Mitochondrial phosphate transport. N-ethylmaleimide insensitivity correlates with absence of beef heart-like Cys42 from the Saccharomyces cerevisiae phosphate transport protein.

The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis. ADP/ATP carriers that copurify have been identified. The PTP from S. cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP. It does not cross-react with anti-sera against beef heart PTP. The CNBr peptide maps of the yeast and beef proteins are very different. The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0. The Km for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide. We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP. The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport. Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.

Discriminant analysis of cellular fatty acids of Candida species, Torulopsis glabrata, and Cryptococcus neoformans determined by gas-liquid chromatography.

We used discriminant analysis of cellular fatty acid compositions determined by gas-liquid chromatography to differentiate yeastlike fungi (a total of 190 strains; including 37 Candida albicans strains, 21 Candida krusei strains, 13 Candida guilliermondii strains, 37 Candida tropicalis strains, 10 Candida pseudotropicalis strains, 24 Candida parapsilosis strains, 32 Torulopsis glabrata strains, and 16 Cryptococcus neoformans strains). Previous results with a standard strain of C. albicans indicated that reproducible fatty acid chromatograms can be obtained with cells grown in a medium of 2% Sabouraud glucose agar at 35 degrees C for between 48 and 72 h. These conditions were also maintained in cultures of the other organisms that we studied. The cellular fatty acid compositions of the organisms were determined quantitatively by gas-liquid chromatography and analyzed by discriminant analysis. The total correct identification expressed as relative peak percent was 95.8% (89.2% for C. albicans to 100% for C. krusei, C. guilliermondii, C. pseudotropicalis, T. glabrata, and C. neoformans). The total correct identification expressed as the common peak (palmitic acid) ratio was 94.7% (87.5% for C. parapsilosis to 100% for C. pseudotropicalis, T. glabrata, and C. neoformans). Both results suggest that cellular fatty acid compositions can be differentiated by this method.

Candidemia and systemic candidiasis.

The growing problem of candidemia and systemic candidiasis reflects the enormous increase in the pool of patients at risk as well as the increased opportunity that exists for Candida sp to invade tissues normally resistant to invasion. Candida sp, as truly opportunistic pathogens, exploit recent technological advances to gain access to the circulation and deep tissues. The increased prevalence of local and systemic disease caused by Candida organisms has resulted in new clinical syndromes, the expression of which depends upon the immune status of the host. These new syndromes include the focal hepatosplenic candidiasis, Candida peritonitis and systemic candidiasis. Management of serious and life-threatening invasive candidiasis remains severely hampered by the lack of reliable diagnostic methods that would allow early detection of both fungemia and tissue invasion by Candida organisms. Amphotericin B remains the cornerstone of effective antifungal therapy in systemic candidiasis. Over the last decade, new principles have emerged, including shorter and lower dosage regimens for catheter-related candidemia. The newer oral azoles may play a useful role in the management of invasive candidiasis.

A novel peroxisomal nonspecific lipid-transfer protein from Candida tropicalis. Gene structure, purification and possible role in beta-oxidation.

We have sequenced the nucleotides of the gene POX18 that encodes PXP-18, a major peroxisomal polypeptide inducible by oleic acid in the yeast Candida tropicalis. POX18 had a single open reading frame of 127 amino acids. Some 33% of the amino acid sequence of the predicted basic polypeptide (13,805 Da), was identical to that of the nonspecific lipid-transfer protein (sterol carrier protein 2) from rat liver. PXP-18, purified to near homogeneity from isolated peroxisomes, had an amino-terminal sequence identical to that of the predicted polypeptide except for the initiator methionine, and had nonspecific lipid-transfer activity comparable to that of its mammalian equivalents. Unexpectedly, PXP-18 lacked the cysteine residue thought to be essential for the activity of this protein in mammals. RNA blot analysis showed that the POX18 gene was expressed exclusively in cells grown on oleic acid, suggesting that PXP-18 has a role in the beta-oxidation of long-chain fatty acids. PXP-18 modulated acyl-coenzyme A oxidase activity at low pH.

Immunochemical analysis of the glucomannan from Candida utilis.

A glucomannan isolated from a Candida utilis mutant having a new chemotype was further studied by inhibition of the homologous precipitin reaction with oligosaccharides obtained from the glucomannan by partial acid hydrolysis and controlled acetolysis. Oligosaccharides having at least two consecutive alpha-(1----2)-linked mannose residues at the non-reducing end and gluco-manno-pentasaccharide were effective inhibitors. Thus, it appears that the glucomannan had two groups of antigenic determinants, one corresponding to the side chains of two, three, and four mannose units connected by alpha-(1----2)-linkage, and the other corresponding to a side chain composed of an O-alpha-D-glucopyranosyl-(1----6)-O-alpha-D-mannopyranosyl- (1----2)-O-alpha-D-mannopyranosyl-(1----2)-D-mannose unit. These results support a probable structure of repeating units for the glucomannan presented previously. The relative susceptibility of intersaccharidic linkages to acid hydrolysis in the mannan is discussed.

