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Elastase and elastase inhibitor from Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger

¹Department of Quality Control, Mathuurayakugyo Co. Ltd, 24-21 Enjo-chou, Syowa-ku, Nagoya 466-0054, Japan ²Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan ³Department of Pulmonary Medicine, National Higashinagoya Hospital, 5-101 Umemorizaka, Meito-ku, Nagoya 465-8620, Japan

Correspondence Toshiaki Nikai nikai{at}ccmfs.meijo-u.ac.jp

Received March 10, 2003
Accepted December 19, 2003

Elastolytic and elastase inhibitory activities were investigated for 13 strains of Aspergillus fumigatus, three strains of Aspergillus flavus and three strains of Aspergillus niger. Nine of the 13 strains of A. fumigatus and all strains of A. flavus demonstrated elastase activity (more than 1 unit ml^–1). Six of the 13 strains of A. fumigatus and all strains of A. flavus expressed elastase inhibitory activity (more than 2 units ml^–1). However, no elastase or elastase inhibitory activities were observed with A. niger. It was also found that crude elastase inhibitors from six strains of A. fumigatus and two strains of A. flavus were stable to heat treatment at 100 °C for 10 min. In addition, human leukocyte elastases were inhibited by crude elastase inhibitors from A. fumigatus and A. flavus; however, no effect was observed on the elastase derived from porcine pancreas.

	Introduction

TOP Introduction Methods Results and Discussion ACKNOWLEDGEMENTS References

 
This laboratory has shown that elastolytic proteinases can be obtained from Aspergillus fumigatus and Aspergillus flavus (Hasegawa et al., 1995, 1997). It has been shown that elastases obtained from Aspergillus spp. (Frosco et al., 1992; Mellon & Cotty, 1995; Monod et al., 1991; Reichard et al., 1990; Rhodes et al., 1990) are pathogenic factors (Iadarola et al., 1998; Frosco et al., 1994; Kolattukudy et al., 1993; Denning et al., 1992).

Protease inhibitors have also been isolated from various sources in the past. For example, some trypsin inhibitors have been isolated from plants; ₂-macroglobulin, antithrombin III and ₁-antitrypsin have been obtained from serum; and leupeptin, antipain, chymostatin and pepstatin have been isolated from actinomycetes. In addition, elasnin (Ohno et al., 1980), elafin (Wiedow et al., 1990), elastatinal (Umezawa et al., 1973) and ONO-5046 (Kawabata et al., 1991) are reported as elastase inhibitors. Elastase inhibitors are inhibitors of leukocyte elastase and promote anti-inflammatory action. The elastase activity of Aspergillus spp. was investigated by the study of Aspergillus culture filtrates in which elastase activity was increased and/or decreased every 3 or 4 days. These results definitively show the presence of elastase inhibitor.

In the present study, the screening and some properties of elastase inhibitors from 13 strains of A. fumigatus, three strains of A. flavus and three strains of Aspergillus niger isolated from sputum samples are reported.

	Methods

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Elastase and elastase inhibitor from A. fumigatus, A. flavus and A. niger.

A. fumigatus, A. flavus and A. niger were identified and isolated from sputum. Elastase was obtained as follows. A sufficient number of fungal spores (A. fumigatus, A. flavus and A. niger) were cultured in 100 ml of a synthetic medium of 1 % yeast carbon base (Difco) containing 1 % elastin (Sigma) as a nitrogen source. The cultures were incubated at 37 °C with shaking at 100 r.p.m. for 12 days.

Elastase inhibitor was obtained by the following method. A sufficient number of fungal spores (A. fumigatus, A. flavus and A. niger) were cultured in 100 ml of synthetic medium of 1 % yeast carbon base containing 1 % Casamino acids (Difco) as a nitrogen source. The cultures were incubated at 37 °C for 7 days.

Assay for elastolytic activity.

