Aspergillus fumigatus does not require fatty acid metabolism via isocitrate lyase for development of invasive aspergillosis

Abstract

Aspergillus fumigatus is the most prevalent airborne filamentous fungus causing invasive aspergillosis in immunocompromised individuals. Only a limited number of determinants directly associated with virulence are known, and the metabolic requirements of the fungus to grow inside a host have not yet been investigated. Previous studies on pathogenic microorganisms, i.e., the bacterium Mycobacterium tuberculosis and the yeast Candida albicans, have revealed an essential role for isocitrate lyase in pathogenicity. In this study, we generated an isocitrate lyase deletion strain to test whether this strain shows attenuation in virulence. Results have revealed that isocitrate lyase from A. fumigatus is not required for the development of invasive aspergillosis. In a murine model of invasive aspergillosis, the wild-type strain, an isocitrate lyase deletion strain, and a complemented mutant strain were similarly effective in killing mice. Moreover, thin sections demonstrated invasive growth of all strains. Additionally, thin sections of lung tissue from patients with invasive aspergillosis stained with anti-isocitrate lyase antibodies remained negative. From these results, we cannot exclude the use of lipids or fatty acids as a carbon source for A. fumigatus during invasive growth. Nevertheless, test results do imply that the glyoxylate cycle from A. fumigatus is not required for the anaplerotic synthesis of oxaloacetate under infectious conditions. Therefore, an antifungal drug inhibiting fungal isocitrate lyases, postulated to act against Candida infections, is assumed to be ineffective against A. fumigatus.

FIG. 1.

Deletion and reintroduction of the isocitrate lyase gene. (A) Scheme of the genomic situation at the acuD locus in the wild type, a complemented deletion mutant, and a deletion mutant. Restriction with EcoRI results in a 7.96-kb fragment for both the wild-type and the complemented strains and a 4.24-kb fragment for the mutant when the probe is hybridized to the upstream region. (B) Southern blot analysis of genomic DNA of the wild-type strain and the independent acuD deletion mutants. The shift of the fragment is indicated by an arrow. (C) Southern blot analysis of the deletion strains complemented with the acuD gene. Bands characteristic of the acuD deletion strain and the wild type are indicated by arrows. (D) Growth analysis of the wild type, an isocitrate lyase mutant, and four complemented strains. WT, wild type; Δ, isocitrate lyase deletion strain; R, complemented strains.

FIG. 2.

Western blot analysis and determination of isocitrate lyase activity. (A) The upper panel shows a Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of cell extracts of isocitrate lyase deletion mutants and the wild type (ΔakuB WT) grown on a glucose-acetate medium. The lower panel shows the respective Western blot analysis performed by use of the specific anti-isocitrate lyase antibody E30F8. As a control, purified isocitrate lyase was loaded (Icl control). Numbers below the panels indicate the specific isocitrate lyase activities which were determined for each extract. (B) Same as for panel A, but in addition to the wild-type and deletion strains, several complemented strains were investigated.

FIG. 3.

Animal model for invasive aspergillosis. (A) Survival curves of cortisone acetate-treated Swiss OF1 mice (in cohorts of 10 mice each) infected with 1 × 10⁵ conidia from the wild type (WT), with the ΔacuD 12C1 deletion strain, with the complemented 12C1RC2 strain, and with PBS as a control. (B) Histopathology of lung tissue sections 5 days after infection. All lung sections shown in panel B derive from the animal experiment described for panel A.

FIG. 4.

Growth analysis of the wild type and an isocitrate lyase deletion mutant on different carbon sources. (A) Representative sections from chamber slide wells are shown. Carbon sources and the times of incubation are indicated on the left. (B) Growth analysis of the wild type and a deletion strain in the presence of 50 mM acetate (A 50) and different concentrations of peptone (P). The growth time was fixed to 7.5 h. (C) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses of the wild type grown on 1% (wt/vol) peptone (lanes 1), 1% (wt/vol) peptone plus 0.5% (wt/vol) olive oil (lanes 2), and 0.5% (wt/vol) olive oil (lanes 3). Lanes C, purified isocitrate lyase taken as a control. The values (mU/mg) mark the isocitrate lyase activities determined from the cell extracts.