Distinguishing Candida species by beta-N-acetylhexosaminidase activity.

A variety of fungi produce the hydrolytic enzyme beta-N-acetylhexosaminidase (HexNAcase), which can be readily detected in assays by using p-nitrophenyl-N-acetyl-beta-D-glucosaminide as a substrate. In the present study we developed a microtiter plate-based HexNAcase assay for distinguishing Candida albicans and Candida dubliniensis strains from other yeast species. HexNAcase activity was detected in 89 of 92 (97%) C. albicans strains and 4 of 4 C. dubliniensis strains but not in 28 strains of eight other Candida species, 4 Saccharomyces cerevisiae strains, or 2 Cryptococcus neoformans strains. The HexNAcase activity in C. albicans and C. dubliniensis was strain specific. All except three clinical C. albicans isolates among the C. albicans strains tested produced enzyme activity within 24 h. These strains did produce enzyme activity, however, after a prolonged incubation period. For two of these atypical strains, genomic DNA at the C. albicans HEX1 gene locus, which encodes HexNAcase, showed nucleotide differences from the sequence of control strains. Among the other Candida species tested, only C. dubliniensis had a DNA sequence that hybridized with the HEX1 probe under low-stringency conditions. The microtiter plate-based assay used in the present study for the detection of HexNAcase activity is a simple, relatively inexpensive method useful for the presumptive identification of C. albicans and C. dubliniensis.

Candida albicans HEX1 gene, a reporter of gene expression in Saccharomyces cerevisiae.

The Candida albicans HEX1 gene was examined as a reporter of gene expression in Saccharomyces cerevisiae. The galactose-inducible S. cerevisiae GAL1-GAL10 promoter was inserted upstream of the C. albicans HEX1 gene, which encodes N-acetylglucosaminidase. The gene was introduced into S. cerevisiae AH22, which has no background N-acetylglucosaminidase activity. Expression of HEX1 in transformed cells was induced significantly by galactose and was repressed by glucose. The HEX1 gene product was functional in S. cerevisiae cells and was targeted to the periplasm. Both untransformed S. cerevisiae cells and cells expressing HEX1 had similar growth curves and cell morphology indicating that expression of N-acetylglucosaminidase was not toxic to the host strain. These results demonstrate that the HEX1 gene can be a useful reporter of gene expression in S. cerevisiae.

Regulation of N-acetylglucosaminidase production in Candida albicans.

The N-acetylglucosaminidase of Candida albicans is a secreted hydrolytic enzyme that contributes to the yeast's virulence. There was a significant increase in the N-acetylglucosaminidase activity of C. albicans cells released from carbon starvation in medium containing N-acetylglucosamine. The increased enzyme activity in N-acetylglucosamine-grown cells correlated with increased transcription of the HEX1 gene, which encodes C. albicans N-acetylglucosaminidase. In contrast, glucose repressed HEX1 transcription, and glucose-grown cells had on average 94-fold lower N-acetylglucosaminidase activities than did N-acetylglucosamine-grown cells. N-acetylglucosaminidase induction in cells grown on N-acetylglucosamine was also repressed by fructose, mannose or galactose, although to a lesser extent than by glucose, and sucrose repressed enzyme production by only 10%. Eighty-eight percent of the enzyme in N-acetylglucosamine-grown cells was localised in the periplasm, and after incubation for 5 h, 30 or 70% of the total enzyme activity was secreted into the medium by yeast or mycelial cells, respectively. The cellular location of the enzyme and the regulation of production by the carbon source indicate a scavenging role for C. albicans N-acetylglucosaminidase.

Differential profiles of soluble proteins during the initiation of morphogenesis in Candida albicans.

Candida albicans is a dimorphic fungus that can grow either as yeast or as mycelia. The mycelial form may be required for tissue penetration and therefore may have a role in pathogenesis. The protein profiles of the cell-free S100 fraction from budding yeast cells and germ tube-forming cells (an early stage of the transition between yeast and mycelia) were evaluated using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yeast growth or germ tube formation was induced in carbon-starved cells at 37 degrees C by either glucose, galactose or N-acetylglucosamine at pH 4.5 or pH 6.7. More than 400 constitutively synthesised polypeptides were identified on 2-D PAGE by silver staining. A few polypeptides which seem to reflect the release from carbon starvation were detected, but no polypeptides unique to either morphology were observed. Fractionation of S100 preparations by polyethylenimine or heparin-agarose affinity chromatography, which have been used to detect DNA-binding proteins, revealed several proteins that were synthesised on the resumption of cell growth or in response to pH difference. Heparin-agarose also bound novel polypeptides in the size range 130-200 kDa that were preferentially synthesised in germ tube-forming cells. These results suggest that any protein factors that might exert a regulatory role early in germ tube formation are of low abundance, and that a minor group of soluble proteins involved in C. albicans morphogenesis may be differentially synthesised.

