Synthesis of 3,6-branched oligomannoside fragments of the mannan from Candida albicans cell wall corresponding to the antigenic factor 4

3-Aminopropyl glycosides of 3,6-branched penta- and hexamannoside fragments of the cell wall mannan from Candida albicans, corresponding to the antigenic factor 4, have been synthesized. Subsequent coupling of both oligosaccharides with BSA using the squarate procedure provided corresponding neoglycoconjugates.     

Synthesis of oligosaccharide fragments of mannan from Candida albicans cell wall and their BSA conjugates

3-Aminopropyl glycosides of alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyra-nosyl-(1-->2)-alpha-D-mannopyranose, alpha-D-mannopyranosyl-(1-->3)-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranosyl-(l -- 2)-alpha-D-mannopyranose, and alpha-D-mannopyranosyl-(1-->2)-[alpha-D-mannopyranosyl-(1-->3)]-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranose were efficiently synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-l-thio-alpha-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-alpha-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-l-thio-alpha-D-mannopyranoside, and 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl bromide. The oligosaccharide chains synthesized correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the third pentasaccharide with bovine serum albumin was prepared using the squarate method.     

Environmental parameters affecting xylitol production from sugar cane bagasse hemicellulosic hydrolyzate by Candida guilliermondii

The bioconversion of xylose to xylitol by Candida guilliermondii FTI 20037 cultivated in sugar cane bagasse hemicellulosic hydrolyzate was influenced by cell inoculum level, age of inoculum and hydrolyzate concentration. The maximum xylitol productivity (0.75 g L⁻¹ h⁻¹) occurred in tests carried out with hydrolyzate containing 54.5 g L⁻¹ of xylose, using 3.0 g L⁻¹ of a 24-h-old inoculum. Xylitol productivity and cell concentration decreased with hydrolyzate containing 74.2 g L⁻¹ of xylose. Received 02 February 1996/ Accepted in revised form 15 November 1996     

Synthesis of oligosaccharide fragments of mannan from <Emphasis Type="Italic">Candida albicans</Emphasis> cell wall and their BSA conjugates

3-Aminopropyl glycosides of α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, α-D-mannopyranosyl-(1→3)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose, and α-D-mannopyranosyl-(1→2)-[α-D-mannopyranosyl-(1→3)]-α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose were efficiently synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-1-thio-α-D-mannopyranoside, and 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl bromide. The oligosaccharide chains synthesized correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the third pentasaccharide with bovine serum albumin was prepared using the squarate method.     

Effect of phosphate buffer concentration on the batch xylitol production by Candida guilliermondii

AIMS: To evaluate the effect of phosphate buffer concentration on growth and xylitol production by Candida guilliermondii FTI 20037. METHODS AND RESULTS: Fermentations runs were carried out in batch mode employing semisynthetic medium supplemented with phosphate buffer at different concentrations (from 200 to 600 mmol l(-1)). The xylitol yield (Y(P/S)) and volumetric productivity (Q(P)) were improved when the fermentation medium was supplemented with phosphate buffer at concentration of 600 mmol l(-1). Under this condition (Y(P/S)) and (Q(P)) values were 0.75 g g(-1) and 0.66 g l(-1) h(-1), respectively, whereas in the absence of the phosphate buffer these values decreased to 0.52 g g(-1) and 0.44 g l(-1)h(-1) respectively. CONCLUSIONS: The use of phosphate buffer at 600 mmol l(-1) promoted an easier pH control during shake flasks fermentation of C. guilliermondii. In addition the xylitol yield and productivity were significantly improved in response to the supplementation of potassium phosphate in the medium. The increase in these parameters could be related to both osmotic effect and pH control. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach provided a method for improving the xylitol production from semisynthetic medium by C. guilliermondii, being possible their use as a simple strategy to achieve efficient fermentation processes employing complex medium such as lignocellulosic hydrolysates.     