Autolytic reduction of the nucleic acid content in Candida utilis.

The nucleic acid content in Candida utilis was autolytically reduced only after previous lyophilization of yeast cells or pretreatment of the cells with ethyl acetate. These pretreatments provided a sufficient permeability of the cell membrane system and thus enabled a reduction of the nucleic acid content. While deep freezing of the cells (-35 degrees C) had no positive effect on the degradation of nucleic acids, lyophilization of yeast cells led to a 75% decrease of the nucleic acid content. Furthermore, a 55% reduction of the nucleic acid content was proceeded without a concomitant loss of proteins. Similar results were obtained by using ethyl acetate.

Distribution of cells in different stages of the cell cycle and some age-related physiological characteristics in dependence on the dilution rate in chemostat cultures of Candida utilis.

Position of cells in their cell cycle was determined microscopically in chemostat cultures of Candida utilis. Proportion of cells in phase G1 decreased in a linear manner from 86% to 58% with dilution rate. Proportion of cells in phase S increased in the same range of D from 5.6 to 13.5% and in the (G2 + M) phase from 8.4 to 28.5%, again linearly. Differential centrifugation was used to separate chemostat cultures to mother and daughter cells. Analyses showed that, relative to mother cells, daughter cells contain 2.1-11.9% more protein and 25.5-34.6% more RNA in dry matter. Their mass is 34.4-5.6% lower and volume is 154-19% smaller.

Continuous growth of Candida utilis under periodic change of growth-limiting substrate.

A periodic change of limitation by glucose and ammonia was effected during continuous cultivation of Candida utilis. Values of feed parameters providing periodic nitrogen limitation were established. Cell biomass yield, macromolecular composition and parameters of individual cells (cell mass, budding percentage and cell-wall polysaccharide per surface square unit) were examined. The cyclic regime was found to result in culture synchrony. Parameters obtained on the basis of cell counts displayed a high sensitivity to changes in limitation where as the remaining parameters were less sensible.

Occurrence of peroxisomal membrane proteins in methylotrophic yeasts grown under different conditions.

We have studied the substructure and polypeptide composition of the peroxisomal membranes in two methylotrophic yeasts in relation to different growth conditions. The results obtained indicated that no significant ultrastructural differences existed between the membranes of variously grown cells. The presence of specific peroxisomal membrane proteins (PMPs) was studied biochemically. On sodium dodecyl sulphate-polyacrylamide gels of purified microbody membranes isolated from methanol-grown Hansenula polymorpha, prominent protein bands were observed at 22, 31, 35, 42, 49 and 51 kD. These proteins were also present when the cells were grown in media containing ethanol and/or ethylamine. Apart from these, several other PMPs were specifically induced under these conditions, namely 24, 29, 37 and 62 kD proteins. The polypeptide composition of peroxisomal membranes from H. polymorpha was compared with that of another methylotroph, Candida biodinii. In the latter organism a specific PMP with a molecular weight of 23 kD was induced during growth on D-alanine instead of ammonium sulphate as the nitrogen source.

Immunochemical and structural analysis of the cell wall mannan as the basis of the taxonomic reidentification of a yeast strain.

The immunochemical properties and structural features of D-mannans from two Candida parapsilosis strains were studied. Weak cross-reactivity of D-mannans with antisera produced to C. parapsilosis strains was observed, as well as significant differences in the mannans structures were found by means of methylation analysis, acetolysis and 1H-NMR spectroscopy. In view of the discrepancies found, new taxonomic identification of one strain was carried out. On the basis of assimilation of potassium nitrate and formation of hat-shaped spores as well as other differences found by investigation of physiological characteristics, one strain of C. parapsilosis was reidentified as Hansenula anomala. This work demonstrates that immunochemical and structural investigations of cell-wall polysaccharide components can serve as a basis for taxonomic identification of yeast strains.

Peroxisomal protein import is conserved between yeast, plants, insects and mammals.

We have previously demonstrated that firefly luciferase can be imported into peroxisomes of both insect and mammalian cells. To determine whether the process of protein transport into the peroxisome is functionally similar in more widely divergent eukaryotes, the cDNA encoding firefly luciferase was expressed in both yeast and plant cells. Luciferase was translocated into peroxisomes in each type of organism. Experiments were also performed to determine whether a yeast peroxisomal protein could be transported to peroxisomes in mammalian cells. We observed that a C-terminal segment of the yeast (Candida boidinii) peroxisomal protein PMP20 could act as a peroxisomal targeting signal in mammalian cells. These results suggest that at least one mechanism of protein translocation into peroxisomes has been conserved throughout eukaryotic evolution.

Cell-constituent polyamines in Candida species and new biotyping of Candida albicans, Candida tropicalis and Candida parapsilosis strains.

