Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45267-0529, USA1
Author for correspondence: Judith C. Rhodes. Tel: +1 513 558 0130. Fax: +1 513 558 2289. e-mail: judith.rhodes{at}uc.edu
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ABSTRACT |
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Keywords: PKA, phosphodiesterase inhibitor, glucose repression
Abbreviations: IBMX, 3-isobutyl-1-methylxanthine; PDE, phosphodiesterase; PKA, protein kinase A
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INTRODUCTION |
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The cAMP/PKA pathway of S. cerevisiae has been proposed as the mechanism by which the cell detects changes in glucose availability and adjusts its metabolic machinery accordingly. To maintain metabolic efficiency, the presence of glucose in the medium stimulates the PKA pathway, which leads to repression of genes involved in metabolizing non-glucose carbon sources (Thevelein, 1994 ). Cells with increased PKA activity, due to deletion of the PKA regulatory subunit, exhibit an inability to grow on non-fermentable, non-repressing carbon sources such as glycerol and ethanol, as well as the weakly fermentable, weakly repressing carbon source galactose (Toda et al., 1987
). These mutants also fail to sporulate in response to nutritional stress (Malone, 1990
). Mutants with reduced PKA activity express genes normally associated with growth on non-glucose carbon sources, and they sporulate on rich medium, which usually suppresses sporulation in wild-type cells (Casperson et al., 1985
). The effects of PKA signalling have also been studied in Aspergillus species. In Aspergillus niger, strains that overexpress the catalytic subunit of PKA exhibit a slower growth rate relative to wild-type cells (Bencina et al., 1997
). They display a similar phenotype to that of wild-type A. niger grown on glucose-containing medium supplemented with cAMP (Bencina et al., 1997
). However, strains of Aspergillus nidulans that overexpress the catalytic subunit of PKA exhibit no alterations with regard to growth rate when grown on medium containing glucose (Shimizu & Keller, 2001
). Although features of PKA signalling have been reported in these two Aspergillus species, the PKA pathway in Aspergillus fumigatus has not been studied.
A. fumigatus is an opportunistic pathogen of humans that plays a key role in the compost cycle in nature. To grow in the lung, the organism must be able to sense the change in environment and translate that change into an appropriate metabolic response. It has recently been shown that transcript levels of the PKA regulatory subunit are elevated when the fungus grows in the presence of endothelial cells, suggesting that PKA may play a role in signalling that environmental change (Rhodes et al., 2001 ). This would be compatible with data from the fungal plant pathogens Ustilago maydis, Magnaporthe grisea and Colletotrichum trifolii, as well as the human pathogens Cryptococcus neoformans and Candida albicans, in which PKA signalling has been shown to be required for wild-type virulence (DSouza et al., 2001
; DSouza & Heitman, 2001
; Kronstad et al., 1998
; Kruger et al., 1998
; Sonneborn et al., 1999
; Xu et al., 1997
). Because little is known of the importance of this pathway and the effects it may have on the growth of A. fumigatus, the response of the organism to agonists and antagonists of the cAMP/PKA pathway was studied on media containing different carbon sources.
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METHODS |
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Radial growth assay.
Conidia were harvested with sterile distilled water, filtered through Miracloth (Calbiochem) and counted in a haemocytometer. Ten microlitres of a conidial suspension (103 conidia µl-1) were spotted onto Aspergillus minimal medium with 10 mM sodium nitrate as the nitrogen source (ASP MM) (Cove, 1966 ) plates or on to ASP MM plates containing cAMP, cAMP analogues or phosphodiesterase (PDE) inhibitors. Triplicate plates of all media were inoculated. Indicated carbon sources were used at 1% final concentrations. cAMP, cAMP analogues and the PDE inhibitors 3-isobutyl-1-methylxanthine (IBMX) and theophylline were used at concentrations between 1 and 10 mM. Vehicle controls were performed with the appropriate amount of the respective solvent. Dose-response studies were used to choose suboptimal levels of cAMP and IBMX for experiments in which the compounds were combined to test for additive activity. All chemicals were obtained from Sigma.