Candida guilliermondii isolated from HIV-infected human secretes a 50 kDa serine proteinase that cleaves a broad spectrum of proteinaceous substrates

Non-albicans Candida species cause 35-65% of all candidemias in the general population, especially in immunosuppressed individuals. Here, we describe a case of a 19-year-old HIV-infected man with pneumonia due to a yeast-like organism. This clinical yeast isolate was identified as Candida guilliermondii through mycological tests. C. guilliermondii was cultivated in brain heart infusion medium for 48 h at 37 degrees C. After sequential centrifugation and concentration steps, the free-cell culture supernatant was obtained and extracellular proteolytic activity was assayed firstly using gelatin-SDS-PAGE. A 50 kDa proteolytic enzyme was detected with activity at physiological pH. This activity was completely blocked by 10 mM phenylmethylsulphonyl fluoride (PMSF), a serine proteinase inhibitor, suggesting that this extracellular proteinase belongs to the serine proteinase class. E-64, a strong cysteine proteinase inhibitor, and pepstatin A, a specific aspartic proteolytic inhibitor, did not interfere with the 50 kDa proteinase. Conversely, a zinc-metalloproteinase inhibitor (1,10-phenanthroline) restrained the proteinase activity released by C. guilliermondii by approximately 50%. Proteinases are a well-known class of enzymes that participate in a vast context of yeast-host interactions. In an effort to establish a functional implication for this extracellular serine-type enzyme, we investigated its capacity to hydrolyze some serum proteins and extracellular matrix components. We demonstrated that the 50 kDa exocellular serine proteinase cleaved human serum albumin, non-immune human immunoglobulin G, human fibronectin and human placental laminin, generating low molecular mass polypeptides. Collectively, these results showed for the first time the ability of an extracellular proteolytic enzyme other than aspartic-type proteinases in destroying a broad spectrum of relevant host proteins by a clinical species of non-albicans Candida.     

Metabolic behavior of immobilized Candida guilliermondii cells during batch xylitol production from sugarcane bagasse acid hydrolyzate

Candida guilliermondii cells, immobilized in Ca-alginate beads, were used for batch xylitol production from concentrated sugarcane bagasse hydrolyzate. Maximum xylitol concentration (20.6 g/L), volumetric productivity (0.43 g/L. h), and yield (0.47 g/g) obtained after 48 h of fermentation were higher than similar immobilized-cell systems but lower than free-cell cultivation systems. Substrates, products, and biomass concentrations were used in material balances to study the ways in which the different carbon sources were utilized by the yeast cells under microaerobic conditions. The fraction of xylose consumed to produce xylitol reached a maximum value (0.70) after glucose and oxygen depletion while alternative metabolic routes were favored by sub-optimal conditions.     

Statistical screening method for selection of important variables on xylitol biosynthesis from rice straw hydrolysate by Candida guilliermondii FTI 20037

Fourteen assays were conducted to study the influence of different variables, namely xylose concentration, inoculum level, agitation speed and nutrient supplementation, on xylitol biosynthesis by Candida guilliermondii FTI 20037. The maximum predicted values for xylitol yield (0.65 g g^–1) and xylitol productivity (0.66 g l^–1 h^–1) can be attained with rice straw hydrolysate containing 60 g xylose l^–1 without supplementation of ammonium sulfate, calcium chloride and rice bran extract, using 5 g inoculum l^–1, at 250 rpm. Xylose concentration and inoculum level were selected for further optimization studies.     

The Candida albicans histidine kinase Chk1p: Signaling and cell wall mannan

Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p (SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/chk1Δ null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Δ or sho1Δ mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GLC/MS and GPC MALLS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 (chk1Δ null), the cek1Δ null, and the double mutant consisted only of low Mw mannan. The sho1Δ null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Δ, chk1Δ, and the double sho1/chk1Δ null mutant lacking high and intermediate Mw mannan than in the sho1Δ null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.     

