Department of Entomology, 4112 Plant Science Building, University of Maryland, College Park, MD 20742-4454, USA1
Author for correspondence: Raymond J. St Leger. Tel: +1 301 405 5402. Fax: +1 301 314 9290. e-mail: rl106{at}umailsrv0.umd.edu
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ABSTRACT |
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Keywords: Metarhizium anisopliae, Aspergillus fumigatus, Neurospora crassa, protease production, pH
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INTRODUCTION |
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From the context of hostpathogen interactions ammonia is a highly toxic agent, which has been shown to inhibit growth in cell lines (Doyle & Butler, 1990 ) and is the toxin responsible for immunosuppression, pathology and death of sheep infested with Lucilia cuprina larvae (Guerrini, 1997
). Helicobacter pylori, the major causative agent of human active gastritis, survives the gastric acidity by producing ammonia, which also acts as a toxin (Chen et al., 1997
).
Several researchers have examined the relationship between acid production and virulence in fungi, with contrasting results. Thus while mutants were used to demonstrate a role for oxalic acid in pathogenicity of the plant pathogen Sclerotinia sclerotiorum on Phaseolus vulgaris (Godoy et al., 1990 ), oxalic acid hyperproduction in mutants of the entomopathogenic fungus Beauveria bassiana did not affect virulence (Bidochka & Khachatourians, 1993
). Likewise some strains of M. anisopliae produce a carbohydrate-dependent acidifying activity but are not altered in pathogenicity compared to acid non-producers (Robert & Al-Aidroos, 1985
). In this study we investigated the influence that M. anisopliae, A. fumigatus and Neurospora crassa have on the pH of their environment and how these pH changes influence production of proteases.
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METHODS |
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Transfer cultures.
Standardized mycelial inocula (5 g wet wt per 100 ml) from 24 h Sabouraud dextrose broth cultures were incubated with shaking (100 r.p.m.) at 28 °C for 4 h in 20 ml minimal medium (0·02% KH2PO4, 0·01% MgSO4, 0·2 p.p.m. FeSO4, 1·0 p.p.m. ZnSO4, 0·02 p.p.m. MnMoO4, 0·02 p.p.m. CuSO4 and 0·02 p.p.m. MnCl2, pH 6·5) supplemented with either insect cuticle, an amino acid, or an alternative carbon source (see Table 2). After set times, protease production, ammonia production and pH were measured. To check for non-fungal ammonia release, un-inoculated culture media were incubated with each assay.
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Growth in a nutrient medium.
The basal medium used was 1% yeast extract (Difco). Cultures (100 ml) were inoculated with 1x107 spores. The pH of the medium was adjusted to 4 or 8 with 1 M HCl or NaOH. All cultures were incubated for 72 h at 28 °C, and ammonia production, pH and growth were measured over this time. In experiments to differentiate between alterations in ammonia formation due to carbon repression and changes in pH, yeast extract medium containing 1% glucose was buffered with 0·1 M citric acid/phosphate buffer at pH 4 or 7.
Analyses.
Ammonia (NH3 and ) was assayed by the colorimetric method of Chaney & Marbach (1962)
and absorbance readings were measured in a Spectra MAX plus plate reader (Molecular Devices). For calculation of ammonia formed in yeast extract medium, the concentration (11·5 mmol l-1) in an uninoculated broth was subtracted from that in an inoculated broth of the same type. Dry weight was determined by filtration of 5 ml aliquots of cultures over Millipore filters of constant weight (type AP 40), washing twice with equal volumes of water, and drying at 105 °C. The identities and quantities of organic acids in spent culture media were determined by capillary electrophoresis with a Bio-Rad BioFocus 3000, employing indirect detection at 230 nm. The background electrolyte was sodium phthalate (5 mM) with cetyltrimethylammonium bromide (0·5 mM) as the osmotic flow modifier; it was prepared daily, adjusted to pH 5·6 and filtered before use. The capillary was a 50 cmx 50 µm i.d. bare silica which had been purged with 0·1 M NaOH, washed with deionized water, then purged with background electrolyte. Background electrolyte was replaced by high-pressure purging before each sample run. Samples were introduced by electrokinetic injection at 10 kV for 5 s. Run conditions of 15 kV with a polarity of - to + at 20 °C resolved standard acids (oxalic, succinic, acetic) in less than 5 min and an entire run was completed in 10 min. BioFocus Integration software was used to generate calibration curves for the standard acids and quantify the concentrations of organic acids in fungal filtrates. Samples were diluted with deionized water to bring acid concentrations within the linear response range of the calibration curves.
The concentration of oxalic acid produced by mycelium grown on agar was determined by the method of Bidochka & Khachatourians (1993) . Four-day cultures and surrounding agar (2·5 cm in diameter) were homogenized in 5 ml 0·1 M HCl, centrifuged (4000 g for 20 min) and the pH of the supernatants adjusted to 5 with 1 M KOH. Oxalic acid in supernatants was quantified by a test kit using enzymic analysis (Sigma Diagnostics). Protease activity versus Suc-Ala-Ala-Pro-Phe-p-nitroanilide was measured as described previously (St Leger et al., 1994
).
Calculations.
The expected pH at different concentrations of ammonia was calculated from the HendersonHasselbach equation using a pKa value of 9·27 and /NH3 ratios at each pH from Emerson et al. (1975)
: pH=pK+log(NH3/
).
Mutagenesis of M. anisopliae strain 2575.
The alkylating reagent ethyl methanesulphonate (EMS) was used in a procedure adapted from that of Al-Aidroos & Seifert (1980) . One millilitre of a spore suspension (1x107 conidia ml-1) was added to 9 ml potassium phosphate buffer (pH 7·0, 0·05 M) containing 100 µl EMS (Sigma). Treatments were carried out at 28 °C for 7·5 h in a shaking water bath, which gave 10% survival. Samples (1 ml) were removed and dispensed into buffer containing 10% (w/v) sodium thiosulphate. After incubation for 45 min, spores were diluted to 1x103 ml-1 and 1 ml samples were dispersed on selection media (1% yeast extract, 2% agar, 0·01% bromocresol purple; adjusted to pH 4·5, 6·3 or 7·0 with 0·1 M HCl), which were subsequently incubated at 27 °C. Colonies that lacked, or had reduced or increased zones of surrounding blue colour were removed and screened twice on solid pH-detection medium before critical study in liquid culture.
Protease activity.
Protease degradation in solid media was measured by clearing zones (produced by degradation of milk proteins) in pH-indicator medium (0·1% yeast extract, 2% agar, 0·01% bromocresol purple; adjusted to pH 5·2) containing 1% skimmed milk. M. anisopliae produces a similar range of subtilisins and trypsin-like proteases in cultures containing skimmed milk as it does in those containing insect cuticle (St Leger et al., 1987 , 1994
).
pH measurements on agar plates.
pH gradients through bacterial colonies have been measured using microelectrodes (Robinson et al., 1991 ). Similarly, the pH of fungal colonies and the surrounding agar was measured using a PHR-146 micro pH electrode (Lazar Research Laboratories). Images of Petri dishes were generated using an HP Scanjet 6200Cse flatbed scanner at 300 d.p.i. The generated files were imported into Corel Draw 7 and the colours adjusted to compensate for scanning distortion. Finally the individual images were collated in Powerpoint 97.
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RESULTS |
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Colonies of wild-type strains 549 and 2575, and the Acid(+) mutants of 2575 grown on yeast extract plus glucose at pH 6·3 (purple colour) showed an overall acidification of the medium, with a yellow halo extending approximately 12 mm from the edge of the colonies, in keeping with the generation of organic acids from carbohydrates. In contrast, the Acid(-) mutants produced no halo (data not shown).
