Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus

BACKGROUND: Although Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp. RESULTS: Here we describe the construction of plasmid vectors that can be used to regulate gene expression in A. fumigatus using a simple co-transfection approach. Vectors were generated in which the tetracycline transactivator (tTA) or the reverse tetracycline transactivator (rtTA2(s)-M2) are controlled by the A. nidulans gpdA promoter. Dominant selectable cassettes were introduced into each plasmid, allowing for selection following gene transfer into A. fumigatus by incorporating phleomycin or hygromycin into the medium. To model an essential gene under tetracycline regulation, the E. coli hygromycin resistance gene, hph, was placed under the control of seven copies of the TetR binding site (tetO(7)) in a plasmid vector and co-transfected into A. fumigatus protoplasts together with one of the two transactivator plasmids. Since the hph gene is essential to A. fumigatus in the presence of hygromycin, resistance to hygromycin was used as a marker of hph reporter gene expression. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the tetO(7)-hph reporter gene, and the addition of doxycycline to the medium suppressed hygromycin resistance in a dose-dependent manner. Similarly, transformants were identified in which expression of rtTA2(s)-M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the tetO(7)-hph reporter gene were within non-toxic ranges for this organism, and low-iron medium was shown to reduce the amount of doxycycline required to accomplish regulation. CONCLUSIONS: The vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in A. fumigatu

Effects of doxycycline on hygromycin sensitivity of a strain expressing rtTA in conjunction with the -reporter gene

<p><b>Copyright information:</b></p><p>Taken from "Doxycycline-regulated gene expression in the opportunistic fungal pathogen "</p><p>BMC Microbiology 2005;5():1-1.</p><p>Published online 13 Jan 2005</p><p>PMCID:PMC546209.</p><p>Copyright © 2005 Vogt et al; licensee BioMed Central Ltd.</p> Ten thousand conidia were spotted onto the center of a plate containing 750 μg/ml hygromycin and the indicated concentrations of doxycycline (μg/ml) and colony diameter was measured with time. Below: levels of RNA levels in the rtTA transformant by Northern blot analysis. Total RNA isolated from overnight cultures grown in the presence of 0, 5 or 50 μg/ml of doxycycline was fractionated by agarose gel electrophoresis, transferred to nylon membranes, and probed with a P-labeled probe

Schematic overview of the tetracycline-regulated gene expression system used in this study

<p><b>Copyright information:</b></p><p>Taken from "Doxycycline-regulated gene expression in the opportunistic fungal pathogen "</p><p>BMC Microbiology 2005;5():1-1.</p><p>Published online 13 Jan 2005</p><p>PMCID:PMC546209.</p><p>Copyright © 2005 Vogt et al; licensee BioMed Central Ltd.</p> The hygromycin resistance gene, , is under the control of seven copies of the TetR binding sequence, , linked to a minimal promoter (Pmin). In the tTA-dependent expression system (A), the tTA protein (green circles) promotes hygromycin resistance of by binding to the promoter and activating transcription of the gene. Incorporation of doxycycline into the medium prevents tTA from binding to , resulting in hygromycin sensitivity due to the absence of expression. The reverse tTA system (B) takes advantage of a reverse tetracycline transactivator, rtTA (blue circles), that binds to the promoter only in the presence of doxycycline. In this case, rtTA promotes hygromycin resistance of only when grown in the presence of doxycycline

Effects of doxycycline on the hygromycin sensitivity of two strains that express tTA (p444) in conjunction with the -reporter gene (p482)

<p><b>Copyright information:</b></p><p>Taken from "Doxycycline-regulated gene expression in the opportunistic fungal pathogen "</p><p>BMC Microbiology 2005;5():1-1.</p><p>Published online 13 Jan 2005</p><p>PMCID:PMC546209.</p><p>Copyright © 2005 Vogt et al; licensee BioMed Central Ltd.</p> (A): Ten thousand conidia from the tTA-1 or tTA-2 transformants were spotted onto the center of a plate containing 1 mg/ml hygromycin and the indicated concentrations of doxycycline (μg/ml) and colony diameter was measured with time. (B): Doxycycline regulation of RNA levels in the tTA-1 and tTA-2 strains by Northern blot analysis. Total RNA isolated from overnight cultures grown in the presence of 0–200 μg/ml doxycycline was fractionated by agarose gel electrophoresis, transferred to nylon membranes, and probed with a P-labeled probe. (C): Hybridization intensity in each lane of the Northern blot in (B) was normalized to levels of the SYBR-green II-stained rRNA by phosphorimager analysis and is shown as a percentage of the levels seen in the tTA-1 transformant in the absence of doxycycline

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress

We have examined the contribution of metacaspases to the growth and stress response of the opportu-nistic human mould pathogen, Aspergillus fumiga-tus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like pro-grammed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DcasA/DcasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated pro-teolytic activity against caspase substrates, phos-phatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phos-phatidylserine exposure was not observed in sta-tionary phase cultures of the DcasA/DcasB mutant, although caspase activity and viability was indi-stinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sen-sitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DcasA/DcasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contrib-utes to the apoptotic-like loss of membrane phos-pholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress

A Fungus-Specific Ras Homolog Contributes to the Hyphal Growth and Virulence of Aspergillus fumigatus

The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The ΔrasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBΔ113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited ∼65% survival compared to ∼10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae