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

Abstract

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 virulenceassociated 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 the pyrG gene, encoding orotidine-5-phosphate decarboxylase, and the pksP gene, encoding a polyketide synthase involved in both pigment biosynthesis and virulence of A. fumigatus, were fused with the egfp gene. The PpksP-egfp construct was integrated via homologous recombination into the genomic pksP locus. EGFP production was analyzed by fluorescence spectrometry, Western blot analysis, and fluorescence microscopy. Differential gene expression in A. fumigatus was 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-egfp expression was also detected in hyphae of germinating conidia isolated from the lungs of immunocompromised mice. This finding indicates that the pksP gene can also be expressed in hyphae under certain conditions and, furthermore, that the pksP gene might also contribute to invasive growth of the fungus.

The Cyclic AMP-Dependent Protein Kinase A Network Regulates Development and Virulence in Aspergillus fumigatus

“…By using the enhanced green fluorescent protein, it was shown that the pksP gene was expressed during sporulation. Interestingly, this gene was also expressed in outgrowing hyphae isolated from the lungs of infected immunocompromised mice (29). This finding suggested that certain stress conditions have an effect on pksP expression.…”

The Aspergillus fumigatus Transcriptional Regulator AfYap1 Represents the Major Regulator for Defense against Reactive Oxygen Intermediates but Is Dispensable for Pathogenicity in an Intranasal Mouse Infection Model

“…The PCR product generated was cloned into plasmid pCR2.1 (Invitrogen, The Netherlands). The DNA fragment encoding the Afyap1 gene was reisolated by cutting the recombinant plasmid obtained with BamHI and cloning the DNA fragment into plasmid pUCGH (34) to give plasmid pOtef-Afyap1-eGFP.…”

EstB-Mediated Hydrolysis of the Siderophore Triacetylfusarinine C Optimizes Iron Uptake of Aspergillus fumigatus

“…Therefore, a 2.2-kb SmaI-SacI egfp-encoding fragment was subcloned from plasmid pUCGH (10) into the compatible EcoRV-SacI sites of plasmid pGEM-5zf(ϩ) (Promega), yielding plasmid pGfp. The 5Ј-flanking region and the open reading frame of estB were PCR amplified by using oligonucleotides oEstAf9 and oEstAf11 (including a restriction enzyme site for NcoI, which replaces the stop codon).…”