH 7.5), and MEP was eluted with 50 mM sodium phosphate buffer, pH 7.5. Fractions with proteolytic activity were pooled and dialyzed against 50 mM sodium acetate buffer, pH 5.0. No proteolytic activity was found in subsequent fractions eluted with higher sodium phosphate concentrations. Step 2. CM-Sephadex, 0.5 g, was added to the dialysate, and the mixture was shaken for 5 min. The CM-Sephadex was transferred to a column and washed with 50 mM acetate buffer, pH 5.0. The MEP was eluted from the column with 150 mM sodium acetate, pH 6.5. Fractions with enzymatic activity were identified and pooled. Step 3. After reduction of the volume to 2 ml by ultrafiltration (Ultracent-30 system; Bio-Rad), the enzyme solution was further purified by gel filtration on a 30-cm column of polyacrylamide P60 gel in 100 mM sodium acetate, pH 6.5. Finally, the MEP was concentrated to 1.0 mg/ml by ultrafiltration and stored at -20°C. Proteolytic assays. Proteolytic activity was measured on

kCDa

1

2

3

4

5

and

described in Materials and Methods in the absence and in the presence of PMSF (10 p.g/ml)

6

97

azocollagen at different pH values in sodium acetate buffer (50 mM; pH 4 to 6.5), in Tris-HCl buffer (50 mM; pH 6.5 to 9), and in glycine-NaOH buffer (100 mM; pH 9 to 12). From a suitably diluted protease solution in buffer at 30°C, 1 ml was added to 10 mg of substrate. After incubation for 30 min with continuous shaking at 37°C, the reaction was stopped by removing the insoluble collagen by centrifugation. The A520 of the colored supematant was measured. One unit of enzyme activity produced an increase in absorbance of 0.001 per min at pH 7.5. Activity against elastin was measured with elastin-Congo red as substrate at a concentration of 10 mg/ml in 50 mM Tris-HCl, pH 7.5. The reaction was stopped by removing the substrate by centrifugation. The A495 of the orange supernatant was measured. Effect of detergents, organic solvent, reducing agents, organic ions, phosphate, divalent cations, and proteinase inhibitors. The actions of detergents (sodium dodecyl sulfate [SDS], Triton X-100, and Tween 80) as well as organic solvents (dimethyl sulfoxide and ethanol) and reducing agents (2-mercaptoethanol and dithiothreitol) were tested in the 0.1 to 5% concentration range. The actions of organic ions (citrate, carbonate, and acetate) and phosphate were tested at 1 to 100 mM. Divalent cations (Mg2+, Cu2+, Ca2+, Mn2+ Zn2+, Co2+, Pb2+, Hg2+, and Ni2+) were

66_ t .-

30..

FIG. 2. SDS-PAGE (9% gels) of MEP preparations stained with Coomassie brilliant blue R250 (lanes 1 to 5) and periodic acid-Schiff reagent (lane 6). Lanes 1 and 2, profile of secreted protein of CBS 144-89 and A18, respectively, after 48 h of growth at 30°C in collagen medium. The proteins of 1 ml of culture supernatant were precipitated with trichloroacetic acid before loading on the gel. Lane 3, protein profile of active fractions eluted from hydroxylapatite chromatography. Lanes 4 and 6, purified MEP after P60 chromatography. Lane 5, molecular mass markers: phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 42 kDa; and bovine carbonic anhydrase, 30.0 kDa. Arrow indicates ALP (33 kDa).

TABLE 1. Purification of the extracellular MEP of A. fumigatus A18 Purification step

Total Total Total vol protein enzyme

(ml)

(jig

Purifi-

Yield Sp act cation activity (%) (units/jig) (fold) (U)

Culture supernatant 1,800 28,900 45,720 100 Step I, hydroxylapatite 30 912 28,900 62 elution 10 230 17,600 40 Step II, CM-Sephadex elution 10 220 17,000 38 Step III,a P60 gel filtration

1.6 31.6

1.0 19.7

76.5

48.4

77.3

48.6

a Elution from a P60 gel column eliminated residual brown pigments without further purification.

Downloaded from http://iai.asm.org/ on June 7, 2018 by guest

A 18 A 18 CBS CBS 144-89 144-89 FIG. 1. Proteolytic activity of A. fiumigatus CBS 144-89 and A18 in collagen medium after 48 h of growth at 30°C. Collagenic (left)

VOL. 61, 1993

A. FUMIGATUS METALLOPROTEASE

4101

-300

6000

CO

._

0

200 ._

._

0

4)

a.

