(1,3)beta-Glucan synthase activity from the filamentous Ascomycete Neurospora crassa was purified... more (1,3)beta-Glucan synthase activity from the filamentous Ascomycete Neurospora crassa was purified 1300-fold to a specific activity of 14,000 nmol glucose incorporated/min per mg protein. Hyphae were disrupted and crude membrane fractions obtained by high-speed centrifugation. Membrane fractions were extracted with Tergitol NP-40 and a second high-speed particulate fraction was obtained. Enzyme activity was solubilized with (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and octyl-beta-D-glucoside from Tergitol-extracted membrane preparations. Solubilized enzyme activity was purified by product entrapment and recovered by low-speed centrifugation through a layer of sucrose. SDS-PAGE analysis revealed 2 proteins of 165 and 100 kDa as likely candidates for subunits of the (1,3)beta-glucan synthase enzyme complex. 5-Azido-[32P]UDP-glucose was photo-crosslinked to UDP-glucose-binding proteins in each fraction of the purification procedure. Autoradiograms of SDS-PAGE gels revealed a single protein of 165 kDa enriching with enzyme activity and labeling with the substrate analog. Photoincorporation of 5-azido-[32P]UDP-glucose by the 165 kDa protein was competed by 0.25 mM UDP-glucose (80%) and TDP-glucose (65%) while ADP-glucose (27%), CDP-glucose (36%), and GDP-glucose (8%) where less effective. These results were similar to in vitro inhibition of enzyme activity by the same compounds. These data strongly suggest that the 165 kDa protein is a substrate-binding subunit of (1,3)beta-glucan synthase.
A new screening technique has been developed for the rapid identification of Neurospora crassa mu... more A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance...
Saponins are steroidal or terpenoid-based glycosides with surface active properties. A steroidal ... more Saponins are steroidal or terpenoid-based glycosides with surface active properties. A steroidal saponin, CAY-1, with a molecular weight of 1243.35 Da, was isolated and purified to homogeneity from commercially available dry, ground fruit of Capsicum frutescens. CAY-1 was shown to be a potent fungicide for the germinating conidia of Aspergillus flavus, A. fumigatus, A. parasiticus and A. niger with species-dependent LD90 values between 3 and 20 microM. Activity against some Aspergillus species was affected by the test medium used. In vitro assays, CAY-1 was effective against Pneumocystis carinii (IC50): 9.5 microM) and Candida albicans (IC90: 6.2 microM). CAY-1 had no effect on the viability of the nongerminating conidia of the two filamentous fungi, P. carinii and C. albicans, nor on the conidial type of Fusarium oxysporum. It was ineffective against the bacteria Enterobacter agglomerans, Bacillus subtilis, Escherichia coli and Staphylococcus aureus. CAY-1 was not cytotoxic to A 54...
... 6). 134 De Lucca et al. Medical Mycology, 40, 131–137 Page 5. Discussion ... Others have anti... more ... 6). 134 De Lucca et al. Medical Mycology, 40, 131–137 Page 5. Discussion ... Others have antifungal properties. For example, extracts of Eriocephalus africanus L., Felicia erigeroides DC and Helichrysum crispum (L.) D. Don, inhibited C. albicans growth [13]. ...
ABSTRACT β(1-3)Glucan synthase activity of Neurospora crassa was rendered soluble by treatment of... more ABSTRACT β(1-3)Glucan synthase activity of Neurospora crassa was rendered soluble by treatment of protoplast membranes from either a protoplast-forming strain (os-1) or wild-type with 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 100 mM glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 mM sodium fluoride. Additions of 1 mM dithiothreitol, 200 mM inorganic phosphate, 10 μM GTP, and 0.5M glycerol increased the amount of enzyme activity solubilized (∼50% of recovered enzyme activity was soluble). Solubilized glucan synthase from both os-1 and wild-type catalyzed the formation of linear, unbranched β(1-3)glucan; Kmapp and Vmax were similar to those of the particulate enzyme activity.
Alkaline phosphomonoesterase (EC 3.1.3.1) activity from Blastocladiella emersonii, while displayi... more Alkaline phosphomonoesterase (EC 3.1.3.1) activity from Blastocladiella emersonii, while displaying typically broad substrate specificity for phosphorylated organic compounds, exhibited nearly complete substrate preference for N-acetylglucosamine-6-phosphate over N-acetylglucosamine-1-phosphate. Enzyme in zoospore extracts was purified 43-fold by differential centrifugation followed by gel filtration (Sephadex G-200) and then by ion-exchange chromatography (diethylaminoethyl-cellulose). The partially purified enzyme displayed an apparent molecular weight (Sephadex G-200) of approximately 170,000. The activity of partially purified enzyme exhibited a pH optimum of pH 8.5, did not require a metal divalent cation, but was inhibitable by ethylenediaminetetraacetic acid. During the life cycle of the organism, the specific activity of the phosphatase decreased slightly during germination and early exponential growth but then increased about 4.5-fold during sporulation. B. emersonii alkali...
CAY-1, a novel saponin from Capsicum frutescens (commercially known as cayenne pepper) was invest... more CAY-1, a novel saponin from Capsicum frutescens (commercially known as cayenne pepper) was investigated to determine its in vitro antifungal activity, mechanism of action and mammalian cell cytotoxicity. CAY-1 was active against 16 different fungal strains, including Candida spp. and Aspergillus fumigatus [minimum inhibitory concentrations (MIC) ranging from 4 to 16 microg ml(-1)], and was especially active against Cryptococcus neoformans (90% inhibition at 1 microg ml(-1)). Synergistic activity was also observed between CAY-1 and amphotericin B against Candida albicans and A. fumigatus. No significant cytotoxicity was demonstrated when CAY-1 was tested against 55 mammalian cell lines at up to 100 microg ml(-1). Importantly, CAY-1 appears to act by disrupting the membrane integrity of fungal cells.
Proceedings of the National Academy of Sciences of the United States of America, 1980
Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is the major macromolecular constituent of... more Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is the major macromolecular constituent of Blastocladiella emersonii cell walls. Zoospores do not possess a wall nor do they contain sufficient total hexosamine to account for the chitin content of the wall abruptly formed during germination. UDPGlcNAc, both the endproduct of hexosamine biosynthesis and the substrate for chitin synthesis, is present in zoospores in sufficient concentration to inhibit the first hexosamine pathway-specific enzyme activity. Net chitin accumulates in register with dry weight during exponential growth, but does not accumulate appreciably during the succeeding sporulation phase. Predicted relationships among net rates of chitin synthesis, UDPGlcNAc concentrations, and UDP plus UTP concentrations throughout the life cycle are explored, as are the assumptions upon which the predictions were based. We find that the sensitivity of the first hexosamine pathway-specific enzyme to endproduct inhibition is no...
2-Deoxy-D-glucose-6-phosphate ketol-isomerase (EC 2.6.1.16) forms glucosamine-6-phosphate and glu... more 2-Deoxy-D-glucose-6-phosphate ketol-isomerase (EC 2.6.1.16) forms glucosamine-6-phosphate and glutamate from fructose-6-phosphate and glutamine and plays an important role in chitin synthesis in fungi. We have established a new assay for fungal ketol-isomerase activity that is amenable to high throughput screening to identify enzyme inhibitors. Aspergillus fumigatus crude lysate was incubated with substrates and after incubation, reactions were terminated. Glutamate dehydrogenase, nitro blue tetrazolium chloride, phenazine methosulfate and beta-NAD were added and the amount of glutamate formed by ketol-isomerase activity was determined by measuring OD585nm. A feedback inhibitor, UDP-N-acetylglucosamine, of fungal ketol-isomerase was successfully detected by this assay (IC50=0.48 mM). In a pilot scale screening, an active extract from an extremophilic bacterium was found, and the extract showed antifungal activity against A. fumigatus, Candida albicans and C. glabrata.
Heterologous expression of plant genes may serve as an important alternative for producing plant ... more Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a pro...
The human fungal pathogen, Candida albicans, has three putative histidine kinases showing homolog... more The human fungal pathogen, Candida albicans, has three putative histidine kinases showing homology to those of plants, bacteria and other fungi. We have constructed a homozygous deletion strain and a hemizygous reconstituted strain of one of these histidine-kinase-encoding genes, COS-1, in C. albicans. Neither strain showed any growth defect in a number of liquid media nor increased resistance or sensitivity to a number of antifungal drugs. Importantly, we show that the COS-1 homozygous disruption strain had significantly reduced virulence in a systemic murine model of candidosis. Thus, COS-1 appears to be an in vivo virulence factor and may represent a novel target for the development of antifungal drugs.
Extracts of the biomasses and fermentation broths of 217 extremophilic microorganisms isolated fr... more Extracts of the biomasses and fermentation broths of 217 extremophilic microorganisms isolated from a number of locales were screened for antifungal activity using whole-cell and mechanism-based in vitro assays. Importantly, eleven broth extracts had activity against several Candida species and Aspergillus fumigatus in whole-cell in vitro assays. One broth specifically inhibited (1,3)beta-glucan synthase activity and four specifically inhibited ketol-isomerase activity, suggesting a mode of action of the antifungal compound(s) present in these extracts. The extract from one thermophile, a novel species of Pseudomonas, was fractionated, an active compound purified and its structure determined. The compound was identified as pyochelin, a previously identified iron-binding compound with heretofore undescribed antifungal activity. To our knowledge, this is the first report demonstrating that extremophiles synthesize compounds that have antifungal activity.
Proceedings of the National Academy of Sciences, 1980
Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is the major macromolecular constituent of... more Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is the major macromolecular constituent of Blastocladiella emersonii cell walls. Zoospores do not possess a wall nor do they contain sufficient total hexosamine to account for the chitin content of the wall abruptly formed during germination. UDPGlcNAc, both the endproduct of hexosamine biosynthesis and the substrate for chitin synthesis, is present in zoospores in sufficient concentration to inhibit the first hexosamine pathway-specific enzyme activity. Net chitin accumulates in register with dry weight during exponential growth, but does not accumulate appreciably during the succeeding sporulation phase. Predicted relationships among net rates of chitin synthesis, UDPGlcNAc concentrations, and UDP plus UTP concentrations throughout the life cycle are explored, as are the assumptions upon which the predictions were based. We find that the sensitivity of the first hexosamine pathway-specific enzyme to endproduct inhibition is not constant throughout the life cycle; sensitivity is very high in the zoospore phase, decreases dramatically during germination, remains very low through the growth phase, and increases gradually to the zoospore level during sporulation. The organism appears to have evolved endproduct regulation in this case as an adaptation to "hard-times" phases of the life cycle-i.e., as a safeguard against overproduction of end product (UDPGlcNAc) when its utilization in cell wall (specifically chitin) synthesis is curtailed. Conversely, the organism effectively discards this mode of regulation during "good times," when the demands for end product are evidently greater than endproduct inhibition would otherwise permit.
Proceedings of the National Academy of Sciences, 1976
De novo construction of a chitinous cell wall accompanies Blastocladiella emersonii zoospore germ... more De novo construction of a chitinous cell wall accompanies Blastocladiella emersonii zoospore germination. At least an order of magnitude increase in total hexosamine occurs during germination. This increase is into polymer (chitin) and occurs on schedule in the presence of cycloheximide. Uridine-5'-diphospho-N-acetylglucosamine (UDPGlcNAc), both the end product of hexosamine biosynthesis and a substrate for chitin biosynthesis, is a potent inhibitor of the activity of the first pathway-specific enzyme of hexosamine biosynthesis in zoospore extracts. Certain uridine nucleotides, not perceptibly influencing the activity of the first enzyme per se, counteract the inhibitory effects of UDPGlcNAc. The concentration of UDPGlcNAc in the zoospore is sufficient to act as an inhibitor of the enzyme, but the amount of UDPGlcNAc is sufficient, by over an order of magnitude, to account for the chitin synthesized during germination. Both the production of UDPGlcNAc and its utilization for chitin synthesis appear to be post-translationally regulated in zoospores and during zoospore germination.
(1,3)beta-Glucan synthase activity from the filamentous Ascomycete Neurospora crassa was purified... more (1,3)beta-Glucan synthase activity from the filamentous Ascomycete Neurospora crassa was purified 1300-fold to a specific activity of 14,000 nmol glucose incorporated/min per mg protein. Hyphae were disrupted and crude membrane fractions obtained by high-speed centrifugation. Membrane fractions were extracted with Tergitol NP-40 and a second high-speed particulate fraction was obtained. Enzyme activity was solubilized with (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and octyl-beta-D-glucoside from Tergitol-extracted membrane preparations. Solubilized enzyme activity was purified by product entrapment and recovered by low-speed centrifugation through a layer of sucrose. SDS-PAGE analysis revealed 2 proteins of 165 and 100 kDa as likely candidates for subunits of the (1,3)beta-glucan synthase enzyme complex. 5-Azido-[32P]UDP-glucose was photo-crosslinked to UDP-glucose-binding proteins in each fraction of the purification procedure. Autoradiograms of SDS-PAGE gels revealed a single protein of 165 kDa enriching with enzyme activity and labeling with the substrate analog. Photoincorporation of 5-azido-[32P]UDP-glucose by the 165 kDa protein was competed by 0.25 mM UDP-glucose (80%) and TDP-glucose (65%) while ADP-glucose (27%), CDP-glucose (36%), and GDP-glucose (8%) where less effective. These results were similar to in vitro inhibition of enzyme activity by the same compounds. These data strongly suggest that the 165 kDa protein is a substrate-binding subunit of (1,3)beta-glucan synthase.
A new screening technique has been developed for the rapid identification of Neurospora crassa mu... more A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance...
Saponins are steroidal or terpenoid-based glycosides with surface active properties. A steroidal ... more Saponins are steroidal or terpenoid-based glycosides with surface active properties. A steroidal saponin, CAY-1, with a molecular weight of 1243.35 Da, was isolated and purified to homogeneity from commercially available dry, ground fruit of Capsicum frutescens. CAY-1 was shown to be a potent fungicide for the germinating conidia of Aspergillus flavus, A. fumigatus, A. parasiticus and A. niger with species-dependent LD90 values between 3 and 20 microM. Activity against some Aspergillus species was affected by the test medium used. In vitro assays, CAY-1 was effective against Pneumocystis carinii (IC50): 9.5 microM) and Candida albicans (IC90: 6.2 microM). CAY-1 had no effect on the viability of the nongerminating conidia of the two filamentous fungi, P. carinii and C. albicans, nor on the conidial type of Fusarium oxysporum. It was ineffective against the bacteria Enterobacter agglomerans, Bacillus subtilis, Escherichia coli and Staphylococcus aureus. CAY-1 was not cytotoxic to A 54...
... 6). 134 De Lucca et al. Medical Mycology, 40, 131–137 Page 5. Discussion ... Others have anti... more ... 6). 134 De Lucca et al. Medical Mycology, 40, 131–137 Page 5. Discussion ... Others have antifungal properties. For example, extracts of Eriocephalus africanus L., Felicia erigeroides DC and Helichrysum crispum (L.) D. Don, inhibited C. albicans growth [13]. ...
ABSTRACT β(1-3)Glucan synthase activity of Neurospora crassa was rendered soluble by treatment of... more ABSTRACT β(1-3)Glucan synthase activity of Neurospora crassa was rendered soluble by treatment of protoplast membranes from either a protoplast-forming strain (os-1) or wild-type with 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 100 mM glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 mM sodium fluoride. Additions of 1 mM dithiothreitol, 200 mM inorganic phosphate, 10 μM GTP, and 0.5M glycerol increased the amount of enzyme activity solubilized (∼50% of recovered enzyme activity was soluble). Solubilized glucan synthase from both os-1 and wild-type catalyzed the formation of linear, unbranched β(1-3)glucan; Kmapp and Vmax were similar to those of the particulate enzyme activity.
Alkaline phosphomonoesterase (EC 3.1.3.1) activity from Blastocladiella emersonii, while displayi... more Alkaline phosphomonoesterase (EC 3.1.3.1) activity from Blastocladiella emersonii, while displaying typically broad substrate specificity for phosphorylated organic compounds, exhibited nearly complete substrate preference for N-acetylglucosamine-6-phosphate over N-acetylglucosamine-1-phosphate. Enzyme in zoospore extracts was purified 43-fold by differential centrifugation followed by gel filtration (Sephadex G-200) and then by ion-exchange chromatography (diethylaminoethyl-cellulose). The partially purified enzyme displayed an apparent molecular weight (Sephadex G-200) of approximately 170,000. The activity of partially purified enzyme exhibited a pH optimum of pH 8.5, did not require a metal divalent cation, but was inhibitable by ethylenediaminetetraacetic acid. During the life cycle of the organism, the specific activity of the phosphatase decreased slightly during germination and early exponential growth but then increased about 4.5-fold during sporulation. B. emersonii alkali...
CAY-1, a novel saponin from Capsicum frutescens (commercially known as cayenne pepper) was invest... more CAY-1, a novel saponin from Capsicum frutescens (commercially known as cayenne pepper) was investigated to determine its in vitro antifungal activity, mechanism of action and mammalian cell cytotoxicity. CAY-1 was active against 16 different fungal strains, including Candida spp. and Aspergillus fumigatus [minimum inhibitory concentrations (MIC) ranging from 4 to 16 microg ml(-1)], and was especially active against Cryptococcus neoformans (90% inhibition at 1 microg ml(-1)). Synergistic activity was also observed between CAY-1 and amphotericin B against Candida albicans and A. fumigatus. No significant cytotoxicity was demonstrated when CAY-1 was tested against 55 mammalian cell lines at up to 100 microg ml(-1). Importantly, CAY-1 appears to act by disrupting the membrane integrity of fungal cells.
Proceedings of the National Academy of Sciences of the United States of America, 1980
Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is the major macromolecular constituent of... more Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is the major macromolecular constituent of Blastocladiella emersonii cell walls. Zoospores do not possess a wall nor do they contain sufficient total hexosamine to account for the chitin content of the wall abruptly formed during germination. UDPGlcNAc, both the endproduct of hexosamine biosynthesis and the substrate for chitin synthesis, is present in zoospores in sufficient concentration to inhibit the first hexosamine pathway-specific enzyme activity. Net chitin accumulates in register with dry weight during exponential growth, but does not accumulate appreciably during the succeeding sporulation phase. Predicted relationships among net rates of chitin synthesis, UDPGlcNAc concentrations, and UDP plus UTP concentrations throughout the life cycle are explored, as are the assumptions upon which the predictions were based. We find that the sensitivity of the first hexosamine pathway-specific enzyme to endproduct inhibition is no...
2-Deoxy-D-glucose-6-phosphate ketol-isomerase (EC 2.6.1.16) forms glucosamine-6-phosphate and glu... more 2-Deoxy-D-glucose-6-phosphate ketol-isomerase (EC 2.6.1.16) forms glucosamine-6-phosphate and glutamate from fructose-6-phosphate and glutamine and plays an important role in chitin synthesis in fungi. We have established a new assay for fungal ketol-isomerase activity that is amenable to high throughput screening to identify enzyme inhibitors. Aspergillus fumigatus crude lysate was incubated with substrates and after incubation, reactions were terminated. Glutamate dehydrogenase, nitro blue tetrazolium chloride, phenazine methosulfate and beta-NAD were added and the amount of glutamate formed by ketol-isomerase activity was determined by measuring OD585nm. A feedback inhibitor, UDP-N-acetylglucosamine, of fungal ketol-isomerase was successfully detected by this assay (IC50=0.48 mM). In a pilot scale screening, an active extract from an extremophilic bacterium was found, and the extract showed antifungal activity against A. fumigatus, Candida albicans and C. glabrata.
Heterologous expression of plant genes may serve as an important alternative for producing plant ... more Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a pro...
