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.