C

0)

100

0 15

20

25

30

35

40

45

50

55

60

Fractions (2 ml) FIG. 3. Column chromatogram of MEP on hydroxylapatite. Fractions 1 to 25 and 25 to 58 were eluted with 50 and 300 mM phosphate buffer (Pi), pH 7.0, respectively (step gradient). The first protein peak contained all proteolytic activity. No protein and no proteolytic activity were found in fractions 1 to 15 eluted with 50 mM phosphate buffer.

studied at 1 and 10 mM. The inhibitors (4-amidinophenyl)methanesulfonyl fluoride, iodoacetamide, EDTA, antipain, leupeptin, chymostatin, phosphoramidon, E64, soybean trypsin inhibitor, a2-macroglobulin, and aprotinin were dissolved in water. 1,10-Phenanthroline, PMSF, and pepstatin were dissolved in ethanol. Each compound was added to 1 ml of culture supernatant or to 1 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 0.5 Fg of purified enzyme. The mixture was incubated for 10 min at 30°C, and assays were performed after the mixture was added to 10 mg of azocollagen. An appropriate control without inhibitor was assayed simultaneously. Protein concentration. Protein concentrations were measured by the method of Bradford (2). Electrophoresis. Protein extracts were analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) by the method of Laemmli (9) with a separation gel of 9% polyacrylamide. Gels were stained with either Coomassie brilliant blue R-250 or, for glycoproteins, the periodic acid-Schiff reagent (28). Human sera. Two serum pools from 50 patients with aspergilloma and 50 healthy individuals were used as well as 10 human individual serum samples positive for aspergilloma and 10 human serum samples from patients with candidiasis. All serum samples were kindly provided by J.-P. Bouchara (CHU, Angers, France). Immunoblotting. After electrophoresis of MEP, the protein was transferred onto nitrocellulose membranes and probed with human sera, as described previously (10). RESULTS Proteolytic activities in collagen culture. After 48 h of growth in collagen medium, A. fimigatus A18 showed 30% of the collagenolytic activity of the wild-type strain CBS

144-89 but no elastinolytic activity (Fig. 1), confirming previous studies by Tang et al. (24) in which the gene encoding ALP was disrupted in another strain of A. fumigatus. In the presence of PMSF, the elastinolytic activity of CBS 144-89 was totally inhibited and the collagenolytic activity was reduced to the rate of that of the A18 mutant strain. This residual proteolytic activity was inhibited 100% by EDTA (1 mM), 1,10-phenanthroline (1 mM), and phosphoramidon (10 ,Lg/ml), and the collagenic activity of the wild-type strain was 100% inhibited by a combination of EDTA and PMSF (Fig. 1). These results demonstrated that A. fiumigatus CBS 144-89 secretes one or several MEPs, inhibited by EDTA and 1,10-phenanthroline, and that the

0 cm I-.

.0

0

0

a.

C.) 0

Fractions (2 ml) FIG. 4. Elution profile of MEP from a CM-Sephadex column with 150 mM sodium acetate buffer, pH 6.5.

Downloaded from http://iai.asm.org/ on June 7, 2018 by guest

0

4102

MONOD ET AL.

INFECT. IMMUN. 0.025

CM 0

0.02

m0 *t

0.015

._

co 0.01 Cu 8

S..

0.005

13

pH FIG. 5. Effect of pH on activity of A. fumigatus MEP with azocollagen as substrate in acetate, Tris-HCl, and glycine-NaOH buffer.

ALP gene disruption had no effect on MEP secretion. An inhibition profile comparable to that of CBS 144-89 was observed for the 10 wild-type strains of A. fumigatus tested (data not shown). Isolation of a 40-kDa A. fiumigatus-secreted MEP. We tried to isolate MEPs secreted by A. fumigatws from strain A18 since this strain and the parental strain CBS 144-89 showed similar profiles of secreted proteins, except that of ALP (Fig. 2, lanes 1 and 2). The purification steps and yields are summarized in Table 1. A 48-fold purification from the

0.05

culture medium, with a 38% overall recovery, was obtained. Elution profiles of proteolytic activity from hydroxylapatite and CM-Sephadex are shown in Fig. 3 and 4, respectively. The purified enzyme, MEP, migrated as a single protein band with an estimated molecular mass of 40 kDa in SDSPAGE (Fig. 2, lane 4). This enzyme appeared to be one major protein secreted by A. fiumigatus in collagen medium (Fig. 2, lanes 1 and 2). It is also the major band in the protein profile of the fractions containing proteolytic activity eluted from hydroxylapatite (Fig. 2, lane 3).