Immune responsiveness of a novel peptidoglycan conjugate prepared from surface Candida immunogens: mannan and CR3-related protein

Candida albicans expresses a CR3-related protein (CR3-RP) antigenically, structurally and functionally related to human adhesion glycoprotein, also known as Mac-l, the iC3b receptor, or complement receptor type 3. The purpose of the present study was to analyze the immunogenic properties of a novel CR3-RP glycoconjugate in a rabbit model. Cell-mediated responses revealed immunoenhancement triggered by CR3-RP glycoconjugate with respect to expression of IL-2 receptor subunit CD25 on B-lymphocytes and inductive increase of the CD4(+)/CD8(+) ratio compared with unconjugated cell wall mannan (P<0.001). Active immunization with the CR3-RP glycoconjugate resulted in an effective IgM-IgG isotypic switch even after the second booster. Altogether, it could be assumed that the novel peptide glycoconjugate is a prospective antigenic candidate for further Candida vaccine design.     

Isolation of a Candida albicans mutant with reduced content of cell wall mannan and deficient mannan phosphorylation

A rough-colony mutant of Candida albicans was isolated after ultraviolet mutagenesis. The mutant contained approximately half the normal amount of the cell wall mannan, the acetolysis pattern of which was indistinguishable from that of the wild-type counterpart. However, the extent of phosphorylation in the mutant mannan was about 12% of the value for wild type.     

Optimization of D-xylose to xylitol biotransformation by Candida guilliermondii cells permeabilized with Triton X-100

Abstract

The effect of NADP⁺ and glucose-6-phosphate (G6P) on the biotransformation of D-xylose to xylitol by cells of Candida guilliermondii permeabilized with surfactant Triton X-100 was evaluated. The experimental runs were performed with 12 g L^?1 of permeabilized cells and a reaction medium composed of Tris–HCl buffer (0.1 M pH 7), D-xylose (57 g L^?1), and MgCl₂.6H₂O (5 mM). The levels of NADP⁺ (from 0.0 to 1.7 mM) and G6P (from 0.00 to 0.17 M) were varied according a 2²-full factorial composed design. Under optimized conditions (NADP⁺ 0.5 mM and 0.05 M G6P), the xylitol volumetric productivity (Q_P) and yield factor (Y_P/S) predicted were 1.86 ± 0.03 g L^?1 h^{? 1} and 0.64 ± 0.03 g g^?1, respectively. These values were 94% and 19% higher than those obtained with unpermeabilized cells under fermentation conditions (0.97 g L^?1 h^?1 and 0.53 g g^?1, respectively). On the basis of the results, it can be concluded that xylitol production by biotransformation with cells of C. guilliermondii permeabilized with Triton X-100 is a promising alternative to the fermentative process.     

A close temporal and spatial correlation between cell growth,cell wall synthesis and the activity of enzymes of mannan synthesis in Acetabularia mediterranea

The synthesis of cell wall mannan and the activities of guanosine-diphosphate-mannose-pyrophosphorylase (EC2.7.7.13) and mannan synthetase were studied during the development of nucleate and enucleated cells of the alga Acetabularia mediterranea. The activities of both enzymes are relatively high as long as the cells grow and synthesize mannans. With termination of growth and mannan synthesis, the activities of both enzymes, but especially of mannan synthetase, drop to a low value. Furthermore, the activities of both enzymes are distributed in the cell along an apical-basal gradient. High activities are present in the apical regions of the cell where growth and mannan synthesis mainly occur, whereas in the basal region, growth, mannan synthesis and the activity of the two enzymes are slight. Since the in vitro activity of GDP-Man-pyr is at least 100 times higher than that of mannan synthetase, it was concluded that mannan synthetase activity is the limiting factor in mannan synthesis. This conclusion is supported by the determined pool sizes of Fru 6-P, Man 6-P, Man 1-P and GDP-Man during the development of the cells. The control of mannan synthesis and with it cell wall formation and growth through the regulation of mannan synthetase activity is discussed.Abbreviations DD dark-dark regime - Fru 6-P fructose-6-phosphate - GDP-Man guanosine-diphosphate-mannose - GDP-Manpyr GDP-diphosphate-mannose-pyrophosphorylase - GTP guanosine-triphosphate - LD light-dark regime - Man 1-P mannose-1-phosphate - Man 6-P mannose-6-phosphate - TCA trichloracetic acid     

Molecular organization of the cell wall of Candida albicans and its relation to pathogenicity

