Extracellular proteases have been suggested to be virulence factors in invasive aspergillosis. Since serine protease gene-disrupted mutants retain virulence, other proteases are suspected to be also involved in the degradation of lung structural material. An elastinolytic neutral metalloprotease was purified 320-fold from the extracellular fluid of Aspergilus fumigatus grown on elastin by affinity chromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-75. The molecular mass was determined to be 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No carbohydrate was attached to this metalloprotease, and its first 22 N-terminal amino acids did not show any homology with the known metalloproteases. The enzyme was completely inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. Zn2+ and, to a lesser extent, Co2' reversed the inhibition caused by 1,10-phenanthroline. The protease hydrolyzed the peptide bonds His-Leu, Ala-Leu, Tyr-Leu, Gly-Phe, and Phe-Phe in the B chain of insulin. Synthetic substrate Abz-Ala-Ala-Phe-Phe-pNA could be used for the * Corresponding author. Mailing address:

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg ⅐ ATP ؉ AMP 7 Mg ⅐ ADP ؉ ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.Pseudomonas aeruginosa is a dominant pathogen in the respiratory tract of cystic fibrosis patients (35). Unlike other bacterial infections, P. aeruginosa is more difficult to control through antibiotic therapy (26). To survive in the hostile environment of the human body, this pathogen utilizes an impressive arsenal of weapons (30). Macrophages constitute the first line of defense against infections, and the ability of P. aeruginosa to kill macrophages and other phagocytic cells such as mast cells may explain this bacterium's capability to persist and disseminate in the host. It is well known that P. aeruginosa can avoid phagocytosis by encapsulating itself with an exopolysaccharide coating, called alginate, which confers on the nonmucoid cells a mucoid phenotype (11,27,31). Earlier, it was demonstrated that a mucoid, alginate-producing strain of P. aeruginosa isolated from the lungs of a cystic fibrosis patient secretes nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase (AK), 5Ј-nucleotidase, and ATP-modifying enzymatic activities that can modulate the external ATP level of macrophages and enhance their cell death through P2Z receptor activation (40). The P2Z receptor is responsible for ATPdependent cell death of macrophages through the formation of membrane pores permeable to molecules of up to 900 daltons in size (4, 37).The role of individual enzymes secreted by the mucoid strain of P. aeruginosa (40) in the killing of macrophages is, however, unknown. In this article, we report the role of a sing...

The function of the long propeptides of fungal proteinases is not known. Aspergillus fumigatus produces a 33-kDa serine proteinase of the subtilisin family and a 42-kDa metalloproteinase of the thermolysin family. These extracellular enzymes are synthesized as preproenzymes containing large amino-terminal propeptides. Recombinant propeptides were produced in Escherichia coli as soluble fusion proteins with glutathione S-transferase or thioredoxin and purified by affinity chromatography. A. fumigatus serine proteinase propeptide competitively inhibited serine proteinase, with a K i of 5.3 ؋ 10 ؊6 M, whereas a homologous serine proteinase from A. flavus was less strongly inhibited and subtilisin was not inhibited. Binding of metalloproteinase propeptide from A. fumigatus to the mature metalloenzyme was demonstrated. This propeptide strongly inhibited its mature enzyme, with a K i of 3 ؋ 10 ؊9 M, whereas thermolysin and a metalloproteinase from A. flavus were not inhibited by this propeptide. Enzymatically inactive metalloproteinase propeptide complex could be completely activated by trypsin treatment. These results demonstrate that the propeptides of the fungal proteinases bind specifically and inhibit the respective mature enzymes, probably reflecting a biological role of keeping these extracellular enzymes inactive until secretion.Many proteinases are synthesized as preproenzymes consisting of a typical signal peptide followed by the propeptide and the mature protein (1). Several functions have been proposed for the propeptide. The propeptide may be required for aiding the proper folding or secretion of the mature proteinase (7,8,28,34), anchoring the proteinase to the membrane and/or maintaining the proteinase in an inactive state until it is released from the cell (33). In several cases, the propeptide plays multiple roles. For instance, the propeptide of a bacterial ␣-lytic proteinase was reported to be involved in the correct folding of mature protein in vivo and in vitro (2, 3, 29-31) and in the inhibition of the activity of the mature proteinase (2). The propeptides of a number of eukaryotic proteinases have been shown to act as strong inhibitors, but their role in folding has not yet been examined. For example, the propeptides of the aspartyl proteinases, pepsin and cathepsin D, inhibit the mature enzymes (11); the propeptide region of the metalloproteinase, carboxypeptidase A, strongly inhibits the mature carboxypeptidase A (27); and a 62-amino-acid pro region of a cysteine proteinase, cathepsin B, strongly inhibits cathepsin B (9). Propeptides of fungal proteinases have not been tested as inhibitors of the respective mature enzymes. During the last several years, extracellular proteinases from different fungal species have been cloned and characterized (10, 13-15, 20, 25, 26, 32, 33). Some of them have large propeptides that in some cases approach the size of the mature enzyme (15,20,32). However, little is known about their function. In this paper, we describe expression of propeptides of a serine pro...

An extracellular elastinolytic metalloproteinase, purified from Aspergillus fumigatus isolated from an aspergillosis patient/and an internal peptide derived from it were subjected to N-terminal sequencing. Oligonucleotide primers based on these sequences were used to PCR amplify a segment of the metalloproteinase cDNA, which was used as a probe to isolate the cDNA and gene for this enzyme. The gene sequence matched exactly with the cDNA sequence except for the four introns that interrupted the open reading frame. According to the deduced amino acid sequence, the metalloproteinase has a signal sequence and 227 additional amino acids preceding the sequence for the mature protein of 389 amino acids with a calculated molecular mass of 42 kDa, which is close to the size of the purified mature fungal proteinase. This sequence contains segments that matched both the N terminus of the mature protein and the internal peptide. A. fumigatus metalloproteinase contains some of the conserved zinc-binding and active-site motifs characteristic of metalloproteinases but shows no overall homology with known metalloproteinases. The cDNA of the mature protein when introduced into Escherichia coli directed the expression of a protein with a size, N-terminal sequence, and immunological cross-reactivity identical to those of the native fungal enzyme. Although the