Aspergillus fumigatusCgrA is the ortholog of a yeast nucleolar protein that functions in ribosome synthesis. To determine how CgrA contributes to the virulence of A. fumigatus, a ⌬cgrA mutant was constructed by targeted gene disruption, and the mutant was reconstituted to wild type by homologous introduction of a functional cgrA gene. The ⌬cgrA mutant had the same growth rate as the wild type at room temperature. However, when the cultures were incubated at 37°C, a condition that increased the growth rate of the wild-type and reconstituted strains approximately threefold, the ⌬cgrA mutant was unable to increase its growth rate. The absence of cgrA function caused a delay in both the onset and rate of germination at 37°C but had little effect on germination at room temperature. The ⌬cgrA mutant was significantly less virulent than the wild-type or reconstituted strain in immunosuppressed mice and was associated with smaller fungal colonies in lung tissue. However, this difference was less pronounced in a Drosophila infection model at 25°C, which correlated with the comparable growth rates of the two strains at this temperature. To determine the intracellular localization of CgrA, the protein was tagged at the C terminus with green fluorescent protein, and costaining with propidium iodide revealed a predominantly nucleolar localization of the fusion protein in living hyphae. Together, these findings establish the intracellular localization of CgrA in A. fumigatus and demonstrate that cgrA is required for thermotolerant growth and wild-type virulence of the organism.Aspergillus fumigatus is a saprophytic filamentous fungus that inhabits soil, water, and organic debris, where it has an essential role in the recycling of carbon and nitrogen (37). The organism propagates itself by the release into the air of high concentrations of asexual spores (conidia), which are unavoidably inhaled on a daily basis (26,46). Since the conidia are efficiently cleared by normal defenses, their inhalation is of minor consequence to healthy individuals. However, in the absence of adequate host immunity, the conidia germinate into highly invasive hyphae that cause severe lung damage and eventually disseminate to other organs. Patients with depressed immunity are at increased risk for infection with A. fumigatus, and the prognosis for invasive disease is very poor in these individuals (40). Of particular concern is the rising incidence of aspergillosis, a situation that has arisen as a consequence of aggressive cancer treatments and the widespread use of potent immunosuppressive regimens that support organ transplantation (33,44,54,58).Since A. fumigatus conidia are no more prevalent in the environment than the spores of some nonpathogenic molds (34), it is generally assumed that the organism has unique features that allow it to survive in humans, and thermotolerance has long been suspected to play a role (37). As a major component of the biomass in a self-heating compost pile, A. fumigatus has evolved mechanisms that allow it to grow we...

SummaryWe have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like programmed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DcasA/DcasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the DcasA/DcasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sensitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DcasA/DcasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.

Background: Although Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.

The ability to thrive at 37 degrees C is characteristic of all human pathogens and has long been suspected to play a role in the pathogenesis of aspergillosis. As a thermotolerant fungus, Aspergillus fumigatus is capable of growth at temperatures that approach the upper limit for all eukaryotes, suggesting that the organism has evolved unique mechanisms of stress resistance that may be relevant to its ability to adapt to the stress of growth in the host. High temperature is a strain on many biological systems, particularly those involved in complex macromolecular assemblies such as ribosomes. This review will discuss the relationship between thermotolerance and virulence in pathogenic fungi, emphasizing the link to ribosome biogenesis in A. fumigatus. Future work in this area will help determine how rapid growth is accomplished at elevated temperature and may offer new avenues for the development of novel antifungals that disrupt thermotolerant ribosome assembly.