Deletion of the Aspergillus fumigatus lysine biosynthesis gene lysF encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis

Abstract

Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality. In order to determine the importance of lysine biosynthesis for growth and pathogenicity, the A. fumigatus lysF gene, encoding a homologue of the A. nidulans homoaconitase LysF, was cloned and characterized. Cosmid cosGTM encoding lysF complemented a lysF mutant of Aspergillus nidulans. A. fumigatus lysF was deleted, resulting in a lysine-auxotroph. This phenotype was complemented to the wild-type by supplementation of the medium with both L-lysine and alpha-aminoadipic acid, or transformation using cosmid cosGTM. To study the virulence of the lysF deletion mutant of A. fumigatus, a low-dose intranasal mouse infection model of invasive aspergillosis was optimized for immunosuppressed BALB/c mice, allowing the application of an infection dose as low as 5 x 10(3) conidia per mouse. In this murine model, the Delta lysF mutant was avirulent, suggesting that lysine biosynthesis, or at least a functional homoaconitase, is important for survival of A. fumigatus in vivo and a potential target for antifungal drugs.

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

“…The murine low-dose model for invasive aspergillosis (45) as optimized by Liebmann et al (34) for BALB/c mice was used. Briefly, 18-to 20-g female BALB/c mice were immunosuppressed with 100 mg of cyclophosphamide (Sigma, Taufkirchen, Germany) per ml on days Ϫ4, Ϫ1, 2, 5, 8, and 11 prior to and after infection on day 0.…”

Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant

“…For instance, biosynthesis of lysine (14,15), aromatic amino acids (16), and folate (17); siderophore-dependent acquisition of iron (18); and transporter-mediated uptake of zinc (19) or calcium (20) are essential for the fungus to grow within the lungs and, therefore, determine its pathogenic effects. On the other hand, any sources of nitrogen in pulmonary tissue have not yet been clearly identified, despite many studies addressing N me-tabolism (21)(22)(23)(24)(25)(26), and there are indications that A. fumigatus endures nitrogen starvation during intrapulmonary growth (27).…”

Riboflavin and pantothenic acid biosynthesis are crucial for iron homeostasis and virulence in the pathogenic mold Aspergillus fumigatus

“…The strongly attenuated virulence of Δ riboB and Δ panA mutants in all tested virulence models demonstrates that both riboflavin and pantothenic acid biosynthesis are not only essential for invasion (intranasal infection model) but also during dissemination (intravenous infection model). In contrast, lysine biosynthesis was previously found to be essential exclusively for invasion but not during dissemination [10,11]. Virulence analysis of auxotrophs was previously tested in the closely related Aspergillus nidulans during the 1970s.…”

“…Using a combination of comparative genomics, transcriptomics and metabolic flux analysis, we recently identified 64 fungal-specific targets including metabolic enzymes participating in amino acid, lipid and vitamin biosynthesis [8]. Of these, 18 targets have already been validated in the literature, including enzymes participating in the biosynthesis of aromatic amino acids [9], lysine [10,11], histidine [12] and cysteine/methionine [13] and in the biosynthesis of lipids, including phospholipids [14], fatty acids [6] and oxylipins [15]. However, the assessment of fungal-specific targets in the vitamin biosynthesis pathways and their importance during infection has not been well established.…”