Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡.

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.

Increasing Long-Chain Dicarboxylic Acid Production in Candida tropicalis by Engineering Fatty Transporters.

Candida tropicalis can metabolize alkanes or fatty acids to produce long-chain dicarboxylic acids (DCAs). Fatty acid transporters located on the cell or peroxisome membrane may play an important role in this process. Using amino acid sequence homologous alignment, two putative proteins, CtFat1p and CtPxa1p, located on the cell and peroxisome membrane were found, respectively. Moreover, single- and double-knockout homologous recombination technology was used to study ctfat1p and ctpxa1p gene effects on DCA synthesis. In comparison to the wild-type strain, long-chain DCA yield decreased by 65.14%, 88.38% and 56.19% after single and double-copy knockout of ctfat1p genes and double-copy knockout of ctpxa1p genes, respectively, indicating that the knockout of ctfat1p and ctpxa1p genes had a significant effect on the conversion of oils and fats into long-chain DCAs by C. tropicalis. However, the yield of long-chain DCAs increased by 21.90% after single-knockout of the ctpxa1p gene, indicating that the single-knockout of the ctpxa1p gene may reduce fatty acid transport to peroxisome for further oxidation. Moreover, to improve the intracellular transport rate of fatty acids, ctfat1p copy number increased, increasing DCA yield by 30.10%. These results may provide useful information for enhancing the production of long-chain DCAs by C. tropicalis.

Characterization of two sugar transporters responsible for efficient xylose uptake in an oleaginous yeast Candida tropicalis SY005.

Microbial conversion of lignocellulosic feedstock to the target bioproduct requires efficient assimilation of its constituent sugars, a large part of which comprises of glucose and xylose. This study aims to identify and characterize sugar transporters capable of xylose uptake in an oleaginous strain of the industrially relevant yeast Candida tropicalis. In silico database mining resulted in two sugar transporter proteins- CtStp1 and CtStp2, containing conserved amino acid residues and motifs that have been previously reported to be involved in xylose transport in other organisms. Several softwares predicted the likelihood of 10-12 transmembrane (TM) helices to be present in both the Stps, while molecular modelling showed 12 TM helices that were organized into a typical structure found in the major facilitator superfamily of transporters. Docking with different sugars also predicted favorable interactions. Heterologous expression in a Saccharomyces cerevisiae strain harboring functional xylose metabolic genes validated the broad substrate specificity of the two Stps. Each transporter supported prominent growth of recombinant S. cerevisiae strains on six sugars including xylose at various concentrations. Expression of CtSTP1 and CtSTP2 along with the xylose metabolic genes in yeast transformants grown in presence of xylose was confirmed by transcript detection. Growth curve and sugar consumption profiles revealed uptake of both glucose and xylose simultaneously by the recombinant yeast strains, though CtStp1 showed relatively less effect of glucose repression in mixed sugars and was a better transporter of xylose than CtStp2. Such glucose-xylose utilizing efficient transporters can be effective tools for developing co-fermenting yeasts through genetic engineering in future, with noteworthy applications in renewable biomass utilization.

Identification and functional characterization of a lipid droplet protein CtLDP1 from an oleaginous yeast Candida tropicalis SY005.

Proteins residing in lipid droplets (LDs) of organisms exhibit diverse physiological roles. Since the LD proteins of yeasts are largely unexplored, we have identified a putative LD protein gene, CtLDP1 in the oleaginous yeast Candida tropicalis SY005 and characterized its function. The increased lipid accumulation in SY005 could be correlated with enhanced (~2.67-fold) expression of the CtLDP1 after low-nitrogen stress. The N-terminal transmembrane domain similar to perilipin proteins and the amphipathic α-helices predicted in silico, presumably aid in targeting the CtLDP1 to LD membranes. Heterologous expression of CtLDP1-mCherry fusion in Saccharomyces cerevisiae revealed localization in LDs, yet the expression of CtLDP1 did not show significant effect on LD formation in transformed cells. Molecular docking showed favourable interactions of the protein with sterol class of molecules, but not with triacylglycerol (TAG); and this was further experimentally verified by co-localization of the mCherry-tagged protein in TAG-deficient (but steryl ester containing) LDs. While oleic acid supplementation caused coalescence of LDs into supersized ones (average diameter = 1.19 ± 0.12 µm; n = 160), this effect was suppressed due to CtLDP1 expression, and the cells mostly exhibited numerous smaller LDs (average diameter = 0.46 ± 0.05 µm; n = 160). Moreover, CtLDP1 expression in pet10Δ knockout strain of S. cerevisiae restored multiple LD formation, indicating functional complementation of the protein. Overall, this study documents functional characterization of an LD-stabilizing protein from an oleaginous strain of Candida genus for the first time, and provides insights on the characteristics of LD proteins in oleaginous yeasts for future metabolic engineering.

