Predicting essential genes for identifying potential drug targets in Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus (Af) is a ubiquitous and opportunistic pathogen capable of causing acute, invasive pulmonary disease in susceptible hosts. Despite current therapeutic options, mortality associated with invasive Af infections remains unacceptably high, increasing 357% since 1980. Therefore, there is an urgent need for the development of novel therapeutic strategies, including more efficacious drugs acting on new targets. Thus, as noted in a recent review, "the identification of essential genes in fungi represents a crucial step in the development of new antifungal drugs". Expanding the target space by rapidly identifying new essential genes has thus been described as "the most important task of genomics-based target validation". RESULTS: In previous research, we were the first to show that essential gene annotation can be reliably transferred between distantly related four Prokaryotic species. In this study, we extend our machine learning approach to the much more complex Eukaryotic fungal species. A compendium of essential genes is predicted in Af by transferring known essential gene annotations from another filamentous fungus Neurospora crassa. This approach predicts essential genes by integrating diverse types of intrinsic and context-dependent genomic features encoded in microbial genomes. The predicted essential datasets contained 1674 genes. We validated our results by comparing our predictions with known essential genes in Af, comparing our predictions with those predicted by homology mapping, and conducting conditional expressed alleles. We applied several layers of filters and selected a set of potential drug targets from the predicted essential genes. Finally, we have conducted wet lab knockout experiments to verify our predictions, which further validates the accuracy and wide applicability of the machine learning approach. CONCLUSIONS: The approach presented here significantly extended our ability to predict essential genes beyond orthologs and made it possible to predict an inventory of essential genes in Eukaryotic fungal species, amongst which a preferred subset of suitable drug targets may be selected. By selecting the best new targets, we believe that resultant drugs would exhibit an unparalleled clinical impact against a naive pathogen population. Additional benefits that a compendium of essential genes can provide are important information on cell function and evolutionary biology. Furthermore, mapping essential genes to pathways may also reveal critical check points in the pathogen's metabolism. Finally, this approach is highly reproducible and portable, and can be easily applied to predict essential genes in many more pathogenic microbes, especially those unculturable.

Protein kinase A and fungal virulence: a sinister side to a conserved nutrient sensing pathway.

Diverse fungal species are the cause of devastating agricultural and human diseases. As successful pathogenesis is dependent upon the ability of the fungus to adapt to the nutritional and chemical environment of the host, the understanding of signaling pathways required for such adaptation will provide insights into the virulence of these pathogens and the potential identification of novel targets for antifungal intervention. The cAMP-PKA signaling pathway is well conserved across eukaryotes. In the nonpathogenic yeast, S. cerevisiae, PKA is activated in response to extracellular nutrients and subsequently regulates metabolism and growth. Importantly, this pathway is also a regulator of pathogenesis, as defects in PKA signaling lead to an attenuation of virulence in diverse plant and human pathogenic fungi. This review will compare and contrast PKA signaling in S. cerevisiae vs. various pathogenic species and provide a framework for the role of this pathway in regulating fungal virulence.

HacA-independent functions of the ER stress sensor IreA synergize with the canonical UPR to influence virulence traits in Aspergillus fumigatus.

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacA(i), or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireA(Δ10). Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus.

The virulence of the opportunistic fungal pathogen Aspergillus fumigatus requires cooperation between the endoplasmic reticulum-associated degradation pathway (ERAD) and the unfolded protein response (UPR).

The filamentous fungal pathogen Aspergillus fumigatus secretes hydrolytic enzymes to acquire nutrients from host tissues. The production of these enzymes exerts stress on the endoplasmic reticulum (ER), which is alleviated by two stress responses: the unfolded protein response (UPR), which adjusts the protein folding capacity of the ER, and ER-associated degradation (ERAD), which disposes of proteins that fail to fold correctly. In this study, we examined the contribution of these integrated pathways to the growth and virulence of A. fumigatus, focusing on the ERAD protein DerA and the master regulator of the UPR, HacA. A ΔderA mutant grew normally and showed no increase in sensitivity to ER stress. However, expression of the UPR target gene bipA was constitutively elevated in this strain, suggesting that the UPR was compensating for the absence of DerA function. To test this, the UPR was disrupted by deleting the hacA gene. The combined loss of derA and hacA caused a more severe reduction in hyphal growth, antifungal drug resistance and protease secretion than the loss of either gene alone, suggesting that DerA and HacA cooperate to support these functions.  Moreover, the ΔderA/ΔhacA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasted the wild type virulence of ΔderA and the reduced virulence of the ΔhacA mutant. Taken together, these data demonstrate that DerA cooperates with the UPR to support the expression of virulence-related attributes of A. fumigatus.

Divergent Protein Kinase A isoforms co-ordinately regulate conidial germination, carbohydrate metabolism and virulence in Aspergillus fumigatus.

