Article: article from journal or magazin.
Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus.
Publication types: Journal Article ; Research Support, Non-U.S. Gov't Publication Status: ppublish
Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.
Amino Acid Sequence, Animals, Aspergillosis/etiology, Aspergillosis/pathology, Aspergillus fumigatus/enzymology, Aspergillus fumigatus/genetics, Base Sequence, Cloning, Molecular, Collagenases/genetics, DNA, Complementary/genetics, DNA, Fungal/genetics, Genes, Fungal, Lung Diseases, Fungal/etiology, Lung Diseases, Fungal/pathology, Male, Metalloendopeptidases/genetics, Mice, Molecular Sequence Data, Mutation, Plasmids/genetics, Restriction Mapping, Virulence/genetics
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