Isolation, activity and immunological characterisation of a secreted aspartic protease, CtsD, from Aspergillus fumigatus.
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.
Vickers
Imelda
I
National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland Maynooth, Co. Kildare, Ireland.
Reeves
Emer P
EP
Kavanagh
Kevin A
KA
Doyle
Sean
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000145:classification / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism*; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003470:Culture Media / Q000032:analysis; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D015870:Gene Expression; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000276:immunology; D002479:Inclusion Bodies / Q000378:metabolism; D007814:Larva / Q000382:microbiology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009036:Moths / Q000382:microbiology*; D010436:Pepstatins / Q000494:pharmacology; D010802:Phylogeny; D017510:Protein Folding; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000145:classification / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015996:Survival Rate; D014774:Virulence
D003470:Culture Media; D007074:Immunoglobulin G; D010436:Pepstatins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin