Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan1
Author for correspondence: Toshiyuki Mio. Tel: +81 467 47 2242. Fax: +81 467 46 5320. e-mail: toshiyuki.mio{at}roche.com
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ABSTRACT |
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Keywords: GNA1, glucosamine-6-phosphate acetyltransferase, antifungal target, Candida albicans, virulence
Abbreviations: GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; ManNAc, N-acetylmannosamine
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INTRODUCTION |
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UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential precursor of peptidoglycan and lipid A in bacteria (Holtje & Schwarz, 1985 ; Park, 1987
; Raetz, 1987
), and of chitin and glycoproteins in yeast (Cabib et al., 1982
; Herscovics & Orlean, 1993
). In the yeast Saccharomyces cerevisiae, four enzymes are involved in the reaction from fructose 6-phosphate to UDP-GlcNAc: (i) glutamine:fructose-6-phosphate amidotransferase (Watzele & Tanner, 1989
; Smith et al., 1996
); (ii) glucosamine-6-phosphate acetyltransferase (Mio et al., 1999
); (iii) phosphoacetylglucosamine mutase (Hofmann et al., 1994
); and (iv) UDP-GlcNAc pyrophosphorylase (Mio et al., 1998
). The gene for glutamine:fructose-6-phosphate amidotransferase has been cloned by complementing the gcn1 mutation, and its expression is increased several fold by
-factor (Whelan & Ballou, 1975
; Watzele & Tanner, 1989
). AGM1, which suppresses the growth defect caused by a pgm1 pgm2 double mutation, is a phosphoacetylglucosamine mutase gene (Boles et al., 1994
; Hofmann et al., 1994
). In previous papers, we reported that UAP1 and GNA1 genes encode UDP-GlcNAc pyrophosphorylase and glucosamine-6-phosphate acetyltransferase, respectively (Mio et al., 1998
, 1999
). The genes for UDP-GlcNAc biosynthesis are conserved between S. cerevisiae and C. albicans. In addition, disruption of each of the S. cerevisiae genes GFA1 (Watzele & Tanner, 1989
), GNA1, AGM1 and UAP1 was lethal, suggesting that enzymes involved in UDP-GlcNAc biosynthesis are potential targets for antifungal agents.
On the other hand, a GlcNAc catabolic pathway has been reported in C. albicans. GlcNAc can be transported into the cells by GlcNAc permease and converted to fructose 6-phosphate by the sequential action of GlcNAc kinase, GlcNAc-6-phosphate deacetylase and glucosamine-6-phosphate deaminase (Singh & Datta, 1979 ; Gopal et al., 1982
; Datta et al., 1989
). Therefore, C. albicans gna1 null mutants should grow in the presence of GlcNAc. In this work, we obtained C. albicans heterozygous and homozygous gna1
null mutants and characterized their phenotypes. In addition, we show the effect of a mutation in GNA1 on virulence in a mouse model of candidiasis.
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METHODS |
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Assay for glucosamine-6-phosphate acetyltransferase (EC 2.3.1.4).
Glucosamine-6-phosphate acetyltransferase activity was determined by measurement of CoA produced. Since CoA reacts with 2-nitrobenzoic acid and releases 4-nitrothiophenolate (Gehring et al., 1996 ; Riddles et al., 1983
), an assay was performed in a 50 µl reaction mixture containing 50 mM Tris/HCl (pH 7·5), 5 mM MgCl2, 150 µM glucosamine 6-phosphate, 150 µM acetyl-CoA, 10% (v/v) glycerol and 70 µg of crude extract obtained from the mutants. After incubation at 37 °C for 20 min, the reaction was terminated by adding a solution containing 50 mM Tris/HCl (pH 7·5) and 6·4 M guanidine hydrochloride, and then 50 µl of a solution containing 50 mM Tris/HCl (pH 7·5), 1 mM EDTA and 20 µM 2-nitrobenzoic acid. The amount of CoA produced by glucosamine-6-phosphate acetyltransferase was estimated from that of 4-nitrothiophenolate by measuring the absorbance at 412 nm.
Systemic infection of mice.
Stationary-phase C. albicans cells were harvested from the YPDGlcNAc cultures. The cells were washed with sterile distilled water, suspended in saline and counted with a haemocytometer. Cell suspensions (0·2 ml) containing 1x105, 1x106 and 1x107 cells were then injected intravenously into mice. Each test strain was injected into five mice.
Quantification of C. albicans in infected tissue.
Cell suspensions (0·2 ml) containing 1x105, 1x106 and 1x107 cells were injected intravenously into mice. The number of C. albicans cells in the kidney and liver was examined 3 d after infection.
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RESULTS |
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To examine the effect of GNA1 disruption on glucosamine-6-phosphate acetyltransferase, the activity of the enzyme from mutants was measured. When incubated with glucosamine 6-phosphate and acetyl-CoA, the crude extract from wild-type CAI4 produced 38·9±3·9 µM CoA from acetyl-CoA. In contrast, the homozygous gna1 null mutant (CGM1210-1) showed a large reduction in glucosamine-6-phosphate acetyltransferase activity (2·0±0·4 µM CoA released), and the activity of the heterozygous gna1
mutant (CGM12-1) retained about 40% of wild-type CAI4 activity (14·2±2·9 µM CoA released), indicating a gene dosage effect.
Effect of GNA1 disruption on morphology and growth
In S. cerevisiae, ScGNA1-deficient cells exhibited morphological defects: most of the yeast cells swelled and then often lysed (Mio et al., 1999 ). The role of GNA1 on C. albicans growth was investigated. CGM1210-1 grew and formed hyphae in YPD + GlcNAc (Fig. 2b
), and the growth rate was indistinguishable from that of wild-type (CAF2) or CGM12-1 (the doubling times for CAF2, CGM12-1 and CGM1210-1 were 71, 64 and 73 min, respectively) (Fig. 2a
). When CGM1210-1 was cultured on YPD, most of the cells dramatically enlarged, swelled and became defective in cell separation (Fig. 2b
). This phenotype was similar to that caused by depletion of ScGNA1, suggesting that C. albicans GNA1 is also essential for growth and the synthesis of UDP-GlcNAc.
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DISCUSSION |
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The avirulence of the homozygous gna1 null mutant indicates that GNA1 is important for the virulence of C. albicans. In addition, the heterozygous gna
mutant displayed an intermediate virulence phenotype, suggesting that the reduced virulence of gna1
mutants is associated with the loss of glucosamine-6-phosphate acetyltransferase activity.
The homozygous gna1 null mutant is not defective in growth rate and hyphal formation in the presence of enough GlcNAc. In addition, the calcofluor susceptibility of the mutants is similar to that of the wild-type, suggesting that the mutants synthesize chitin from exogenous GlcNAc in an amount sufficient for growth (data not shown). These facts suggest that the homozygous gna
null mutant does not survive in host animals because of an insufficient level of GlcNAc in the tissues.
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ACKNOWLEDGEMENTS |
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Received 20 December 1999;
revised 2 March 2000;
accepted 16 March 2000.