Radial colony growth was monitored for cultures of A. fumigatus and A. niger at 37 °C every 12 h for 48 to 60 h. Colony diameter was measured at each time point with callipers. Growth rates, in µm h-1, were based on colony diameters measured at 24 and 48 h, except for acetate, which used measurements taken at 48 and 60 h. All experiments were performed a minimum of two times, and results from representative experiments are presented.
Enzyme activity assays.
Fifty millilitre aliquots of ASP MM+1% glucose were inoculated with 1x108 conidia and cultures were incubated at 37 °C, 225 r.p.m., for 12 h. Because glycerol does not support robust germination, conidia were allowed to germinate in ASP MM+1% glucose for 6 h before being washed twice with ASP MM+1% glycerol and transferred to 50 ml ASP MM+1% glycerol. These cultures were then grown at 37 °C, 225 r.p.m. for 12 h. At the end of the growth period, hyphae were treated with cAMP at a final concentration of 10 mM and collected by vacuum filtration after 0, 5, 15 or 30 min continued incubation. Hyphae were washed quickly with ice-cold 0·8% NaCl, frozen in liquid nitrogen, and crushed with a mortar and pestle to obtain cell extracts. Samples were then resuspended in extraction buffer (25 mM Tris/HCl pH 7·4, 10 mM MgCl2, 150 mM NaCl, 1 mM DTT, 1 mM EDTA) and incubated on ice for 30 min. The homogenate was centrifuged at 10000 g for 10 min at 4 °C, and the supernatant was used in the assay. PKA enzyme activity was measured by kemptide phosphorylation (colorimetric PKA assay, Spinzyme format; Pierce) using the manufacturers protocol with the following modification: no activator solution (500 µM cAMP) was used in the assay to measure the amount of PKA activity in the extracts. Purified bovine cardiac PKA-C (Pierce) was used as a standard. The amount of phosphorylated product was measured spectrophotometrically at 570 nm in a 96-well plate and normalized to the amount of total protein used in the assay. Protein was measured using the BCA assay (Pierce) as described by the manufacturer. Triplicate cultures were processed for cell extracts under each experimental condition. Each experiment was repeated and results from representative experiments are shown.
Statistical analysis.
Statistical significance was determined by one tailed, paired t-test for means with a 95% confidence interval. Values are expressed as the mean±SEM. Statistical tests were performed using Microsoft Excel.
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RESULTS AND DISCUSSION |
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Previous studies in A. niger demonstrated a reduction in growth when glucose-containing medium was supplemented with cAMP. Likewise, strains of A. niger that overexpress the catalytic subunit of PKA also show growth inhibition (Bencina et al., 1997 ). To determine whether PKA signalling plays a role in A. fumigatus growth, colonies of A. fumigatus were grown on minimal medium with different carbon sources, with or without cAMP.
