Purification and properties of the elastase from Aspergillus fumigatus

Frosco, MaryBeth; Chase, Theodore; Macmillan, James D.

doi:10.1128/iai.60.3.728-734.1992

Cited by 79 publications

(43 citation statements)

References 26 publications

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“…Interestingly, similar results have been obtained by us [33] and other workers [26,[34][35][36] using more complex procedures. The molecular mass of the purified enzyme was estimated to be between 20 and 35 kDa [35] and more precisely 32-33 kDa [26,33,34,36]; minor bands with molecular masses lower than 30 kDa were demonstrated to be self-degradation products of the enzyme [26,34,36]. Moreover, these results obtained using different strains of A. fumigatus suggest that this 32-33-kDa serine proteinase is highly conserved in this pathogen.…”

Section: Fumigatus Various Enzyme Activities Were Charac-supporting

confidence: 88%

Extracellular fibrinogenolytic enzyme of Aspergillus fumigatus: substrate-dependent variations in the proteinase synthesis and characterization of the enzyme

Bouchara¹

1993

FEMS Immunology and Medical Microbiology

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To get a better understanding of the role of the previously reported fibrinogenolytic enzyme of Aspergillus fumigatus, we investigated the in vitro conditions of enzyme synthesis and attempted to characterize it. Modification of the nitrogen source did not influence the extracellular serine-proteinase profile, but resulted in important quantitative differences in the yields in batch cultures. The enzyme synthesis appeared to be an inducible phenomenon in A. fumigatus since it was initiated exclusively in the presence of proteins or protein hydrolysate. Free amino acids or inorganic nitrogen compounds could not promote significant enzyme production. Moreover, peptone at a concentration of 0.1% appeared to be the best inducer of enzyme synthesis. Conversely, modification of the carbon source did not affect fungal growth or enzyme synthesis. However, the production of chymotrypsin was highly sensitive to the carbohydrate level in the culture medium and, with peptone as nitrogen source, highest yields were obtained in the presence of 0.3 or 0.5% glucose. Culture filtrates of A. fumigatus CBS 113.26 grown with peptone or nitrate as nitrogen source were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the protein patterns suggested for the proteinase a molecular mass of 33 kDa which was confirmed by chromatographic purification of the enzyme through (N alpha-CBZ)-D-phenylalanine agarose.

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Section: Fumigatus Various Enzyme Activities Were Charac-supporting

confidence: 88%

Extracellular fibrinogenolytic enzyme of Aspergillus fumigatus: substrate-dependent variations in the proteinase synthesis and characterization of the enzyme

Bouchara¹

1993

FEMS Immunology and Medical Microbiology

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“…It has also been demonstrated that the prote01ytic activity in the brain of infected mice increases proportionally with the growth of A. fumigatus [3]. An extracellular alkaline protease (ALP), a serine protease of the subtilisin family, has been isolated from clinical sources of A. fumigatus [4][5][6]. This protease of 33 kDa was totally inhibited by PMSF, antipain, chymostatin, and /3-2-macroglobulin [5].…”

Section: Introductionmentioning

confidence: 99%

Virulence of alkaline protease-deficient mutants of Aspergillus fumigatus

Monod¹

1993

FEMS Microbiology Letters

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The gene encoding the secreted alkaline protease, a suspected virulence factor of Aspergillus fumigatus, was inactivated by gene disruption. The disruption was performed by transformation of a pathogenic strain of the fungus with a linear DNA fragment carrying the gene from which the central part was replaced by the selectable Escherichia coli hygromycin B dominant resistance marker. Two transformants were shown to produce no alkaline protease. Restriction fragment analysis of the DNA of these two transformants was consistent for chromosomal integration of the disrupted gene by homologous recombination. Both isogenic alkaline protease-producing and non-producing A. fumigatus strains invaded lung tissues, causing comparable mortality in immunosuppressed mice. A significant residual proteolytic activity observed in alkaline protease non-producing strain cultures could play a role in the invasion of the tissues by the fungus.

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“…In addition, when cultivated in a medium containing human fibrinogen as sole nitrogen source, A fumigatus produced an extracellular fibrinogenolytic enzyme [7]. This exoprotease, which has been identified to be a chymotrypsin-like serine protease [24], degraded elastin [17,331 and collagen [30]. Its ability to detach Vero ceils in culture from plastic substrate or to induce the detachment of human epithelial cells from basement membrane was also reported [33, 341. Destruction of the extracellular matrix proteins could contribute to the pathogenesis of the disease [27,28].…”

Section: Introductionmentioning

confidence: 99%

Interaction between Aspergillus fumigatus and basement membrane laminin: Binding and substrate degradation

Tronchin

Bouchara

Larcher

et al. 1993

Biology of the Cell

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Aspergillus fumigatus, the causative agent of human aspergillosis, binds to and degrades basement membrane laminin. Using immunoelectron microscopy, laminin binding appeared to be associated with the cell wall expansions of resting conidia, and progressively extended to the outer electron dense layer of the conidial wall during the germination process. Labeling of thin sections revealed numerous binding sites in the cytoplasm, whereas the inner cell wall and the plasma membrane were not labeled. Attachment of A fumigatus conidia on microtiter plates coated with laminin and its fragments P1 and E8 was also investigated. Conidia cells showed good adhesion to wells coated with laminin. As indicated by inhibition experiments, the interaction was specific and fragment P1 represented the major binding site on the laminin molecule. In addition, since A fumigatus produced an extracellular serine protease, we determined the susceptibility of laminin to this enzyme. We demonstrated that protease extract was capable to degrade laminin in solution as well as in tissue sections. The laminin cleavage products were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All the three chains were extensively degraded within 1 h. Treatment of the crude protease extract with the enzyme inhibitors, phenylmethylsulfonyl-fluoride and chymostatin, blocked the degradation of laminin, indicating a chymotrypsin-like serine protease activity. Immunofluorescence microscopy of cryostat sections of mouse and rat kidneys treated with the protease extract showed widespread loss of laminin epitopes from basement membranes. Enzyme treatment also removed immunoreactivity from lungs as observed after immunoperoxidase performed on paraffin sections. Binding and proteolytic degradation of laminin may together facilitate initial interaction of A fumigatus with the host tissues.

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Purification and properties of the elastase from Aspergillus fumigatus

Cited by 79 publications

References 26 publications

Extracellular fibrinogenolytic enzyme of Aspergillus fumigatus: substrate-dependent variations in the proteinase synthesis and characterization of the enzyme

Extracellular fibrinogenolytic enzyme of Aspergillus fumigatus: substrate-dependent variations in the proteinase synthesis and characterization of the enzyme

Virulence of alkaline protease-deficient mutants of Aspergillus fumigatus

Interaction between Aspergillus fumigatus and basement membrane laminin: Binding and substrate degradation

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