3

0

0.04

co.2

8

0.03

0.02

Cu

0

25

30

35

40

45

50

55

6

65

T [0C] FIG. 6. Effect of temperature on MEP activity. A. fumigatus MEP, 0.5 ,g in 20 F±l of 50 mM sodium acetate buffer (pH 7.5), was maintained at 4°C (A) or preincubated in a sealed tube for 10 min at the assay temperature in the presence (A) or the absence (-) of glycerol. Enzymatic activity was measured as described in Materials and Methods by adding the pretreated enzyme to azocollagen in 50 mM Tris-HCl buffer, pH 7.5.

Downloaded from http://iai.asm.org/ on June 7, 2018 by guest

w

A. FUMIGATUS METALLOPROTEASE

VOL. 61, 1993 TABLE 2. Effects of different compounds on purified MEP

Compound&

Concn

% Inhibition

0.1-1.0% 0.1-1.0% 0.1-1.0% 1.0-5.0% 1.0-5.0%

25-50 0

0.1-1.0% 0.1-1.0%

65-100 100

A

4103

B

Detergent SDS Tween 80 Triton X-100 Ethanol DMSO Reducing agent 2-Mercaptoethanol DTT

1-10 mM 10-100 mM 10-100 mM 10-100 mM

Divalent metal chloride Mg, Ca Mn Zn, Cu, Co, Pb, Hg, Ni

1-10 mM 1-10 mM 1-10 mM

Protease inhibitor Class specific PMSF APMSF Iodoacetamide EDTA 1,10-Phenanthroline

10-50 ,u.g/ml 10-50 jLg/ml 10-50 4g/ml 1-10 mM 1-10 mM

Microbial origin Antipain Leupeptin Chymostatin Pepstatin Phosphoramidon E64

10-50 pg/ml 10-50 ,ug/ml 10-50 ,ug/ml 10-50 ,ug/ml 10-50 tLg/ml 1 mg/ml

High molecular weight SBTI a2-Macroglobulin Aprotinin

10-50 pg/ml 10-50 pg/ml 10-50 pg/ml

100 0-30 0-25 0

a DMSO, dimethyl sulfoxide; DTT, dithiothreitol; APMSF, (4-amidinophenyl)-methanesulfonyl fluoride; SBTI, soybean trypsin inhibitor.

FIG. 7. Immunoblot analysis of MEP (arrow) probed with pools of sera from patients with aspergilloma (A) and from healthy individuals (B). Sera were diluted 1:1,000.

vents (ethanol and dimethyl sulfoxide) and nonionic detergents (Tween 80 and Triton X-100) tested had no significant effects on protease activity (Table 2). A. fimigatus MEP was very sensitive to divalent cations. The activity was totally inhibited in the presence of 1 mM each Cu21, Zn2+, Co2+, Pb2+, Ni2+, and Hg2+. In contrast, the enzyme was not inhibited by 10 mM Mg2+ or Ca2+. Different protease inhibitors were also tested on purified MEP. The enzyme was totally inactivated by EDTA, 1,10-phenanthroline, and phosphoramidon, confirming our preliminary observations with activities observed in the culture supernatant. MEP was also inhibited by the nonspecific inhibitor a2-macroglobulin. In contrast, inhibitors specific for serine, cysteine, and aspartate proteases, such as PMSF, (4-amidinophenyl)methanesulfonyl fluoride, antipain, leupeptin, chymostatin, soybean trypsin inhibitor, aprotinin, and iodoacetamide, had no effect. Immunoblot analysis with sera from patients with aspergilloma showed that MEP was antigenic. This result was obtained with a pool of positive patient sera at a 1:1,000 dilution (Fig. 7). When probed individually, each of the 10 aspergilloma patient serum samples reacted positively with MEP, whereas serum samples from all candidiasis patients were negative even at a 1:100 dilution (not shown). DISCUSSION

Properties of MEP. Periodic acid-Schiff-positive staining indicated that MEP was a glycoprotein (Fig. 2, lane 6). An isoelectric point of 5.5 was estimated by isoelectric focusing. The enzyme was stable for at least 1 week at 4°C and for 4 months at -20°C in 100 mM sodium acetate buffer, pH 7.0. MEP showed a plateau of activity between pH 6.5 and 9.0 (Fig. 5). Protein substrate and glycerol had stabilizing effects on the enzyme. This was demonstrated by the results shown in Fig. 6. When the enzyme, maintained at 4°C, was added to the substrate preincubated at the assay temperature, maximal activity was observed between 45 and 50°C. In contrast, a 10-min pretreatment of the enzyme at 50°C resulted in 100% of loss of activity, whereas a similar treatment at 450C reduced the activity to 45%. MEP could be stabilized at 45°C by 20% (vol/vol) glycerol. The effects of different compounds used to characterize MEP are summarized in Table 2. The enzyme was inhibited by citrate (1 mM) and by reducing agents such as 2-mercaptoethanol (1%) and dithiothreitol (1 mM). The organic sol-