The effect of cetylpyridinium chloride (CPC) on the cell surface hydrophobicity and adherence of Candida albicans to human buccal epithelial cells in vitro.

PURPOSE: This study examined the effects of cetylpyridinium chloride (CPC) on cell surface hydrophobicity (CSH) and adherence of blastospores of Candida albicans (MEN strain) to human buccal epithelial cells (BEC) in vitro. METHODS: The effect of CPC treatment of either C. albicans blastospores or BEC on their subsequent adherence was determined using 35SO4 labelled blastospores in association with a Percoll gradient. The effects of CPC treatment of blastospores on their CSH was determined using Hydrophobic Interaction Chromatography. RESULTS: Treatment of exponential and stationary phase blastospores with CPC (50 micrograms mL-1) for 0.5-30 minutes, or with CPC (0.5-50 micrograms mL-1) for 15 minutes resulted in significant reductions in both blastospore CSH and adherence to BEC in vitro. No correlation was apparent (r < 0.8) between reduced CSH and reduced blastospore adherence following treatment with CPC (0.5-50 micrograms mL-1). Significantly reduced adherence of C. albicans (stationary or exponential growth phases) to human BEC was also observed following treatment of BEC with CPC (50 micrograms mL-1) for 0.5-30 minutes or with CPC (0.5-50 micrograms mL-1) for 15 minutes. Antiadherence effects were observed at both sub and super-minimum inhibitory concentrations of CPC. CONCLUSIONS: It is suggested that, whilst the ability of CPC to reduce the CSH of C. albicans may contribute to its reduced adherence to human BEC in vitro, reduced CSH is only one of several possible factors that contribute to the observed antiadherence effects.

Differential extraction of N-acetylglucosaminidase and trehalase from the cell envelope of Candida albicans.

Dithiothreitol (DTT) extraction of N-acetylglucosaminidase and trehalase from intact Candida albicans ATCC 10261 cells was monitored as an index of cell envelope porosity during N-acetylglucosamine-induced morphogenesis. Trehalase, which is secreted into the cell envelope during starvation and bud-formation, displayed similar extraction kinetics in starved, germ tube-forming, and bud-forming cells, indicating that the mother cell wall remains largely unchanged during morphogenic outgrowth and that the porosity of bud and mother cell walls is similar. N-acetylglucosaminidase, which is secreted specifically during morphogenesis, was released eightfold more rapidly from germ tube-forming than bud-forming cells, reflecting major differences in porosity between bud and germ tube. In addition, by assaying DTT extracts and extracted cell residues, it was found that the total extracellular N-acetylglucosaminidase activity increased 2- to 2.5-fold during DTT treatment. Thus, DTT unmasks a cryptic form of N-acetylglucosaminidase. The cryptic activity was associated with the cell wall fraction.

Primary interactions of three quaternary ammonium compounds with blastospores of Candida albicans (MEN strain).

The absorption of three quaternary ammonium compounds (QAC), cetylpyridinium chloride, cetrimide and benzalkonium chloride, onto the surface of blastospores of Candida albicans (MEN strain) was examined at room temperature. Equilibrium uptake occurred in less than 30 seconds for cetylpyridinium chloride and cetrimide whereas 5 min contact time was required for benzalkonium chloride. The adsorption of all three agents may be mathematically described as Langmuirian and hence a concentration-dependent formation of drug-monolayer on the surface of the blastospore occurred. From this the number of molecules adsorbed onto the surface of a single blastospore was calculated to be 1.33 x 10(12), 3.17 x 10(12) and 2.32 x 10(12) for cetylpyridinium chloride, cetrimide and benzalkonium chloride, respectively. These dissimilarities are most likely due to differences in the orientations of both the cationic nitrogen atom and the accompanying lipophilic portions of each QAC at the blastospore surface. Relating these observations to the known antiadherence effects of cetylpyridinium chloride and cetrimide, it can be concluded that monolayer coverage of the blastospore surface with QAC does not account for the observed reduced adherence. This suggests that the anti-adherence effects are due to either direct interaction with, or steric blockade of, adhesions on the blastospore surface.

