Disruption of a nonribosomal peptide synthetase in Aspergillus fumigatus eliminates gliotoxin production

Abstract

The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in DeltagliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the DeltagliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the DeltagliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.

FIG. 1.

Disruption of A. fumigatus NRPS gene gliP. (A) Representation of the gliP locus in wild-type and ΔgliP mutant strains. In ΔgliP mutant strain ARC2, a 3.8-kb internal section of the gliP coding region was replaced with the 3.1-kb pyrG gene from A. parasiticus. The ApaI restriction enzyme was used to digest chromosomal DNA, which was probed with the 1.0-kb probe as indicated. (B) PCR confirmation of gliP gene disruption. Lanes: L, DNA ladder; N, water control; A5, ectopic transformant strain ARC5; A2, ΔgliP mutant strain ARC2; WT, wild-type strain AF293. Group 1, amplification of internal gliP amplicon absent in ΔgliP mutant strain ARC2 because this sequence was replaced with pyrG via homologous recombination. Groups 2 and 3, 5′ and 3′ flanking regions, respectively. Amplification products in ΔgliP mutant strain ARC2 indicate successful homologous recombination at the gliP locus. (C) Southern analysis of A. fumigatus transformants. Lanes are the same as those in panel B.

FIG. 2.

HPLC analysis of the A. fumigatus strains used in this study. Chloroform extracts of CFs from Czapek Dox broth cultures grown for 72 h at 37°C. Gliotoxin and unknown metabolite peaks are identified. Panels: A, gliotoxin standard, 1 mg/ml; B, wild-type AF293; C, ectopic transformant ARC5; D, ΔgliP mutant strain ARC2 (lacks the peak at approximately 8.3 min, which demonstrates elimination of gliotoxin production in this mutant).

FIG. 3.

Scanning electron microscopy of conidia from the A. fumigatus strains used in this study. Panels: A, ectopic transformant ARC5; B, ΔgliP mutant strain ARC2; C, wild-type strain AF293. No change in conidial surface morphology is apparent.

FIG. 4.

Inhibition of ionomycin-dependent degranulation of RBL-2H3 mast cells by wild-type CF. CFs were added to RBL-2H3 mast cells, which were then incubated for 2 h. Mast cells were then stimulated for 1 h with 0.1 ml of 1 μM ionomycin. Significant inhibition of ionomycin-dependent degranulation is shown in wild-type CF versus the CF from ΔgliP mutant strain ARC2. *, P < 0.05 compared to wild-type (WT) CF plus ionomycin and a medium-alone control.

FIG. 5.

Inhalational neutropenic murine model of IA. Outbred ICR mice were used to assess the role of gliotoxin in fungal pathogenesis. Mice were immunosuppressed by i.p. injection of cyclophosphamide (250 mg/kg) and s.c. injection of cortisone acetate (250 mg/kg) 2 days prior to infection and injection of 200 mg/kg cyclophosphamide and 250 mg/kg cortisone acetate 3 days postinoculation. Mice were inoculated by exposure to a 10⁹-conidium/ml suspension for 1 h in an inhalation chamber. A log rank test was used for pairwise comparisons of survival levels among the strain groups. P value for comparison between ARC2 and wild-type (WT) AF293, 0.642.