Intracellular amines from three Candida species were extracted and chemically derivatized by a modification of the Seiler procedure. The results of qualitative determinations of 13 amines in 20 strains each of Candida albicans, Candida tropicalis and Candida parapsilosis are presented. This report is the first to describe the detection of amines other than the classical spermine, spermidine, putrescine and cadaverine in yeasts and fungi. Characteristic profiles due to the presence or absence of particular amines in the Candida species studied are demonstrated. Although these could not be used as strict differential markers at the species level, biotyping schemes based on amines are proposed to differentiate strains of C. albicans, C. tropicalis and C. parapsilosis.

An extended QBASIC program for the normalization and computation of whole-cell protein profiles and the application to clinically important Candida species.

The computer program described enables a rapid calculation of relative molecular masses of proteins from different yeasts and microbial pathogens, by interpolation from a molecular weight calibration curve that comprises stepwise linear regression between the protein bands produced by the internal standards. A similarity matrix can then be produced, taking into account variations between calculated molecular masses caused by small differences in bandwidths and/or positions of specific protein bands. This program has been applied to different Candida species and the similarity data obtained further analysed numerically utilizing CLUSTAN II on a SPERRY 1100 multi-processor.

Gas chromatographic assessment of alcoholyzed fatty acids from yeasts: a new chemotaxonomic method.

An alternative chemotaxonomic method to methanolysis was developed for gas chromatographic assessment of fatty acids in whole yeast cells. Clinical and reference strains of the medically important yeasts Candida albicans, Torulopsis glabrata, and Saccharomyces cerevisiae were cultured for 48 h at 26 degrees C. Cellular lysis and transesterification were then performed with ethanol, propanol, butanol, or methanol. The relative recovery rates for cellular fatty acids, including the volatile acids C10:0 and C12:0, were similar after alcoholysis with ethanol, propanol, or butanol, while methanolysis gave lower recoveries of volatile fatty acids. Thus, after ethanolysis, the recovery of C10:0 acid (0.1, 1, and 10%) from a defined matrix (lyophilized Actinobacillus actinomycetemcomitans cells) varied from 97 to 102%, while the recovery of C10:0 after methanolysis varied from 49 to 75%. This indicated that with the frequently used methanolysis technique, there is a considerable loss of volatile fatty acids. These acids may be used as marker molecules for taxonomic differentiation between yeasts.

[Isolation of yeast nuclei and characteristics of their lipid composition]. / Vydelinie i kharakteristika lipidnogo sostava iader iz drozhzhei.

Intact nuclei of a high purity degree were isolated from Candida utilis protoplasts, and their lipid content and composition were determined.

SDS-PAGE protein patterns of yeasts from human sources.

Protein patterns of Candida species and other yeasts have been studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Although differences in patterns occur which tend to separate the species, variability between replicate samples is sometimes high. The method cannot be used for speciation of common yeasts from medical sources.

Protein and enzyme electrophoresis profiles of selected Candida species.

The cellular protein profiles and malate dehydrogenases, superoxide dismutases, alkaline phosphatases, and esterases from whole cell extracts of Candida spp. were studied with polyacrylamide gel electrophoresis. We investigated isolates that differed in their ability to assimilate sucrose as the sole carbon source. The protein and enzyme patterns of Candida tropicalis and its sucrose-negative variant "Candida paratropicalis Baker, Salkin, Pincus et D'Amato" were indistinguishable. Although the cellular protein and superoxide dismutase patterns of Candida albicans and its sucrose-negative variant "Candida stellatoidea" were quite similar, differences were noted in the profiles of the other enzymes studied. In addition, the C. stellatoidea isolates were found to be separable, on the basis of their enzyme profiles, into the same two types that have been reported by Kwon-Chung et al. (K.J. Kwon-Chung, B.L. Wickes, and W.G. Merz, Infect. Immun. 56:1814-1819, 1988).

Lipid composition and lectin-mediated agglutination of Candida kefyr cells grown in media enriched with choline or its analogues.

Candida Kefyr cells and their spheroplasts grown in media enriched with choline, N,N'-dimethylethanolamine (DMEA) or ethanolamine (EA) showed decreased concanavalin A (Con A) and ph to hemagglutinin (PHA) mediated agglutination while supplementation with N-monomethylethanolamine (MMEA) increased PHA-mediated agglutination. In all cases, the amount of phospholipid was increased and, consequently, a decrease in the free sterol; phospholipid ratio was observed except in the case of MMEA where this ratio remained almost unchanged. In the cells grown in media enriched with choline. DMEA or MMEA, but not in the EA-supplemented medium, the phosphatidylcholine to phosphatidylethanolamine ratio was increased. Saturation of fatty acids as well as their chain length decreased, which could lead to increased membrane fluidity. No breakpoint in Arrhenius plots of Mg2+ -ATPase of the choline-supplmented cells was observed. Cells grown in media enriched with EA and MMEA did not show any sharp break in Arrhenius plots of Mg2+ -ATPase.