Aspergillus fumigatus Ras homologs regulate vegetative growth, asexual development and virulence

Conidial germination and growth by apical extension are two processes required for the initiation and progression of disease by the opportunistic pathogen Aspergillus fumigatus. The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. Therefore, we sought to determine the requirements for Ras proteins in conidial germination and hyphal development of A. fumigatus. Previously, only one homolog, rasA , had been identified, but nothing is known of its role in growth and development. We have identified a second homolog, rasB, which characterizes a new subclass of Ras genes, found only in fungi that undergo hyphal growth. Unique to the rasB homologs is a conserved amino acid domain found just upstream of the third GTP/GDP binding region. To gain insight into the roles played by RasB in A. fumigatus, dominant active (DA), dominant negative (DN) and deletion mutations were constructed for both Ras homologs.; DArasB expression led to reduced conidiation. Expression of DNrasB slightly delayed the initiation of germination and caused the development of conidiophores in submerged culture, an abnormal event in A. fumigatus. Deletion of A. fumigatus rasB caused decreased germination and growth rates but no significant change in total biomass after 24 hours of growth. Deletion of rasB also created an irregular hyphal morphology characterized by increased branching. Expression of rasBDelta113-135, a mutant lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype. Virulence of the rasB deletion strain was also reduced, as mice infected with this strain exhibited ∼70% survival compared to ∼10% with wild type and reconstituted strains.; In contrast, DArasA expression led to reduced conidiation, malformed conidiophores, and altered mitotic progression. Expression of DN rasA caused a significant reduction in the rate of conidial germination, without further altering development. However, complete deletion of rasA caused slowed germination, nearly absent radial outgrowth, and decreased hyphal mass. Although conidiation was reduced and delayed, the conidial viability was near wild type levels. The DeltarasA mutant was also resistant to protoplast preparation using normal levels of cell wall digestion enzymes. Normal mitotic events in the mutant were also disturbed, as staining of nuclear material revealed many small nuclei that were irregularly positioned throughout the hyphal compartments.; RasA and RasB appear to play distinct, and overlapping, roles in the vegetative growth and asexual development of A. fumigatus. The data in this study, along with the fact that rasB homologs are highly conserved among fungi that undergo hyphal growth, also support the hypothesis that rasB may control signaling modules important to the directional growth of fungal hyphae.