Purification and characterization of two forms of N-acetylglucosaminidase from Candida albicans showing widely different outer chain glycosylation.

Two forms of N-acetylglucosaminidase were purified to homogeneity by ion exchange (TSK DEAE-3SW, Aquapore CX-300) and gel filtration (TSK G4000 SW) HPLC of Candida albicans ATCC 10261 culture filtrates. Synthesis and secretion of N-acetylglucosaminidase were induced by incubating starved yeast cells at 37 degrees C in medium containing N-acetylglucosamine (GlcNAc). The form of the enzyme depended on the cell growth and starvation conditions before GlcNAc induction. N-Acetylglucosaminidase A (32% total carbohydrate, M(r) 85,000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and N-acetylglucosaminidase B (56% carbohydrate, M(r) 132,000 subunit) was isolated from cells grown in yeast extract/peptone/dextrose. The estimated relative molecular masses of the native enzymes, based on Sephacryl S-300 gel filtration were: A form, 350,000; B form, 600,000; A and B forms after endoglycosidase H (endo H) treatment, 180,000. The purified enzymes migrated on SDS polyacrylamide gels as heterogeneous glycoproteins of M(r) centred at approximately 100,000 (A) and approximately 150,000 (B) but were reduced to a single 58,000 band after denaturation with SDS and cleavage of asparagine-linked sidechains by endo H. When the native glycoproteins were treated with endo H, both enzyme forms had three oligosaccharide sidechains of M(r) approximately 3000 that were endo H resistant. Therefore the difference in the size of N-acetylglucosaminidase A and B was due to variations in outer chain glycosylation of endo H-sensitive inner core structures. N-Acetylglucosaminidase was active and stable over a broad pH range with maximum activity against both p-nitrophenylGlcNAc (pNPGlcNAc) and pNPGalNAc at pH 4.0. The kinetic parameters kcat (s-1) and Km (mM) of N-acetylglucosaminidase A using the following substrates were, respectively: pNPGlcNAc, 740, 0.77; pNPGalNAc, 910, 1.26; N,N'-diacetylchitobiose 620, 0.20; and N,N',N"-triacetylchitotriose, 170, 0.044. The enzyme showed substrate inhibition with all substrates above 0.5 mM except for pNPGalNAc.

Molecular cloning and expression of the Candida albicans beta-N-acetylglucosaminidase (HEX1) gene.

beta-N-Acetylglucosaminidase was purified from the spent culture medium of Candida albicans A72 grown in the presence of N-acetylglucosamine (GlcNAc). The N-terminal amino acid sequence of the protein was determined, two degenerate oligonucleotide probes were constructed, and a 3.9-kb BamHI fragment of DNA that hybridized to both probes was subcloned from a lambda EMBL4 library of C. albicans A72 genomic DNA. This fragment of DNA contained the entire beta-N-acetylglucosaminidase (HEX1) gene, which consisted of an open reading frame coding for a polypeptide precursor of 562 amino acids with a putative 22-amino-acid leader sequence. The deduced HEX1 amino acid sequence showed similarity to hexosaminidases from a variety of organisms. Growth of C. albicans on GlcNAc induced transcription of HEX1, resulting in increased specific beta-N-acetylglucosaminidase activity. HEX1 mRNA (2.35 kb) from GlcNAc-grown cells was approximately 200 bp larger than HEX1 mRNA from cells grown on glucose. This size difference was suggested to result from the use of alternative transcription termination sites. The cloned HEX1 gene introduced into C. albicans SGY-243 on a plasmid also responded to GlcNAc induction.

Detection of Candida albicans and other yeasts in blood by PCR.

Primers complementary to the region of genes coding for rRNA in Candida albicans were used in PCRs to detect yeast DNA extracted from blood samples containing various Candida species. One fragment (105 bp) was amplified from all yeasts tested, whereas a second (684 bp) was only amplified when C. albicans DNA was present. The level of sensitivity was 15 +/- 5 (mean +/- standard error) CFU of C. albicans per ml of blood.