The genome of Aspergillus fumigatus encodes two isoforms of the catalytic subunit of the cAMP-dependent Protein Kinase (PKA). Although deletion of the class I isoform, pkaC1, leads to an attenuation of virulence, the function of the class II subunit, PkaC2, was previously uninvestigated. In this report, we demonstrate that both isoforms act in concert to support various physiologic processes that promote the virulence of this pathogen. Whereas pkaC1 and pkaC2 single-deletion mutants display wild-type conidial germination, a double-deletion mutant is delayed in germination in response to environmental nutrients. Furthermore, PkaC1 and PkaC2 interact to positively regulate flux through the carbohydrate catabolic pathway and, consequently, the ΔpkaC1ΔpkaC2 mutant is unable to grow on low glucose concentrations. Importantly, the reduced germinative capacity and inability to utilize glucose observed for the ΔpkaC1ΔpkaC2 strain correlated with an inability of the mutant to establish infection in a murine model. Conversely, overexpression of pkaC2 both promotes the in vitro growth on glucose, and restores the fungal burden and mortality associated with the ΔpkaC1 to that of the wild-type organism. Taken together, these data demonstrate the functional capacity of pkaC2 and emphasize the importance of PKA-mediated metabolic control in the pathogenic potential of A. fumigatus.

A role for the unfolded protein response (UPR) in virulence and antifungal susceptibility in Aspergillus fumigatus.

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The DeltahacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37 degrees C, but cell wall integrity was disrupted at 45 degrees C, resulting in a dramatic loss in viability. The DeltahacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the DeltahacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

Deletion of the protein kinase A regulatory subunit leads to deregulation of mitochondrial activation and nuclear duplication in Aspergillus fumigatus.

Proper regulation of the cyclic AMP-dependent protein kinase (PKA) pathway is required for normal growth and development in many fungi. We have reported that deletion of the PKA regulatory subunit gene, pkaR, in Aspergillus fumigatus leads to defects in germination and a hypersensitivity of conidia to oxidative stress. In this study, we further analyzed the defects of DeltapkaR conidia and found that a large proportion were abnormally larger than wild type. Because swelling and increased susceptibility to oxidative stress are characteristic of germinating conidia, we analyzed the metabolic activity of the conidia by mitochondrial staining. Whereas it required 4 h in rich medium for wild-type mitochondria to become active, DeltapkaR conidia harbored active mitochondria in the absence of a germinant. Furthermore, conidia of the mutant showed a dramatic loss in viability upon short-term storage in water, indicating starvation-induced death. Taken together, our data suggest that PKA activity regulates metabolic activation of resting conidia. Additionally, the DeltapkaR mutant displayed an abnormal abundance of hyphal nuclei and had increased transcript levels of several cell cycle regulatory genes. These data indicate an important role for PKA in the nuclear duplication cycle of A. fumigatus.

Aspergillus fumigatus RasA regulates asexual development and cell wall integrity.

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 DeltarasA 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 DeltarasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The DeltarasA 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.

Impaired ribosome biogenesis disrupts the integration between morphogenesis and nuclear duplication during the germination of Aspergillus fumigatus.

Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37 degrees C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a DeltacgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the DeltacgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although DeltacgrA conidia germinated normally at 22 degrees C, they swelled excessively when incubated at 37 degrees C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.

Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus.

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 DeltaAfatg1 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 DeltaAfatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the DeltaAfatg1 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 DeltaAfatg1 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.

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress.

We 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, DeltacasA/DeltacasB, 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 DeltacasA/DeltacasB 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 DeltacasA/DeltacasB 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.

Aspergillus fumigatus: growth and virulence.

Aspergillus fumigatus is a ubiquitous fungus that plays an important role in carbon and nitrogen recycling in nature. Because A. fumigatus is thermotolerant, it is a predominant organism during the high-temperature phase of the compost cycle. The ability to grow at elevated temperatures and to utilize numerous varied sources of both carbon and nitrogen to support its growth have made A. fumigatus an important opportunistic pathogen of humans as well as a vital part of the nutrient-recycling ecosystem. Data correlating the growth rate and germination potential of A. fumigatus at 37 degrees C with its pathogenic potential suggest that these are related, both when viewed from a population standpoint and when analyzed on a single gene basis. Nutritional versatility has been cited as an important contributor to virulence as well. Indeed, perturbation of pathways involved with nitrogen or carbon sensing has been shown to reduce virulence in animal models, even when in vitro growth rates have not been altered. Therefore, the remarkable ability of A. fumigatus to grow efficiently under a variety of environmental conditions and to utilize a wide variety of substrates to meet its nutritional needs contributes to its role as the predominant mould pathogen of immunocompromised patients.

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

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 DeltapkaR 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.

Nucleolar localization of Aspergillus fumigatus CgrA is temperature-dependent.