We report the isolation and characterization of a MEP secreted by A. fumigatus, as revealed by testing various protease inhibitors. A 48-fold purification with a 38% overall recovery was obtained. During this purification procedure, 35% of the total proteolytic activity was not absorbed on hydroxylapatite. This residual activity, present in the culture supematant after sedimentation of hydroxylapatite, could be recovered by adding a second batch of resin and was attributed to MEP. Thus, it appeared that MEP was the only protease secreted by the A. fumigatus ALP-deficient mutants in collagen medium. Acid proteases identified during the growth of A. fumigatus on protein-based media (14-16) were not found in our experimental conditions since no inhibition of proteolytic activity was noted when the culture supernatant was preincubated in the presence of pepstatin and since no proteolytic activity was detected in acetate buffer at pH 4.0 (data not shown). Several species of Aspergillus are known to secrete MEPs (18-22, 26). On the basis of glycosylation and molecular mass, MEP resembles A. sojae neutral MEP I (19-22). A

Downloaded from http://iai.asm.org/ on June 7, 2018 by guest

Organic ion and phosphate Na citrate Na carbonate Na phosphate Na acetate

0 0 0

4104

INFECT. IMMUN.

MONOD ET AL.

fiumigatus wild strain and in ALP-deficient mutants.

ACKNOWLEDGMENTS We thank J.-P. Bouchara for providing human sera from aspergilloma patients and H. Pooley for critical review of the manuscript and assistance with the English. This work was supported by the Swiss National Foundation for Scientific Research, grant 31-36248.92.

REFERENCES 1. Bodey, G. P., and S. Vartivarian. 1989. Aspergillosis. Eur. J. Clin. Microbiol. Infect. Dis. 8:413-437. 2. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 3. Dell, M. J., P. E. Steele, and J. C. Rhodes. 1993. Abstr. 93rd Gen. Meet. Am. Soc. Microbiol. 1993, F-74, p. 540. 4. Denning, D. W., P. N. Ward, L. E. Fenelon, and E. W. Benbow. 1992. Lack of vessel wall elastinolysis in human invasive pulmonary aspergillosis. Infect. Immun. 60:5153-5156. 5. Frosco, M., T. Chase, and J. D. MacMillan. 1992. Purification and properties of the elastase from Aspergillus fumigatus. Infect. Immun. 60:728-734. 6. Jaton-Ogay, K., M. Suter, R. Crameri, R. Faichetto, A. Faith, and M. Monod. 1992. Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus. FEMS Microbiol. Lett. 92:163-168. 7. Kothary, M. H., T. Chase, and J. D. MacMillan. 1984. Correlation of elastase production by some strains of Aspetgillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice. Infect. Immun. 43:320-325. 8. Kurup, V. P., and A. Kumar. 1991. Immunodiagnosis of as-