Effects of exogenous cAMP
A. fumigatus and A. niger were grown on glucose-containing medium supplemented with cAMP to determine the effect of cAMP on growth rate. Although A. niger displayed a reduction in growth rate (P<0·001) on medium containing cAMP, as previously demonstrated (Bencina et al., 1997 ), there was no growth rate reduction seen with A. fumigatus (Table 1
; Fig. 1
). To test the possibility that A. fumigatus was impermeable to cAMP or was degrading the cAMP, the non-hydrolysable and lipid-soluble cAMP analogues 8-bromo-cAMP and dibutyryl-cAMP were used in place of cAMP. Neither cAMP analogue changed the growth rate of A. fumigatus, but the growth rate of A. niger was significantly reduced by both analogues (Table 1
). The use of PDE inhibitors with intact cells has been reported to result in an increase in intracellular cAMP levels (Beebe & Corbin, 1986
). The addition of either of the PDE inhibitors IBMX or theophylline to the medium led to a reduction in the growth rate of both A. fumigatus and A. niger, suggesting either that cAMP and its analogues were not able to enter the cells of A. fumigatus or that the inhibitors were decreasing growth through another, non-PKA related mechanism. To demonstrate that the decreases in growth rate obtained with cAMP and PDE inhibitors were due to modulation of the cAMP/PKA pathway, rather than to non-specific toxicity, suboptimal concentrations of cAMP and IBMX were used in combination to show an additive effect on growth inhibition (Beebe & Corbin, 1986
). Indeed, on minimal medium plus glucose, the growth rate of A. niger was significantly inhibited by the combination (P<0·05) when compared with the growth rate on cAMP alone and IBMX alone (Table 2
). In contrast, A. fumigatus did not display a significant change in growth rate on medium containing cAMP and IBMX when compared with both alone. Results with theophylline were similar to those obtained with IBMX (data not shown). These results support the interpretation that the decreased growth rate of A. fumigatus when grown in the presence of PDE inhibitors is due to toxicity or some other non-PKA related mechanism, whereas a PKA-specific mechanism appears to be active in A. niger.
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PKA activity
To explore further the hypothesis that the insensitivity of A. fumigatus to the addition of exogenous cAMP may be due to the glucose activation of the cAMP/PKA pathway, kemptide phosphorylation was used to measure PKA activity in cell extracts of A. fumigatus and A. niger. It had been previously shown that strains of A. niger in which the catalytic subunit of PKA is overexpressed, as a consequence of introduction of additional copies of the gene, exhibit increased kinase activity, as determined by kemptide phosphorylation (Bencina et al., 1997 ). A. niger was grown in minimal medium containing glucose and cAMP was added at time zero prior to cell harvest. Cell extracts from these cultures exhibited a 3·5-fold increase in kinase activity over the baseline at 5 and 15 min following cAMP addition, suggesting that PKA activity is increased by the addition of exogenous cAMP (Table 3
). Under the same growth conditions, there was no change in the PKA activity of cell extracts from A. fumigatus, although the basal level of kinase activity for A. fumigatus was approximately sevenfold higher than that of A. niger (Table 3
). The high basal level of activity of A. fumigatus may be near the organisms maximum, preventing further increases in activity stimulated by the addition of exogenous cAMP. This would be reflected in the failure to see a growth rate reduction in A. fumigatus on glucose medium with cAMP.
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Both A. niger and A. fumigatus exhibited increases in kemptide phosphorylation activity under conditions in which they displayed reductions of growth. These data are consistent with the hypothesis that the cAMP-induced growth reduction is modulated through changes in carbon-source-regulated PKA activity. Although the precise mechanism underlying the carbon-source-dependent effect of cAMP remains to be elucidated, it raises interesting questions regarding the disparate regulation between these two Aspergillus species. The pathways and receptors used to sense glucose and other carbon sources may differ between the two species, which may be reflected in their different industrial roles. Although A. fumigatus is an important member of the compost cycle, it has little use in industrial processes whereas A. niger, which is cosmopolitan in distribution, is an important industrial source for citric acid and enzymes, such as amylase (Raper & Fennell, 1965 ).
A. fumigatus responds to the addition of extracellular cAMP only when growing on a carbon source that is non-repressing. Our data supports the interpretation that during growth on glucose, the PKA activity is already near maximal, rendering the organism refractory to addition of exogenous cAMP. However, when basal levels of PKA are low, the organism responds to a pulse of cAMP, and PKA activity transiently increases. The results also suggest that A. fumigatus undergoes carbon catabolite repression in a manner similar to that of other fungi, and that glucose itself or intracellular acidification secondary to the metabolism of glucose results in activation of the cAMP/PKA pathway. The interrelationship between cAMP/PKA signalling and pathogenesis of other fungi and the recent finding that components of the pathway are up-regulated in a model of invasive aspergillosis, when coupled with the data presented here, suggest that PKA signalling is a fruitful area for continued study in A. fumigatus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 March 2002;
accepted 10 April 2002.