Pathogenic fungi must adapt to multiple adverse environmental conditions during the transition from the environment to a mammalian host, one of which is temperature. The ability of Aspergillus fumigatus to grow optimally under conditions of thermal stress requires the nucleolar protein CgrA. In this study, we have determined how temperature affects the intracellular localization of CgrA in A. fumigatus using a green fluorescent protein (GFP)-tagging approach. At 22 degrees C, CgrA was almost exclusively in the nucleolus, with a ratio of nucleolar to cytoplasmic fluorescence of 10:1. At 37 degrees C, the ratio of nucleolar to cytoplasmic fluorescence was reduced fivefold, and increased correspondingly in the cytoplasm. This effect was not seen with the nucleolar protein NopA in wild-type A. fumigatus. However, in a DeltacgrA mutant NopA was delocalized from the nucleolus at 37 degrees C but not at 22 degrees C. These results provide evidence for a temperature-dependent mechanism of intracellular localization for CgrA, and suggest that CgrA facilitates nucleolar compartmentalization of NopA at higher temperature.

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

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.

Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus.

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. RESULTS: Here we describe the construction of plasmid vectors that can be used to regulate gene expression in A. fumigatus using a simple co-transfection approach. Vectors were generated in which the tetracycline transactivator (tTA) or the reverse tetracycline transactivator (rtTA2s-M2) are controlled by the A. nidulans gpdA promoter. Dominant selectable cassettes were introduced into each plasmid, allowing for selection following gene transfer into A. fumigatus by incorporating phleomycin or hygromycin into the medium. To model an essential gene under tetracycline regulation, the E. coli hygromycin resistance gene, hph, was placed under the control of seven copies of the TetR binding site (tetO7) in a plasmid vector and co-transfected into A. fumigatus protoplasts together with one of the two transactivator plasmids. Since the hph gene is essential to A. fumigatus in the presence of hygromycin, resistance to hygromycin was used as a marker of hph reporter gene expression. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the tetO7-hph reporter gene, and the addition of doxycycline to the medium suppressed hygromycin resistance in a dose-dependent manner. Similarly, transformants were identified in which expression of rtTA2s-M2 conferred hygromycin resistance only in the presence of doxycycline. The levels of doxycycline required to regulate expression of the tetO7-hph reporter gene were within non-toxic ranges for this organism, and low-iron medium was shown to reduce the amount of doxycycline required to accomplish regulation. CONCLUSIONS: The vectors described in this report provide a new set of options to experimentally manipulate the level of specific gene products in A. fumigatus.

Disruption of the Aspergillus fumigatus gene encoding nucleolar protein CgrA impairs thermotolerant growth and reduces virulence.

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 Delta 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 Delta cgrA mutant had the same growth rate as the wild type at room temperature. However, when the cultures were incubated at 37 degrees C, a condition that increased the growth rate of the wild-type and reconstituted strains approximately threefold, the Delta 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 degrees C but had little effect on germination at room temperature. The Delta 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 degrees 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 rasA and rasB regulate the timing and morphology of asexual development.

Expression of rasA plays an important role in conidial germination in Aspergillus nidulans. Conidial germination is required to initiate both infection and asexual development in the opportunistic pathogen Aspergillus fumigatus. Therefore, we sought to determine the requirements for Ras proteins in conidial germination and asexual development of A. fumigatus. A second homolog, rasB, has been identified that characterizes a new subclass of Ras genes. Dominant active (DA) and dominant negative (DN) mutations of each gene were introduced into protoplasts as transgenes. DArasA expression led to reduced conidiation, malformed conidiophores, and altered mitotic progression, whereas expression of DNrasA caused a significant reduction in the rate of conidial germination. In contrast, expression of DNrasB slightly delayed the initiation of germination and caused the development of conidiophores in submerged culture. DArasB expression led to reduced conidiation. RasA and RasB appear to play different, but overlapping, roles in the vegetative growth and asexual development of A. fumigatus.

Deletion of the Aspergillus fumigatus gene encoding the Ras-related protein RhbA reduces virulence in a model of Invasive pulmonary aspergillosis.

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 Delta rhbA 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 Delta rhbA 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 Delta rhbA mutant exhibited increased uptake of arginine. In addition, the Delta rhbA 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.

cAMP alteration of growth rate of Aspergillus fumigatus and Aspergillus niger is carbon-source dependent.

cAMP signalling has been shown to be essential for normal growth, morphology and virulence in fungal pathogens of both plants and animals. The effects of exogenous cAMP on the growth of the opportunistic pathogen Aspergillus fumigatus were compared to those of Aspergillus niger, which has previously been demonstrated to respond to extracellular cAMP. Both cAMP and phosphodiesterase inhibitors markedly reduced the radial growth rate of A. niger after 48 h on minimal medium with glucose as the carbon source, whereas the growth of A. fumigatus was not affected by cAMP. However, when glycerol, which does not initiate carbon catabolite repression, was used as a carbon source, cAMP inhibited the radial growth rate of only A. fumigatus (P<0.05). The addition of cAMP to glycerol-minimal medium resulted in a fourfold increase in protein kinase A activity in A. fumigatus cell extracts when compared with pre-treatment samples. The protein kinase A activity in A. fumigatus cell extracts from cultures grown in glucose did not change significantly with the addition of cAMP. These studies demonstrate that although the growth rates of both A. fumigatus and A. niger are sensitive to the addition of exogenous cAMP, the response of each organism is distinct and dependent on the carbon source used.