Jarrod R. Fortwendel

Disruption of the Aspergillus fumigatus Gene Encoding Nucleolar Protein CgrA Impairs Thermotolerant Growth and Reduces Virulence

ABSTRACT Aspergillus fumigatus CgrA 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 ΔcgrA mutant was constructed by targeted gene disruption, and the mutant was reconstituted to wild type by homologous introduction of a functional cgrA gene. The ΔcgrA 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 ΔcgrA 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 ΔcgrA 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.

Differential Effects of Inhibiting Chitin and 1,3-β-d-Glucan Synthesis in Ras and Calcineurin Mutants of Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus must be able to properly form hyphae and maintain cell wall integrity in order to establish invasive disease. Ras proteins and calcineurin each have been implicated as having roles in these processes. Here, we further delineate the roles of calcineurin and Ras activity in cell wall biosynthesis and hyphal morphology using genetic and pharmacologic tools. Strains deleted for three genes encoding proteins of these pathways, rasA (the Ras protein), cnaA (calcineurin), or crzA (the zinc finger transcription factor downstream of calcineurin), all displayed decreased cell wall 1,3-β-d-glucan content. Echinocandin treatment further decreased the levels of 1,3-β-d-glucan for all strains tested yet also partially corrected the hyphal growth defect of the ΔrasA strain. The inhibition of glucan synthesis caused an increase in chitin content for wild-type, dominant-active rasA, and ΔrasA strains. However, this important compensatory response was diminished in the calcineurin pathway mutants (ΔcnaA and ΔcrzA). Taken together, our data suggest that the Ras and calcineurin pathways act in parallel to regulate cell wall formation and hyphal growth. Additionally, the calcineurin pathway elements cnaA and crzA play a major role in proper chitin and glucan incorporation into the A. fumigatus cell wall.

Transcriptional regulation of chitin synthases by calcineurin controls paradoxical growth of Aspergillus fumigatus in response to caspofungin.

ABSTRACT Attenuated activity of echinocandin antifungals at high concentrations, known as the “paradoxical effect,” is a well-established phenomenon in Candida albicans and Aspergillus fumigatus. In the yeast C. albicans, upregulation of chitin biosynthesis via the protein kinase C (PKC), high-osmolarity glycerol response (HOG), and Ca2+/calcineurin signaling pathways is an important cell wall stress response that permits growth in the presence of high concentrations of echinocandins. However, nothing is known of the molecular mechanisms regulating the mold A. fumigatus and its paradoxical response to echinocandins. Here, we show that the laboratory strain of A. fumigatus and five of seven clinical A. fumigatus isolates tested display various magnitudes of paradoxical growth in response to caspofungin. Interestingly, none of the eight strains showed paradoxical growth in the presence of micafungin or anidulafungin. Treatment of the ΔcnaA and ΔcrzA strains, harboring gene deletions of the calcineurin A subunit and the calcineurin-dependent transcription factor, respectively, with high concentrations of caspofungin revealed that the A. fumigatus paradoxical effect is calcineurin pathway dependent. Exploring a molecular role for CnaA in the compensatory chitin biosynthetic response, we found that caspofungin treatment resulted in increased chitin synthase gene expression, leading to a calcineurin-dependent increase in chitin synthase activity. Taken together, our data suggest a mechanistic role for A. fumigatus calcineurin signaling in the chitin biosynthetic response observed during paradoxical growth in the presence of high-dose caspofungin treatment.

Unexpected Link between Metal Ion Deficiency and Autophagy in Aspergillus fumigatus

ABSTRACT Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus Aspergillus fumigatus, we disrupted the A. fumigatus atg1 gene, encoding a serine/threonine kinase required for autophagy. The ΔAfatg1 mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ΔAfatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ΔAfatg1 mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type A. fumigatus, even in the presence of abundant carbon and nitrogen, and the ΔAfatg1 mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.

Deletion of the regulatory subunit of protein kinase A in Aspergillus fumigatus alters morphology, sensitivity to oxidative damage, and virulence

ABSTRACT Aspergillus fumigatus is an important opportunistic fungal pathogen. The cAMP-dependent protein kinase (PKA) signaling pathway plays an important role in regulating morphology, growth, and virulence in a number of fungal pathogens of plants and animals. We have constructed a mutant of A. fumigatus that lacks the regulatory subunit of PKA, pkaR, and analyzed the growth and development, sensitivity to oxidative damage, and virulence of the mutant, along with those of the wild type and a complemented mutant. Both growth and germination rates of the mutant are reduced, and there are morphological abnormalities in conidiophores, leading to reduced conidiation. Conidia from the ΔpkaR mutant are more sensitive to killing by hydrogen peroxide, menadione, paraquat, and diamide. However, the hyphae of the mutant are killed to a greater extent only by paraquat and diamide, whereas they are less susceptible to the effects of hydrogen peroxide. In an immunosuppressed mouse model, intranasally administered conidia of the mutant are significantly less virulent than those of the wild type or a complemented mutant. Unregulated PKA signaling is detrimental to the virulence of A. fumigatus, perhaps through the reduced susceptibility of the mutant to damage by oxidizing agents and reduced growth kinetics.

