The complex chitinolytic system of aspergillus fumigatus

Abstract

Aspergillus fumigatus is one of the most important fungal pathogens of humans and there is an urgent need for new drugs to counter infections caused by A. fumigatus and other pathogenic species. Enzymes of chitin metabolism, and their regulators present novel targets for antifungal agents. During the work described here, the patterns of expression of the chiA1 and chiB1 chitinase genes of A. fumigatus during batch culture were investigated using real-time, reversetranscription PCR. The chiA1 gene, encoding the fungal/plant chitinase ChiA1, was expressed at significant levels throughout the six days of culture. However, the level of expression of chiB1, encoding the fungal/bacterial chitinase ChiB1, was only just detectable on day one but had been induced 1280-fold, to a level similar to that detected for chiA1 expression, by day 6. The results suggest markedly different roles for these enzymes. The gene encoding the transcription factor CreA was cloned and expressed, as a glutathione S-transferase (GST) fusion protein, in Escherichia coli. In electrophoretic mobility shift assays purified GST-CreA, or an A. fumigatus cell extract, bound specifically to putative CreA binding sites upstream of the chiB1 gene. CreA may therefore have a role in the regulation of chitinase activity in A. fumigatus. The effects of a range of compounds on A. fumigatus chitinase activity were determined. The cyclopentapeptides, argadin and argifin (each at 0.6 uM), were potent inhibitors of enzyme activity. The cyclic dipeptides, D-Leu-D-Pro, cyclo-(D-Leu-D-Pro) and cyclo-(L-His-L-Pro) (each at 300 uM), did not inhibit chitinase activity, while the methylxanthines, pentoxyfylline and theophylline, caused significant inhibition at concentrations of 75 uM and 300 uM, respectively. In preliminary expression studies, ChiA1 was fused with GST or maltose-binding protein (MBP) and expressed in E. coli. In addition, ChiA1-His6 peptide was expressed in Pichia pastoris. These constructs will be used in future work which will further explore the complex chitinolytic system of A. fumigatus and which may lead to the exploitation of this system as a target for antifungals.