The human fungal pathogen, Candida albicans, has three putative histidine kinases showing homolog... more The human fungal pathogen, Candida albicans, has three putative histidine kinases showing homology to those of plants, bacteria and other fungi. We have constructed a homozygous deletion strain and a hemizygous reconstituted strain of one of these histidine-kinase-encoding genes, COS-1, in C. albicans. Neither strain showed any growth defect in a number of liquid media nor increased resistance or sensitivity to a number of antifungal drugs. Importantly, we show that the COS-1 homozygous disruption strain had significantly reduced virulence in a systemic murine model of candidosis. Thus, COS-1 appears to be an in vivo virulence factor and may represent a novel target for the development of antifungal drugs.
Extracts of the biomasses and fermentation broths of 217 extremophilic microorganisms isolated fr... more Extracts of the biomasses and fermentation broths of 217 extremophilic microorganisms isolated from a number of locales were screened for antifungal activity using whole-cell and mechanism-based in vitro assays. Importantly, eleven broth extracts had activity against several Candida species and Aspergillus fumigatus in whole-cell in vitro assays. One broth specifically inhibited (1,3)beta-glucan synthase activity and four specifically inhibited ketol-isomerase activity, suggesting a mode of action of the antifungal compound(s) present in these extracts. The extract from one thermophile, a novel species of Pseudomonas, was fractionated, an active compound purified and its structure determined. The compound was identified as pyochelin, a previously identified iron-binding compound with heretofore undescribed antifungal activity. To our knowledge, this is the first report demonstrating that extremophiles synthesize compounds that have antifungal activity.
Proceedings of the National Academy of Sciences, 1980
Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is the major macromolecular constituent of... more Chitin, a homopolymer of N-acetylglucosamine (GlcNAc), is the major macromolecular constituent of Blastocladiella emersonii cell walls. Zoospores do not possess a wall nor do they contain sufficient total hexosamine to account for the chitin content of the wall abruptly formed during germination. UDPGlcNAc, both the endproduct of hexosamine biosynthesis and the substrate for chitin synthesis, is present in zoospores in sufficient concentration to inhibit the first hexosamine pathway-specific enzyme activity. Net chitin accumulates in register with dry weight during exponential growth, but does not accumulate appreciably during the succeeding sporulation phase. Predicted relationships among net rates of chitin synthesis, UDPGlcNAc concentrations, and UDP plus UTP concentrations throughout the life cycle are explored, as are the assumptions upon which the predictions were based. We find that the sensitivity of the first hexosamine pathway-specific enzyme to endproduct inhibition is not constant throughout the life cycle; sensitivity is very high in the zoospore phase, decreases dramatically during germination, remains very low through the growth phase, and increases gradually to the zoospore level during sporulation. The organism appears to have evolved endproduct regulation in this case as an adaptation to "hard-times" phases of the life cycle-i.e., as a safeguard against overproduction of end product (UDPGlcNAc) when its utilization in cell wall (specifically chitin) synthesis is curtailed. Conversely, the organism effectively discards this mode of regulation during "good times," when the demands for end product are evidently greater than endproduct inhibition would otherwise permit.
Proceedings of the National Academy of Sciences, 1976
De novo construction of a chitinous cell wall accompanies Blastocladiella emersonii zoospore germ... more De novo construction of a chitinous cell wall accompanies Blastocladiella emersonii zoospore germination. At least an order of magnitude increase in total hexosamine occurs during germination. This increase is into polymer (chitin) and occurs on schedule in the presence of cycloheximide. Uridine-5'-diphospho-N-acetylglucosamine (UDPGlcNAc), both the end product of hexosamine biosynthesis and a substrate for chitin biosynthesis, is a potent inhibitor of the activity of the first pathway-specific enzyme of hexosamine biosynthesis in zoospore extracts. Certain uridine nucleotides, not perceptibly influencing the activity of the first enzyme per se, counteract the inhibitory effects of UDPGlcNAc. The concentration of UDPGlcNAc in the zoospore is sufficient to act as an inhibitor of the enzyme, but the amount of UDPGlcNAc is sufficient, by over an order of magnitude, to account for the chitin synthesized during germination. Both the production of UDPGlcNAc and its utilization for chitin synthesis appear to be post-translationally regulated in zoospores and during zoospore germination.
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