The Candida albicans plasma membrane and H(+)-ATPase during yeast growth and germ tube formation.

PMA1 expression, plasma membrane H(+)-ATPase enzyme kinetics, and the distribution of the ATPase have been studied in carbon-starved Candida albicans induced with glucose for yeast growth at pH 4.5 and for germ tube formation at pH 6.7. PMA1 expression parallels expression of the constitutive ADE2 gene, increasing up to sixfold during yeast growth and twofold during germ tube formation. Starved cells contain about half the concentration of plasma membrane ATPase of growing cells. The amount of plasma membrane ATPase is normalized prior to either budding or germ tube emergence by the insertion of additional ATPase molecules, while ATPase antigen appears uniformly distributed over the entire plasma membrane surface during both growth phases. Glucose addition rapidly activates the ATPase twofold regardless of the pH of induction. The turnover of substrate molecules per second by the enzyme in membranes from budding cells quickly declines, but the enzyme from germ tube-forming cells maintains its turnover of substrate molecules per second and a higher affinity for Mg-ATP. The plasma membrane ATPase of C. albicans is therefore regulated at several levels; by glucose metabolism/starvation-related factors acting on gene expression, by signals generated through glucose metabolism/starvation which are thought to covalently modify the carboxyl-terminal domain of the enzyme, and possibly by additional signals which may be specific to germ tube formation. The extended period of intracellular alkalinization associated with germ tube formation may result from regulation of proton-pumping ATPase activity coupled with higher ratios of cell surface to effective cytosolic volume.

Yeast-specific DNA probes and their application for the detection of Candida albicans.

Two DNA fragments cloned from the genome of Candida albicans ATCC 10261 may be useful in the rapid diagnosis of disseminated candidosis. One sequence (probe EOB1) was specific for C. albicans (positive hybridisation with 45 strains tested). The second sequence (probe EOB2) detected C. albicans, as well as five other pathogenic Candida spp. and Saccharomyces cerevisiae, but did not react with human or bacterial DNA. Both probes were repetitive sequences in the genome of C. albicans. Probe EOB1 was used to detect, without DNA amplification, 500 C. albicans yeast cells in 1 ml of human blood.

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu-) was transformed by pRC2312 to Leu+ at a frequency of 1.41 x 10(5) colonies per microgram DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura+ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 x 10(3) per microgram DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per microgram DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15 +/- 3 per haploid genome in S. cerevisiae and 2-3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7-12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade- strains of both C. albicans and S. cerevisiae to Ade+. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.

Mechanisms of aggregation accompanying morphogenesis in Candida albicans.

Cultures of Candida albicans yeast cells do not normally aggregate, but extensive aggregation accompanies the induction of mycelial growth, indicating the occurrence of cell surface changes during the yeast to mycelial transition. Aggregation correlated with the formation of germ tubes as did changes in surface charge determined by attachment to ion exchange sepharose beads. Yeast cells of all strains examined were negatively charged and attachment to positively charged (DEAE) sepharose beads increased following germ tube formation. If Mg2+ was present during germ tube formation, a high degree of clumping occurred that could only be dispersed by treatment with protein-disrupting agents. Trypsin, chymotrypsin, SDS, urea, guanidine HCl and dithiothreitol but not EDTA or EGTA caused irreversible dispersal of aggregates, although germ tube aggregates dispersed by treatment with buffers at high pH reaggregated if neutralized or if calcium ions were added. Germ tube cultures produced in divalent cation-deprived medium formed aggregates that were readily dispersed by washing. However, the addition of Mg2+ or other divalent cations (Ca2+, Zn2+, Cu2+, Fe2+) caused immediate aggregation of these cultures. These results suggest that divalent cation crossbridging between opposing anionic sites and protein interactions act synergistically to promote aggregation of C. albicans germ tube cells.

Peanut sensitivity as a cause of burning mouth.

A patient with a history of a burning tongue together with discomfort of the labial and buccal mucosae was given an elimination diet and skin patch tests to determine the allergen in her diet. The patient was identified as being intolerant of an aqueous peanut extract. Three allergens in peanut butter were identified, the one with greatest reactivity being a heat-stable, water-soluble, nonglycosylated protein with a molecular weight in excess of 10 kD. Modification of her diet has resulted in resolution of the oral problem.