Elastolytic activity of the crude enzyme was assayed by the method of diazocoupling and measuring the p-nitroanilide (p-NA) released from 50 mM succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide (STANA) (Peptide Institute Inc., Osaka, Japan) in DMSO used as the substrate (Hasegawa et al., 1997). To determine the extent of STANA digestion, 0.1 ml of the enzyme solution, 0.9 ml of 50 mM Tris/HCl buffer, pH 7.5, and 20 µl of 50 mM STANA solution were incubated for 60 min at 37 °C. The reaction was stopped by adding 1 ml of 10 % trichloroacetic acid (TCA). Then, 0.2 ml of 0.1 % sodium nitrate, 0.5 % ammonium sulfamate and 0.1 % N-(1-naphthyl)ethylenediamine dihydrochloride were added to the solution and the colour that developed was read at 550 nm. One unit is defined as the amount of p-NA released from STANA at 1 min.

Assay for elastase inhibitory activity.

Fifty microlitres of elastase inhibitor solution (crude enzyme) was mixed with the purified elastase from A. flavus and incubated for 15 min at 37 °C prior to the addition of the STANA substrate. Then, 0.9 ml of 50 mM Tris/HCl buffer, pH 7.5, and 20 µl of 50 mM STANA solution were added and incubated for 60 min at 37 °C. The reaction was stopped by adding 1 ml of 10 % TCA. Then, 0.2 ml of 0.1 % sodium nitrate, 0.5 % ammonium sulfamate and 0.1 % N-(1-naphthyl)ethylenediamine dihydrochloride were added to the solution and the colour developed was read at 550 nm. One unit is defined as the minimal dose that did not release p-NA from STANA at 1 min.

	Results and Discussion

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Elastolytic activity.

Each day, 1 ml of the culture broth was filtered aseptically through a 0.22 µm pore-size membrane filter. The cell-free culture supernatant was used as the source of crude enzyme and the elastolytic activity of the culture supernatant was measured. Nine of the 13 strains of A. fumigatus demonstrated elastase activity (more than 1 unit ml^–1). Data from six strains are shown in Fig. 1; these strains exhibited elastase inhibitory activity (more than 2 units ml^–1). In addition, all strains of A. flavus demonstrated elastase activity (Fig. 2). However, no elastase activity was observed with A. niger.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Elastolytic activity of A. fumigatus strains. Solid lines: •, AFU-2; , AFU-3; , AFU-5. Broken lines: •, AFU-6; , AFU-9; , AFU-12.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Elastolytic activity of A. flavus (AFL) and A. niger (AN) strains. Solid lines: •, AFL-1; , AFL-2; , AFL-3. Broken lines: •, AN-1; , AN-2; , AN-3.

Elastase inhibitory activity.

Each day, 1 ml of the culture broth was filtered aseptically through a 0.22 µm pore-size membrane filter. The cell-free culture supernatant was used as the source of crude enzyme and the elastase inhibitory activity of the culture supernatant was measured. Six of the 13 strains of A. fumigatus and two strains of A. flavus showed elastase inhibitory activity (Figs 3 and 4). A. niger was non-responsive as no inhibitory activity was detected.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Elastase inhibitory activity of A. fumigatus strains. Solid lines: •, AFU-2; , AFU-3; , AFU-5. Broken lines: •, AFU-6; , AFU-9; , AFU-12.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Elastase inhibitory activity of A. flavus (AFL) and A. niger (AN) strains. Solid lines: •, AFL-1; , AFL-2; , AFL-3. Broken lines: •, AN-1; , AN-2; , AN-3.

Heat stability of elastase inhibitory activity of culture supernatant from A. fumigatus and A. flavus.

The culture supernatants were heated for 10 min at various temperatures (37, 50, 60, 80 and 100 °C), quickly cooled to 4 °C, and the elastase inhibitory activity on STANA was determined (Fig. 5). All culture supernatants were stable to heat treatment, with 60–95 % of the activity remaining even after heat treatment at 100 °C for 10 min (pH 5.5).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Heat stability of culture supernatants of A. fumigatus (AFU) and A. flavus (AFL) strains. Solid lines: •, AFU-2; , AFU-3; , AFU-5; , AFL-1; , AFL-2. Broken lines: •, AFU-6; , AFU-9; , AFU-12.

