VeA regulates conidiation, gliotoxin production, and protease activity in the opportunistic human pathogen Aspergillus fumigatus

Abstract

Invasive aspergillosis by Aspergillus fumigatus is a leading cause of infection-related mortality in immunocompromised patients. In this study, we show that veA, a major conserved regulatory gene that is unique to fungi, is necessary for normal morphogenesis in this medically relevant fungus. Although deletion of veA results in a strain with reduced conidiation, overexpression of this gene further reduced conidial production, indicating that veA has a major role as a regulator of development in A. fumigatus and that normal conidiation is only sustained in the presence of wild-type VeA levels. Furthermore, our studies revealed that veA is a positive regulator in the production of gliotoxin, a secondary metabolite known to be a virulent factor in A. fumigatus. Deletion of veA resulted in a reduction of gliotoxin production with respect to that of the wild-type control. This reduction in toxin coincided with a decrease in gliZ and gliP expression, which is necessary for gliotoxin biosynthesis. Interestingly, veA also influences protease activity in this organism. Specifically, deletion of veA resulted in a reduction of protease activity; this is the first report of a veA homolog with a role in controlling fungal hydrolytic activity. Although veA affects several cellular processes in A. fumigatus, pathogenicity studies in a neutropenic mouse infection model indicated that veA is dispensable for virulence.

Fig 1

DNA and RNA analyses for the verification of the A. fumigatus deletion and overexpression mutants. (A) Schematic representation showing EcoRI sites in the A. fumigatus wild-type veA locus and the veA deletion construct generated to replace veA with the A. fumigatus pyrG gene (pyrG^{A. fum}) utilized as a transformation marker gene. The fragment used as the probe template for Southern blot analysis is also shown. The sizes of the DNA fragments predicted for the Southern blot analysis using this probe are also shown for both the wild-type and veA deletion mutant strains. (B) Southern blot analysis. The veA gene replacement construct was transformed in CEA17. EcoRI-digested genomic DNA of CEA10 wild-type (WT), ΔveA transformant (TSD1.15), and complementation transformant (TSD3.5) was hybridized with the probe shown in panel A. Additional deletion transformants also showed the same band pattern (data not shown). The extra band in the analysis of the complementation strain indicates integration of the wild-type veA allele in the TSD1.15 genomic DNA. Additional complementation transformants were also obtained. Integration of the full complementation cassette in these strains was also confirmed by PCR (data not shown). (C) Diagram showing the veA overexpression DNA cassette (from plasmid pSD21) used in this study. Vertical lines indicate the annealing sites for the primers gpdAF592and veAR399_NotI (Table 2) used in the diagnostic PCR of four veA overexpression transformants shown in panel D, with a predicted PCR product of 1,917 bp. (E) Expression analysis of veA by qRT-PCR using primers veA_qRTPCR_F814 and veA_qRTPCR_R815 (Table 2).

Fig 2

Role of veA in A. fumigatus colony growth and conidiation. (A) Aspergillus fumigatus wild-type (WT), ΔveA, complementation (com), and OEveA (OE) strains were point inoculated on Czapek Dox medium and incubated for 5 days in the dark. (B) Colony diameter measurements. (C) Quantification of conidial production. Cores (16 mm in diameter) were taken 1 cm from the center of the plates and homogenized in water. Spores were counted using a hemocytometer. (D) Relative expression levels of brlA after 72 h in liquid stationary cultures. The Aspergillus fumigatus 18S gene was used as an internal reference gene. Error bars represent standard errors.

Fig 3

veA controls gliotoxin production. Aspergillus fumigatus wild-type (WT), ΔveA mutant, complementation (com), and OEveA (OE) strains were grown in liquid stationary cultures in Czapek Dox medium. Samples were collected for toxin analysis 72 h and 120 h after inoculation and extracted with chloroform as detailed in Materials and Methods. Gliotoxin production in these cultures was analyzed by HPLC. The experiment was done with three replicates. Error bars represent standard errors. Different letters indicate samples that are significantly different (P ≤ 0.05).

Fig 4

Expression of gliZ and gliP is regulated by veA. The transcriptional pattern of gliZ (A) and gliP (B) was evaluated by qRT-PCR. Total RNA was isolated at 48 and 72 h from liquid stationary Czapek Dox cultures incubated in the dark at 37°C. Primer pairs gliZ_qRTPCR_F838 and gliZ_qRTPCR_R839 and gliP_qRTPCR_R841 and gliP_qRTPCR_R841 (Table 2) were used to measure expression of gliZ and gliP, respectively. The relative expression levels were calculated using the 2^−ΔCT method described by Schmittgen and Livak (78). Expression of the A. fumigatus 18S gene was used as internal reference. Values were normalized to that of the wild type at 48 h, which was considered 1. The error bars indicate the ranges for three replicates.

Fig 5

veA controls gliotoxin production through additional mechanisms besides influencing gliZ expression. (A) HPLC analysis of gliotoxin from the Aspergillus fumigatus wild-type (WT) and OEveA and OEgliZ strains after 72 and 120 h of incubation in liquid stationary cultures. (B and C) Transcriptional patterns of gliZ and gliP, respectively. Total RNA was isolated at 48 and 72 h after inoculation. Primer pairs gliZ_qRTPCR_F838 and gliZ_qRTPCR_R839 and gliP_qRTPCR_R841 and gliP_qRTPCR_R841 (Table 2) were used to measure expression of gliZ and gliP, respectively. The relative expression levels were calculated using the 2^−ΔCT method described by Schmittgen and Livak (78). Expression of the A. fumigatus 18S gene was used as an internal reference. Values were normalized to that of the wild type at 48 h, which was considered 1. The error bars indicate the ranges for three replicates.

Fig 6

levels of veA expression are necessary for normal protease activity. (A) Aspergillus fumigatus wild-type (WT), ΔveA, complementation (com), and OEveA (OE) strains were point inoculated on Czapek Dox medium containing 5% skim milk. The plates were grown at 37°C in the dark. Degradation halos indicate protease activity. (B) Quantification of proteolytic activity measured by azocasein assay. The experiment included four replicates and was repeated twice with similar results. Error bars represent standard errors. Different letters indicate statistically significant differences (P ≤ 0.05).

Fig 7

Aspergillus fumigatus VeA preferentially localizes in nuclei. (A) Representation of the strategy to fuse GFP to VeA. The tagged construct was introduced at the veA locus by a double-recombination event. (B) Micrographs showing the subcellular localization of the VeA::GFP fusion described in panel A in A. fumigatus. From left to right, Nomarski images, DAPI images, and green fluorescence images are included. DAPI images were used as a nuclear localization internal control. Scale bars represent 10 μm. Arrows indicate nuclei.

Fig 8

veA is dispensable in Aspergillus fumigatus virulence. (A) Neutropenic CD-1 mice were infected intranasally with conidia from Aspergillus fumigatus wild-type (WT), ΔveA, complementation (com), and OEveA (OE) strains. Survival rates are shown. (B) Fungal burden in mouse lungs measured by qPCR. DNA isolated from whole mouse lungs was used. The experiment included 10 mice per group.