Ammonium assimilation by Candida albicans and other yeasts: a 13N isotope study.

Nuclear magnetic resonance spectra of cultures of Candida albicans incubated in the presence of 15N-labelled ammonium demonstrated that glutamine and glutamate were the only initial products of ammonium assimilation. The nature of the route of assimilation in the yeasts Candida albicans, Saccharomyces cerevisiae, and Candida tropicalis was further examined by the use of the short-lived isotope 13N. [13N]ammonium was generated in the reaction 16O(p,alpha)13N, induced by proton bombardment of water in tandem accelerator. High-pressure liquid chromatography was used to separate and identify the products of assimilation, and radioactivity was detected and corrected for decay, using a computer-linked NaI scintillation detector. In the three yeasts studied, the labelled ammonium was assimilated into the acid-extractable fraction of cell suspensions within 1 min, and over 75% was converted to glutamine and glutamate. Subsequent to exhaustion of the labelled ammonium, the stoichiometry of the distribution of radiolabel was consistent with a net transfer of radiolabel from glutamine to glutamate, confirming the operation of glutamate synthase (EC 1.4.1.14) in these yeasts. Initial assimilation of label was mostly into glutamine (at a maximal rate within 10 s in C. albicans), whereas accumulation in glutamate did not occur at maximal rate until more than 70% of the labelled ammonium had been assimilated (between 30 and 60 s in C. albicans). We conclude that the glutamine synthetase-glutamate synthase pathway is the major route of ammonium assimilation in C. albicans and also in nitrogen-starved cultures of S. cerevisiae and C. tropicalis.

Changes in cell envelope glycoproteins during germ-tube formation of Candida albicans.

Germ-tube formation by Candida albicans induced by N-acetylglucosamine resulted in the appearance of a 43 kD protein in a cell envelope fraction. The protein increased quantitatively in the cell envelope during the emergence of the germ-tube and the amount in the envelope fraction reflected the efficiency of the morphogenesis. The 43 kD protein was labelled by the lactoperoxidase catalysed iodination procedure confirming a surface location for the antigen. Concanavalin A binding to the 43 kD protein demonstrated that this protein contained carbohydrate. Tunicamycin inhibited both germ-tube formation in C. albicans and the appearance of the 43 kD protein in the cell envelope fraction. Instead the presence of tunicamycin resulted in the appearance of a new protein of 39 kD molecular weight in the cell envelope which did not bind concanavalin A. Endoglycosidase H digestion of the 43 kD protein produced a 39 kD protein. Peptide mapping of the 43 kD protein from germ-tube cells and the 39 kD protein from tunicamycin-treated cells indicated that these proteins are homologous.

Effect of calcium ion uptake on Candida albicans morphology.

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.

Fluoride intake of infants in New Zealand.

Since the fluoride (F-) intake of New Zealand infants and young children is not known, a study was designed to determine and compare the F- intake of infants, aged 11 to 13 months, residing in fluoridated (F) and non-fluoridated (NF) areas. Parents of 60 infants duplicated quantitatively and qualitatively all food and drink that the infants ingested during a three-day period. The acid-diffusible F- content in the liquid homogenate was isolated by the HMDS-HCl diffusion technique (Taves, 1968) and measured by a fluoride electrode. The ionic F- in samples of breast milk was measured directly by the electrode. In the F area, the F- content of the food and drinks of 31 subjects ranged from 0.130 to 0.679 mg/kg (mean, 0.320; SD, 0.168); in the NF areas, the F- content of the food and drinks of 29 subjects ranged from 0.036 to 0.281 mg/kg (mean, 0.095; SD, 0.053). The dietary intake ranged from 0.089 to 0.549 mg F/day (0.009-0.056 mg F/kg bw) in the F area, and from 0.038 to 0.314 mg F/day (0.004-0.038 mg F/kg bw) in the NF area. Including F- from tablets and toothpastes, total intake ranged from 0.093 to 1.299 mg F/day (0.009-0.150 mg F/kg bw) and from 0.039 to 0.720 mg F/day (0.004-0.061 mg F/kg bw) in F and NF areas, respectively. The mean dietary intake of infants in the F area was about half the recommended "optimal" range; in the NF areas, the dietary intake was five to seven times less than the optimal.(ABSTRACT TRUNCATED AT 250 WORDS)