pergillosis. Clin. Microbiol. Rev. 4:439-456. 9. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 10. Latge, J. P., M. Moutaouakil, J. P. Debeaupuis, J. P. Bouchara, K. A. Haynes, and M. C. Prevost. 1991. The 18-kDa antigen secreted by Aspergillus fumigatus. Infect. Immun. 59:25862594. 11. Monod, M., S. Paris, J. Sarfati, K. Jaton-Ogay, P. Ave, and J. P. Latge. 1992. Virulence of alkaline protease deficient mutants of Aspergillus fumigatus. FEMS Microbiol. Lett. 106:39-46. 12. Monod, M., G. Togni, L. Rahalison, and E. Frenla 1991. Isolation and characterization of an extracellular alkaline protease of Aspergillus fumigatus. J. Med. Microbiol. 35:23-28. 13. Moutaouakil, M., M. Monod, M. C. Prevost, J. P. Bouchara, S. Paris, and J. P. Latge. Identification of the 33 KDa alkaline protease of Aspergillus fumigatus in vitro and in vivo. J. Med. Microbiol., in press. 14. Panneerselvam, M., and S. C. Dhar. 1980. Chromatographic purification and homogeneity of extracellular acid proteinase of Aspergillus fumigatus. Ital. J. Biochem. 29:102-112. 15. Panneerselvam, M., and S. C. Dhar. 1981. Physico-chemical properties of the acid proteinase from A. fumigatus. Ital. J. Biochem. 30:63-74. 16. Reichard, U., S. Buttner, H. Eiffert, F. Staib, and R. Riuchel. 1990. Purification and characterization of an extracellular serine protease from Aspergillus fumigatus and its detection in tissue. J. Med. Microbiol. 33:243-251. 17. Rhodes, J. C., and T. W. Amlung. 1991. The elastinolytic proteinase of Aspergillus flavus is not glycosylated. J. Med. Vet. Mycol. 29:407-411. 18. Rhodes, J. C., T. W. Amlung, and M. S. Miller. 1990. Isolation and characterization of an elastinolytic proteinase from Aspergillus flavus. Infect. Immun. 58:2529-2534. 19. Sekine, H. 1972. Neutral proteinases I and II of Aspergillus sojae. Isolation in homogeneous form. Agric. Biol. Chem. 36:198-206. 20. Sekine, H. 1972. Neutral proteinases I and II of Aspergillus sojae. Some enzymatic properties. Agric. Biol. Chem. 36:207216. 21. Sekine, H. 1972. Some properties of neutral proteinases I and II of Aspergillus sojae as zinc-containing metallo-enzyme. Agric. Biol. Chem. 36:2143-2150. 22. Sekine, H. 1973. Neutral proteinases I and II of Aspergillus sojae: some physicochemical properties and amino acid composition. Agric. Biol. Chem. 37:1945-1952. 23. Staib, F. 1982. Extracellular proteolytic activity of Aspergillus fumigatus strains with septum-like structures in their phialides in serum-albumin agar. Preliminary report. Zentralbl. Bakteriol. Hyg. Abt. 1 Orig. Reihe A 252:279-285. 24. Tang, C. M., J. Cohen, and D. W. Holden. 1992. AnAspegillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity. Mol. Microbiol. 6:1663-1671. 25. Tang, C. M., J. Cohen, T. Krausz, S. Van Noorden, and D. W. Holden. 1993. The alkaline protease of Aspergillusffumigatus is not a virulence determinant in two murine models of invasive pulmonary aspergillosis. Infect. Immun. 61:1650-1656. 26. Tatsumi, H., S. Murakami, R. F. Tsuji, Y. Ishida, K. Murakami, A. Masaki, H. Kawabe, H. Arimura, E. Nakano, and H. Motai. 1991. Cloning and expression in yeast of a cDNA clone encodingAspergillus oryzae neutral protease II, a unique metalloprotease. Mol. Gen. Genet. 228:97-103. 27. Van den Bossche, H., D. W. R. Mackenzie, and G. Cauwenbergh. 1987. Aspergillus and aspergillosis. Plenum Press, New York. 28. Zacharius, R., T. E. Zell, J. H. Morrison, and J. J. Woodlock. 1969. Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30:148-152.

Downloaded from http://iai.asm.org/ on June 7, 2018 by guest

MEP recently was isolated and well characterized from the pathogenic species A. flavus (17, 18). This protease, like neutral protease II of A. oryzae (26) and A. sojae (19-22), differs from MEP by having elastinolytic activity, a high thermotolerance, a smaller molecular mass (approximately 23 kDa), and a lack of glycosylation. Preliminary results of Dell et al. (3) showed the absence of a neutral protease II-like enzyme in A. fumigatus. The mechanisms of tissue invasion by A. fumigatus still remain an open question since no difference of pathogenicity was observed between isogenic ALP-positive and ALPnegative strains (11, 25). It is likely that ALP does not contribute decisively to the ability to invade lung tissues. These results agree with the observation that elastinolysis is not involved in fungal infiltration of blood vessel walls (4). However, it is difficult to explain the ability to invade tissues without an efficient invasive system. The level of MEP secreted by the alp deletion mutant could be high enough to allow a residual proteolytic tissue degradation, which may play a significant role in the virulence of A. fiumigatus. Antibodies against ALP were detected by Western blotting (immunoblotting) analysis in patients suffering from aspergilloma as well as in patients suffering from candidiasis and in healthy controls (13). In contrast to ALP, MEP is an antigen able to discriminate between aspergilloma patients and control individuals. Although MEP has a lower collagenic activity in vitro than ALP, the presence of a significant antigenic response against MEP suggests that MEP is secreted in substantial amounts in vivo and could be responsible for the proteolytic degradation of the lung tissues. To test the role of MEP in the virulence of A. fumigatus, we intend to clone and disrupt the gene encoding MEP in an A.