Differential expression of the Aspergillus fumigatus pksP gene detected invitro and in vivo with green fluorescent protein

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To be able to analyze the expression of putative virulence-associated genes o f A. fumigatus, the use of the enhanced green fluorescent protein (EGFP) as a reporter was established. Two 5 ' sequences, containing the putative pro moters of the PyrG gene, encoding orotidine-5 ' -phosphate decarboxylase, a nd the pksP gene, encoding a polyketide synthase involved in both pigment b iosynthesis and virulence of A. fumigatus, were fused with the egfp gene. T he PpksP-egfp construct was integrated via homologous recombination into th e genomic pksP locus. EGFP production was analyzed by fluorescence spectrom etry, Western blot analysis, and fluorescence microscopy. Differential gene expression in A. fumigatus was observed. Fluorescence derived from the PYR G-EGFP fusion protein was detected during all developmental stages of the f ungus, i.e., during germination, during vegetative growth, in conidiophores , and weakly in conidia. In addition, it was also detected in germinating c onidia when isolated from the lungs of immunocompromised mice. By contrast, PKSP-EGFP-derived fluorescence was not found in hyphae or stalks of conidi ophores but was found in phialides and conidia in vitro when the fungus was grown under standard conditions, indicating a developmentally controlled e xpression of the gene. Interestingly, pksP-egfp expression was also detecte d in hyphae of germinating conidia isolated from the lungs of immuno compro mised mice. This finding indicates that the pksP gene can also be expressed in hyphae under certain conditions and, furthermore, that the pksP gene mi ght also contribute to invasive growth of the fungus.