Abstract
Aspergillus fumigatus is an opportunistic fungal pathogen that infects immunocompromised patients. A putative aspartic protease gene (ctsD; 1425 bp; intron-free) was identified and cloned. CtsD is evolutionarily distinct from all previously identified A. fumigatus aspartic proteases. Recombinant CtsD was expressed in inclusion bodies in Escherichia coli (0.2mg/g cells) and subjected to extensive proteolysis in the baculovirus expression system. Activation studies performed on purified, refolded, recombinant CtsD resulted in protease activation with a pH(opt)4.0 and specific activity=10 U/mg. Pepstatin A also inhibited recombinant CtsD activity by up to 72% thereby confirming classification as an aspartic protease. Native CtsD was also immunologically identified in culture supernatants and purified from fungal cultures using pepstatin-agarose affinity chromatography (7.8 microg CtsD/g mycelia). In A. fumigatus, semi-quantitative RT-PCR analysis revealed expression of ctsD in minimal and proteinaceous media only. Expression of ctsD was absent under nutrient-rich conditions. Expression of ctsD was also detected, in vivo, in the Galleria mellonella virulence model following A. fumigatus infection.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Animals
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Aspartic Acid Endopeptidases / antagonists & inhibitors
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Aspartic Acid Endopeptidases / chemistry
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Aspartic Acid Endopeptidases / classification
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Aspartic Acid Endopeptidases / immunology*
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Aspartic Acid Endopeptidases / isolation & purification*
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Aspartic Acid Endopeptidases / metabolism*
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Aspergillus fumigatus / enzymology*
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Aspergillus fumigatus / genetics
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Aspergillus fumigatus / pathogenicity*
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Baculoviridae / genetics
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Base Sequence
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Binding Sites
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Chromatography, Affinity
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Cloning, Molecular
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Culture Media / analysis
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Enzyme Activation
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Enzyme-Linked Immunosorbent Assay
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Escherichia coli / genetics
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Evolution, Molecular
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Gene Expression
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Genes, Fungal
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Hydrogen-Ion Concentration
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Hydrolysis
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Immunoglobulin G / immunology
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Inclusion Bodies / metabolism
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Larva / microbiology
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Models, Biological
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Molecular Sequence Data
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Moths / microbiology*
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Pepstatins / pharmacology
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Phylogeny
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Protein Folding
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Protein Sorting Signals
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Recombinant Proteins / antagonists & inhibitors
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Recombinant Proteins / chemistry
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Recombinant Proteins / classification
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Recombinant Proteins / immunology
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Survival Rate
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Virulence
Substances
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Culture Media
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Immunoglobulin G
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Pepstatins
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Protein Sorting Signals
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Recombinant Proteins
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Aspartic Acid Endopeptidases
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pepstatin