VIRULENCE OF AN ASPERGILLOPEPSIN-DEFICIENT MUTANT OF ASPERGILLUS-FUMIGATUS AND EVIDENCE FOR ANOTHER ASPARTIC PROTEINASE LINKED TO THE FUNGAL CELL-WALL

A gene replacement was performed to produce mutants of Aspergillus fum igatus deficient in the aspergillopepsin PEP (E.C. 3.4.23.18). The cor rect replacement of the PEP gene was confirmed by PCR and Southern hyb ridization experiments, whereas the absence of PEP production was demo nstrated by Western blots. The culture supernatant of the transformant s showed no detectable acid proteinase activity, suggesting that there is only one acid proteinase secreted by the fungus. The wild-type str ain and the PEP-deficient mutants invaded tissues to a similar extent and produced comparable mortality in guinea pigs. As PEP represents a third secretory proteinase of A. fumigatus and the other two proteinas es also did not show significant influence on fungal invasiveness, it is probable that secreted proteinases do not contribute decisively to tissue invasion in the pathogenesis of systemic aspergillosis. However , immunofluorescence on A, fumigatus colonies using polyclonal antibod ies to PEP showed a similar pattern for the wild-type and for the muta nts, with a bright fluorescence on young conidiophores, on submerged m ycelium and on the tips of growing aerial mycelium. Conidia and mature aerial hyphae were not fluorescent. This pattern could reflect the ex istence of another crossreacting aspartic proteinase (PEP2) which was found to be sensitive to pepstatin but tightly linked to the fungal ce ll wall.