Elastases from pancreas and leukocytes were incubated for 15 min at 37 °C with elastase inhibitors. Following incubation, 0.9 ml of 50 mM Tris/HCl buffer, pH 7.5, and 20 µl of 50 mM STANA solution were added and incubated for an additional 60 min at 37 °C. The reaction was stopped by adding 1 ml of 10 % TCA followed by 0.2 ml of 0.1 % sodium nitrate, 0.5 % ammonium sulfamate and 0.1 % N-(1-naphthyl)ethylenediamine dihydrochloride. Colour was developed and read at 550 nm. Elastase from pancreas was inhibited 12.1, 11.1 and 6.5 % by A. fumigatus strains AFU-6, AFU-9 and AFU-12, respectively; it was not inhibited by A. fumigatus strains AFU-2, AFU-3 and AFU-5 nor by A. flavus strains AFL-1 and AFL-2. Elastase from leukocytes was inhibited 52.5, 36.8, 23.9, 33.6, 54.6, 27.4 and 15.0 % by A. fumigatus strains AFU-2, AFU-3, AFU-6, AFU-9 and AFU-12 and A. flavus strains AFL-1 and AFL-2, respectively; it was not inhibited by A. fumigatus strain AFU-5.

The purpose of this study was to develop a screening procedure and investigate the biological properties regarding elastase inhibitory activity of culture filtrates. Elastase activity (more than 1 unit ml^–1) was recognized in nine strains of A. fumigatus and three strains of A. flavus. Elastase inhibitory activity (more than 2 units ml^–1) was recognized in six strains of A. fumigatus and two strains of A. flavus, respectively. Since eight strains of Aspergillus demonstrated inhibitory activity against elastase, the possibility exists that the inhibitory activity is related to suppression of the immune system. The results indicate that the elastase inhibitor possesses resistance to the host defence and, as a consequence, could participate in the onset of mycosis. This could be caused by the elastase inhibitor not being susceptible to the host immune defence which would result in physiological conditions conducive to the occurrence of mycosis in the host.

These results also suggest that the activity of the crude elastase inhibitors from A. fumigatus and A. flavus on human leukocyte elastases may be helpful in the treatment of inflammation. More detailed investigations of the properties of the elastase inhibitor are needed in order to refine and purify the inhibitor and to investigate its relationship to mycosis as well as to test its effectiveness against inflammation caused by human leukocyte elastases.

It has been reported that Aspergillus-produced elastase is a definitive virulence factor of aspergillosis (Blanco et al., 2002). Frosco et al. (1992, 1994) and Kolattukudy et al. (1993) reported that elastase is a significant virulence factor to the extent that Frosco et al. (1994) used elastase-specific monoclonal and polyclonal antibodies. Kolattukudy et al. (1993) provided proof of virulence by using an elastase-producing and a non-producing strain (elastase-deficient mutant). Ogawa et al. (1992), Hasegawa et al. (1994) and Ogawa (1997) reported that urinastatin, which contains an enzyme inhibitor, was administered by way of intravenous drip infusion concomitantly with antifungal agents to patients suffering pulmonary aspergillosis. Maesaki et al. (1993) and Sugimura et al. (1994) reported urinastatin treatment of aspergillosis or aspergilloma. The results shown in Figs 1 and 2 confirm that the elastase inhibitor controls the action of elastase in releasing irritating and/or toxic products in vivo.

The results of this investigation indicate that the use of a highly purified and structurally determined elastase inhibitor would be more effective against aspergillosis than urinastatin.

	ACKNOWLEDGEMENTS

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We acknowledge the support of the Ministry of Education, Science, Sports and Culture (High-Tech Research Center Project). We are thankful to Akiko Hirofuzi for technical support.

	References

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