A fungus-specific ras homolog contributes to the hyphal growth and virulence of Aspergillus fumigatus

Abstract

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 DeltarasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBDelta113-135, 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 approximately 65% survival compared to approximately 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.

FIG. 1.

Deletion of A. fumigatus rasB. (A) Graphical representation of the rasB genomic locus from the wild-type, ΔrasB, and reconstituted strains (ΔrasB+rasB). Probes (P) used for Southern blotting are shown as small black boxes. Relative positions of the BamHI restriction sites are given. (B) Genomic Southern blot of wild-type, ΔrasB, and ΔrasB+rasB DNA digested with BamHI. (C) Reverse transcription-PCR of the isogenic set, showing loss and recovery of rasB message RNA in the ΔrasB and reconstituted strains, respectively. The loading control is A. fumigatus gpdA.

FIG. 2.

Germination, radial growth, and biomass of the isogenic set. (A) Graphic representation of germination rates from the wild-type (♦), ΔrasB (▪), and ΔrasB+rasB (▴) strains. Conidia from each strain were inoculated onto coverslips immersed in YG medium at 37°C. (B) Radial growth rates. Conidia (10⁴) from each strain were inoculated onto AMM solid agar and incubated for 48 h at 37°C. (C) Comparison of the total biomass of the isogenic set grown for 24 or 48 h at 37°C.

FIG. 3.

Colony morphology of the wild-type, ΔrasB, and reconstituted strains.

FIG. 4.

Hyphal morphology of the ΔrasB mutant. (A) The ΔrasB mutant grown for 12 h at 37°C with shaking at 250 rpm. (B) Wild-type A. fumigatus grown for 12 h at 37°C and 250 rpm. (C) Enlarged image of the ΔrasB strain grown for 24 h in YG medium at 37°C and 250 rpm.

FIG. 5.

Predicted protein sequences of the RasBΔ113-135 gene product. Sequence alignment of the predicted protein sequences for RasB (top line) and RasBΔ113-135 (bottom line). Conserved Ras protein domains are indicated above the sequences.

FIG. 6.

Phenotype of the rasBΔ113-135 mutant. Hyphal morphology of the rasBΔ113-135 strain after 12 h of growth at 37°C with shaking at 250 rpm. Arrowheads indicate sites of multiple branching.

FIG. 7.

Reduced virulence and fungal burden of the ΔrasB strain. (A) Survival plot comparison of the wild-type (▪), ΔrasB (○), and ΔrasB+rasB (▾) strains employed in a murine model of invasive aspergillosis. (B) QPCR analysis of fungal burden at days 2, 4, and 6 in mice inoculated with the isogenic set. Genomic equivalents were calculated from a standard curve generated from A. fumigatus DNA and normalized to mouse GAPDH. Dots represent duplicate QPCR results for each of three mice per group per day. Bars represent the average genomic equivalents for all mice at each time point.

FIG. 8.

Representative day 6 lesions from the wild-type (A), ΔrasB (B) and ΔrasB+rasB (C) strains. Tissue sections were stained with Grocott silver stain.