Protease production
Protease degradation in solid media was determined by measuring clearing zones in pH indicator medium containing skimmed milk (Fig. 1c, d
). Wild-type strains and Acid(-) mutants of M. anisopliae produced proteolytic clearing zones slightly smaller than the zone of alkalinization (blue halo). Acid(+) strains, which did not result in an overall alkalinization of the agar, likewise did not produce proteolytic clearing zones (Fig. 1c
). To determine if these results may be more generally applicable to fungi, strains of A. fumigatus and N. crassa were tested. A. fumigatus produced a proteolytic clearing zone within the zone of alkalinization resembling that of M. anisopliae. N. crassa produced a thin net-like mycelium on the surface of the agar and a smaller-diameter zone of protein degradation within the alkaline zone it generated (data not shown).
To determine if the failure of M. anisopliae Acid(+) strains to produce protease in agar is due to a single mutation causing acid overproduction, the Acid(+)1 strain was subjected to a second round of mutagenesis with EMS. From a population of 3100 surviving colonies, 13 mutants were identified that resembled wild-type 2575 in not acidifying yeast extract medium at pH 7·0. These strains also produced proteolytic clearing zones similar to the 2575 wild-type strain, confirming that overproduction of acid and non-production of proteases are linked characteristics.
To determine if the failure of M. anisopliae Acid(+) strains to produce protease in agar is due only to acidification of the medium, or if additional pH-independent changes in regulation of production occur, the wild-type and the Acid(+)1 mutant were assayed for enzyme activity 24 h after exponentially growing mycelium was transferred to buffered (pH 4 or pH 7) liquid minimal medium containing 1% insect cuticle as sole carbon and nitrogen source. Wild-type and Acid(+)1 strains produced similar levels of protease activity against the subtilisin substrate Suc-Ala-Ala-Pro-Phe-p-nitroanilide both at pH 7 [wild-type, 115±7 nmol min-1; Acid(+)1, 123±8] and at pH 4 [wild-type, 10±2; Acid(+)1, 10±1]. This suggests that regulation of protease production is not altered in Acid(+)1 compared to the wild-type.
Ammonia and acidic metabolites in nutrient medium
Wild-type A. fumigatus, N. crassa and M. anisopliae, as well as mutants of M. anisopliae selected on pH indicator medium, were evaluated for interrelationship of pH, mycelial dry weight, and generation of organic acids and ammonia (Figs 3 and 4
).
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The mechanism of early alkalinization of the pH of the medium was investigated by following the concentration of ammonia in the medium. Increasing concentrations of ammonia correlated with the increasing medium pH in cultures of M. anisopliae, N. crassa and A. fumigatus. Using 9:1 as the ratio of to NH3 at pH 8·3 (Emerson et al., 1975
), the calculated pH of 3 d cultures of M. anisopliae wild-type strain based on ammonia production (680 µg ml-1) is 10·3. Consequently, production of ammonia is more than sufficient to account for the rise in pH, and presumably additional more acidic components are present in cultures, and/or the cultures contain buffering agents. An assessment of organic acids detected by capillary electrophoresis indicated that the wild-type M. anisopliae produced acetic, succinic and oxalic acids in yeast extract medium (Table 1
). Traces of other acidic metabolites were present; however, these did not closely match the standards and were not positively identified. The Acid(+)1 mutant produced 30-fold higher levels of oxalic acid compared to the wild-type 2575, but other organic acids were not detected. Such enhanced production of oxalic acid may account for the final pH of Acid(+)1 cultures being lower than that of wild-type or Acid(-)1 cultures. The Acid(-)1 mutant produced negligible levels of oxalic acid.
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DISCUSSION |
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In contrast to growth, protease production was reduced by acidification of the medium in Acid(+) mutants, and these traits co-reverted, indicating that they involve a single mutation. In addition, wild-type and Acid(+) mutants produced very similar levels of protease activity in buffered liquid culture containing insect cuticle, demonstrating that regulation of protease production was not altered in the mutants. Tests with solid media permitted simultaneous evaluation of changes in the pH of the medium and in protease production, and their interrelations. Our results show that alkalinization of the medium is closely related to the degradation of extracellular proteins in wild-type and Acid(-) mutants. This suggests that alkalinization is adaptive, facilitating the utilization of a nutrient source, and is linked to our observation that extracellular enzyme activities are only produced at the pH at which they are active (St Leger et al., 1998 ). Similar observations with A. fumigatus and N. crassa suggest that this phenomenon may be widespread.
