Isolation, activity and immunological characterisation of a secreted aspartic protease, CtsD, from Aspergillus fumigatus

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 (0.2 mg/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 opt4.0 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 μg 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.