Candida albicans is one of the most important opportunistic pathogenic fungi. Weakening of the defense mechanisms of the host, and the ability of the microorganism to adapt to the environment prevailing in the host tissues, turn the fungus from a rather harmless saprophyte into an aggressive pathogen. The disease, candidiasis, ranges from light superficial infections to deep processes that endanger the life of the patient. In the establishment of the pathogenic process, the cell wall of C. albicans (as in other pathogenic fungi) plays an important role. It is the outer structure that protects the fungus from the host defense mechanisms and initiates the direct contact with the host cells by adhering to their surface. The wall also contains important antigens and other compounds that affect the homeostatic equilibrium of the host in favor of the parasite. In this review, we discuss our present knowledge of the structure of the cell wall of C. albicans, the synthesis of its different components, and the mechanisms involved in their organization to give rise to a coherent composite. Furthermore, special emphasis has been placed on two further aspects: how the composition and structure of C. albicans cell wall compare with those from other fungi, and establishing the role of some specific wall components in pathogenesis. From the data presented here, it becomes clear that the composition, structure and synthesis of the cell wall of C. albicans display both subtle and important differences with the wall of different saprophytic fungi, and that some of these differences are of utmost importance for its pathogenic behavior.     

Synthesis of two heptasaccharide analogues of the lentinan repeating unit

beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp (18) and the allyl glycoside of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)[-beta-D-Glcp-(1-->6)]-alpha-D-Glcp (29) were synthesized as the analogues of the lentinan repeating heptaose by building the pentasaccharide backbones first, followed by attaching the side chains. 4,6-O-benzylidenated mono-13 or disaccharide 8 were used as the acceptor to ensure the beta linkage in the synthesis of 18, while 4,6-O-benzylidenated disaccharides 21 and 23 were used as the donor and acceptor, respectively, to ensure the beta linkage in the synthesis of 29.     

Bioconversion of Ferulic Acid to 4-Vinylguaiacol and 4-Ethylguaiacol and of 4-Vinylguaiacol to 4-Ethylguaiacol by Halotolerant Yeasts Belonging to the Genus Candida

In order to examine the genesis of the characteristic flavors of soy sauce and miso, seven novel halotolerant yeast strains of two types, which showed convertibility of ferulic acid (FA) to 4-vinylguaiacol (4-VG) and to 4-ethylguaiacol (4-EG), were isolated from miso-koji and miso pastes. Two of these strains were identified as Candida guilliermondii (anamorph of Pichia guilliermondii), and Candida fermentati (anamorph of Pichia caribbica), based on sequence analyses of a partial 26S ribosomal RNA gene and the region of internal transcribed spacers 1 and 2, and the 5.8S ribosomal RNA gene. Moreover, we also found three Candida etchellsii strains which showed convertibility of FA to 4-VG, but not to 4-EG, and two atypical strains of Candida versatilis which showed no convertibility of FA to 4-VG, but did show convertibility of 4-VG to 4-EG from soy sauce mashes. The bioconversion pathway from FA to 4-EG via 4-VG in halotolerant yeasts and bacteria is discussed.     

Two new species in the Pichia guilliermondii clade: Pichia caribbica sp. nov., the ascosporic state of Candida fermentati, and Candida carpophila comb. nov

Pichia caribbica sp. nov. (type strain DBVPG 4519, NRRL Y-27274, CBS 9966) is described as the ascosporic state of Candida fermentati, and Candida guilliermondii var. carpophila (type strain DBVPG 7739, NRRL Y-17905, CBS 5256) is elevated to species status as Candida carpophila comb. nov. These new taxa, which are indistinguishable on the basis of conventional taxonomic criteria, differ from one another and from Pichia guilliermondii by low DNA base sequence relatedness, different electrophoretic karyotypes, and nucleotide divergence in domains D1/D2 of 26S rDNA. Pichia caribbica produces one, rarely two, saturn-shaped ascospores in persistent asci. On the basis of molecular criteria, C. carpophila comb. nov., C. fukuyamaensis, and C. xestobii are conspecific, with the name C. carpophila having taxonomic priority.