The capacity to elevate pH in their locality confirms that fungi produce one or more basic compounds. The pH at the margin of colonies on solid media is greater than the pH at the centre. This presumably reflects greater metabolic activity at growing hyphal points, including oxidative deamination of amino acids leading to ammonia accumulation and diffusion. Analysis of end-products in liquid culture revealed that ammonia was produced during growth on yeast extract at levels sufficient to account for the rise in the pH of the medium. Ammonia production by some fungi during growth on protein as sole carbon source is believed to result from dissimilation of amino nitrogen produced in excess of that required for growth (Jennings, 1989 ). Growing M. anisopliae in the presence of low levels of single amino acids also resulted in high levels of ammonia formation. Complete catabolism of glutamate liberates two ammonia molecules per molecule of glutamate, which could explain the elevated ammonia levels compared with growth on methionine. However, increasing the abundance of amino acids decreased the formation of ammonia, suggesting that in nature, continual formation of ammonia requires release of low levels of amino acids from a protein source. This is contrary to the idea that ammonia is a by-product of excess amino nitrogen during amino acid catabolism and implies either induction of catabolite repressible enzyme(s), presumably deaminases, or regulation of the enzyme activity via substrate inhibition. Regulation of amino acid oxidase in N. crassa is inducible by amino acids and would release ammonia (Sikora & Marzluf, 1982
). However, N. crassa and A. fumigatus also produced ammonia in minimal medium, again contradicting the superabundance of nitrogen theory and implying that the source was internal nitrogenous reserves. Ammonia production by the three fungal species also declined or ceased altogether when amino acids were supplemented with glucose. Glucose may have repressed production of deaminases. It is also possible that the higher growth rate with glucose allowed more complete utilization of ammonia released from amino acid catabolism. In addition, the fungi may have switched from catabolizing amino acids to catabolizing sugars for the production of nitrogen-non-containing polymers, thereby decreasing free ammonia production.
Previous studies with bacteria (Mekalanos, 1992 ), C. albicans (De Bernardis et al., 1998
) and M. anisopliae (St Leger et al., 1998
) have focused on pH as an environmental cue in the host niche, serving to regulate the differential gene expression needed to survive within that niche. However, filamentous fungi are also able to modify the environmental pH, and these modifications may have a function related to exploitation of resources rather than just being by-products of metabolic activity. Ammonia production by M. anisopliae is tightly regulated by amino acids, which as protein degradation products could serve as signals for the presence of proteinaceous nutrients in the environment. Generation of ammonia by N. crassa or A. fumigatus is more loosely regulated and occurs whenever they are in nutrient-deprived conditions, presumably from degradation of endogenous compounds. Given that ammonium is linked with nitrogen catabolite repression (Arst & Cove, 1969
), there are presumably additional regulatory mechanisms that allow protease production in the presence of ammonia.
Most of the ammonia produced by fungi will be transformed to ammonium in solution. However, at higher pH a larger proportion of the ammonium becomes toxic unionized ammonia; the approximate ratios of to NH3 will be 1800:1 at pH 6, and 9:1 at pH 8·3. Ammonia is very toxic because it is lipid soluble and raises intracellular pH, thus inhibiting protein synthesis and enzyme activity (Doyle & Butler, 1990
). Measurement of the pH of insect cuticle infected with M. anisopliae demonstrated an overall increase compared to unaffected cuticles, but the precise pH at sites of infection by M. anisopliae and A. fumigatus remains to be determined. Further research will indicate whether acid/base production by M. anisopliae and A. fumigatus in vivo is a factor in their virulence to animal hosts. As regards saprophytes such as N. crassa, proteases can work as important factors in scavenging nutrients during saprotrophy.
The results of this study suggest that ammonia may have functions related to regulation of the microenvironment and may represent a previously unconsidered virulence factor in diverse fungi with the potential to harm tissues and disturb the hosts immune system.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 March 1999;
revised 17 May 1999;
accepted 8 June 1999.