Heat shock protein 90 is required for conidiation and cell wall integrity in Aspergillus fumigatus.

ABSTRACT Heat shock protein 90 (Hsp90) is a eukaryotic molecular chaperone. Its involvement in the resistance of Candida albicans to azole and echinocandin antifungals is well established. However, little is known about Hsp90s function in the filamentous fungal pathogen Aspergillus fumigatus. We investigated the role of Hsp90 in A. fumigatus by genetic repression and examined its cellular localization under various stress conditions. Failure to generate a deletion strain of hsp90 suggested that it is essential. Genetic repression of Hsp90 was achieved by an inducible nitrogen-dependent promoter (pniiA-Hsp90) and led to decreased spore viability, decreased hyphal growth, and severe defects in germination and conidiation concomitant with the downregulation of the conidiation-specific transcription factors brlA, wetA, and abaA. Hsp90 repression potentiated the effect of cell wall inhibitors affecting the β-glucan structure of the cell wall (caspofungin, Congo red) and of the calcineurin inhibitor FK506, supporting a role in regulating cell wall integrity pathways. Moreover, compromising Hsp90 abolished the paradoxical effect of caspofungin. Pharmacological inhibition of Hsp90 by geldanamycin and its derivatives (17-AAG and 17-DMAG) resulted in similar effects. C-terminal green fluorescent protein (GFP) tagging of Hsp90 revealed mainly cytosolic distribution under standard growth conditions. However, treatment with caspofungin resulted in Hsp90 accumulation at the cell wall and at sites of septum formation, further highlighting its role in cell wall stress compensatory mechanisms. Targeting Hsp90 with fungal-specific inhibitors to cripple stress response compensatory pathways represents an attractive new antifungal strategy.

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 ΔrasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBΔ113-135, 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 ∼65% survival compared to ∼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.

Deletion of the Aspergillus fumigatus Gene Encoding the Ras-Related Protein RhbA Reduces Virulence in a Model of Invasive Pulmonary Aspergillosis

ABSTRACT Aspergillus fumigatus is the predominant mold pathogen in patients who lack functional innate immunity. The A. fumigatus rhbA gene was first identified as a transcript that was upregulated when the organism was grown in the presence of mammalian cells. To gain insight into the function of rhbA in the growth and pathogenesis of A. fumigatus, we constructed a strain that lacks a functional rhbA gene. The ΔrhbA mutant showed a significant reduction in virulence compared to the virulence of the wild type in a mouse model of invasive aspergillosis. Complementation of the deletion with the wild-type gene restored full virulence. Although the ΔrhbA mutant grew as well as the wild type on solid medium containing the rich nitrogen source ammonium, the growth of the mutant was impaired on medium containing poor nitrogen sources. Like the Saccharomyces cerevisiae rhb1 mutant, the ΔrhbA mutant exhibited increased uptake of arginine. In addition, the ΔrhbA strain underwent asexual development in submerged cultures, even under ammonium-excess conditions. Growth of the mutant with poor nitrogen sources eliminated both the arginine uptake and submerged asexual development phenotypes. The mutant showed enhanced sensitivity to the TOR kinase inhibitor rapamycin. These findings establish the importance of rhbA for A. fumigatus virulence and suggest a role for rhbA in nutrient sensing.

Localization and activity of the calcineurin catalytic and regulatory subunit complex at the septum is essential for hyphal elongation and proper septation in Aspergillus fumigatus.

Calcineurin, a heterodimer composed of the catalytic (CnaA) and regulatory (CnaB) subunits, plays key roles in growth, virulence and stress responses of fungi. To investigate the contribution of CnaA and CnaB to hyphal growth and septation, ΔcnaB and ΔcnaAΔcnaB strains of Aspergillus fumigatus were constructed. CnaA colocalizes to the contractile actin ring early during septation and remains at the centre of the mature septum. While CnaBs septal localization is CnaA‐dependent, CnaAs septal localization is CnaB‐independent, but CnaB is required for CnaAs function at the septum. Catalytic null mutations in CnaA caused stunted growth despite septal localization of the calcineurin complex, indicating the requirement of calcineurin activity at the septum. Compared to the ΔcnaA and ΔcnaB strains, the ΔcnaAΔcnaB strain displayed more defective growth and aberrant septation. While three Ca2+‐binding motifs in CnaB were sufficient for its association with CnaA at the septum, the amino‐terminal arginine‐rich domains (16‐RRRR‐19 and 44‐RLRKR‐48) are dispensable for septal localization, yet required for complete functionality. Mutation of the 51‐KLDK‐54 motif in CnaB causes its mislocalization from the septum to the nucleus, suggesting it is a nuclear export signal sequence. These findings confirm a cooperative role for the calcineurin complex in regulating hyphal growth and septation.

Aspergillus fumigatus RasA Regulates Asexual Development and Cell Wall Integrity

ABSTRACT The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The ΔrasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The ΔrasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The ΔrasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.