Disruption of theAspergillus fumigatusGene Encoding Nucleolar Protein CgrA Impairs Thermotolerant Growth and Reduces Virulence

Abstract

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...

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

“…Larvae of G. mellonella have been used previously to distinguish between Aspergillus species producing different levels of the immunosuppressive agent gliotoxin [7] and insects have also been used to quantify the role of rel A gene in contributing to the virulence of P. aeruginosa [16] and the virulence of mutants of A. fumigatus lacking the nucleolar protein Cgr A [11]. There is increasing attention being focused on the use of insects for measuring changes in microbial virulence -a process which has time and cost benefits to the researcher as well as ethical benefits in reducing the need to use mammals for this form of testing [3].…”

“…G. mellonella have also been utilised as a model for studying the virulence of Cryptococcus neoformans [10]. Bhabhra et al [11] have used an insect model to assess the virulence of mutants of A. fumigatus lacking the nucleolar protein Cgr A. In addition larvae of silkworms have been utilised to measure the efficacy of antibiotics in killing bacteria and a positive correlation with the results obtained from murine studies has been demonstrated [12].…”

The virulence of the opportunistic fungal pathogenAspergillus fumigatusrequires cooperation between the endoplasmic reticulum-associated degradation pathway (ERAD) and the unfolded protein response (UPR)

“…Both inserts were gel-purified and 10 μg of each was used to transform wt-ΔakuA 36 protoplasts as previously described. 37 Loss of the derA gene was confirmed by Southern blot analysis of genomic DNA isolated from hygromycin resistant monoconidial isolates. Probe 1 (Fig.…”

Drosophila and Galleria insect model hosts

“…First, an Aspergillus fumigatus mutant strain lacking CgrA, a key thermotolerance regulator, was hypovirulent in mice but fully virulent in Toll-deficient Drosophila. 47 Therefore, because flies are infected and maintained at 29°C, certain aspects of fungal virulence in mammals may not be accurately modeled in this organism; Galleria, which can be maintained at 37°C, the mammalian physiologic temperature, may be used instead, taking into account however that increasing the temperature of Galleria to 37°C itself alters cellular and humoral immune responses. 48,49 Second, the alb1-deficient Aspergillus fumigatus mutant, which is hypovirulent in mice and flies, 11,50 was hypervirulent in Galleria, in which it appears to trigger dysregulated immunopathology.…”