Differential Expression of the Aspergillus fumigatus pksPGene Detected In Vitro and In Vivo with Green Fluorescent Protein

ABSTRACTAspergillus fumigatusis 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 of A. fumigatus, the use of the enhanced green fluorescent protein (EGFP) as a reporter was established. Two 5′ sequences, containing the putative promoters of thepyrGgene, encoding orotidine-5′-phosphate decarboxylase, and the pksPgene, encoding a polyketide synthase involved in both pigment biosynthesis and virulence ofA. fumigatus, were fused with the egfpgene. The PpksP-egfpconstruct was integrated via homologous recombination into the genomicpksPlocus. EGFP production was analyzed by fluorescence spectrometry, Western blot analysis, and fluorescence microscopy. Differential gene expression in A. fumigatuswas observed. Fluorescence derived from the PYRG-EGFP fusion protein was detected during all developmental stages of the fungus, i.e., during germination, during vegetative growth, in conidiophores, and weakly in conidia. In addition, it was also detected in germinating conidia when isolated from the lungs of immunocompromised mice. By contrast, PKSP-EGFP-derived fluorescence was not found in hyphae or stalks of conidiophores but was found in phialides and conidia in vitro when the fungus was grown under standard conditions, indicating a developmentally controlled expression of the gene. Interestingly,pksP-egfpexpression was also detected in hyphae of germinating conidia isolated from the lungs of immunocompromised mice. This finding indicates that thepksPgene can also be expressed in hyphae under certain conditions and, furthermore, that the pksPgene might also contribute to invasive growth of the fungus.