Antifungal effects of a 1,3,4-thiadiazole derivative determined by cytochemical and vibrational spectroscopic studies.

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 µg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and ß(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of ß(1→3) glucans and ß(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses.

Effect of quorum sensing molecules and natamycin on biofilms of Candida tropicalis and other yeasts isolated from industrial juice filtration membranes.

AIMS: Cells limit the cell number of dense biofilms by releasing self-inhibitory molecules. Here, we aim to assess the effectiveness of yeast quorum sensing (QS) molecules and the antifungal agent natamycin against yeast biofilms of strains commonly isolated from fruit juice ultrafiltration membranes. METHODS AND RESULTS: Yeast QS molecules, such as tyrosol, 2-phenylethanol and farnesol, were detected by solvent extraction and HS-SPME GC-MS in Candida tropicalis cultures. The effect of QS molecules on mono- and multispecies biofilms formed by Rhodotorula mucilaginosa, C. tropicalis, Candida krusei and Candida kefyr was evaluated by plate count and epifluorescence microscopy. Farnesol caused a decrease in cell number and disrupted mono- and multispecies yeast biofilms during adhesion (0·6 mmol l-1 ). 2-phenyl ethanol 1·2 mmol l-1 stimulated biofilm density and increased cell number in both mono- and multispecies biofilms, while tyrosol did not show effects when tested against C. tropicalis biofilms (0·05-1·2 mmol l-1 ). Natamycin caused a strong decrease in cell number and disruption of biofilm structure in C. tropicalis biofilms at high concentrations (0·3-1·2 mmol l-1 ). The combination of farnesol 0·6 mmol l-1 and natamycin at 0·01 mmol l-1 , the maximum concentration of natamycin accepted for direct addition into fruit juices, effectively reduced cell counts and disrupted the structure of C. tropicalis biofilms. CONCLUSION: Farnesol 0·6 mmol l-1 significantly increased the inhibition exerted by natamycin 0·01 mmol l-1 (~5 ppm) reducing biofilm development from juice on stainless steel surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: These results support the use of QS molecules as biofilm inhibitors in beverages and would certainly inspire the design of novel preservative and cleaning products for the food industry based on combinatory approaches.

A rapid microwave-assisted phosphoric-acid treatment on carbon fiber surface for enhanced cell immobilization in xylitol fermentation.

A rapid microwave-assisted phosphoric-acid treatment (MP) was applied to modify the surface of carbon fibers (CFs) for cell immobilization and fermentation. After the novel treatment, the surface of CFs subjected to MP (CF-MP) contained the CPO and COP bonds and the surface area and hydrophilicity were enhanced, which improved the cell adhesion and growth compared with the raw CF. Because of these structural advantages, the biocompatibilities of the CF-MP as a cell carrier were improved according to the immobilization behavior of microorganisms and batch fermentation. The optimized immobilization efficiency of Candida tropicalis on the CF-MP was improved to 0.587 g g-1, and the maximum xylitol yield and volumetric productivity were 67.15% and 1.16 g h-1 L-1, respectively, which were substantially higher than that in case of the untreated CF. This indicated that the biocompatibility of CF improved after the favorable MP surface treatment, resulting in the enhancement of immobilized efficiency and xylitol fermentation.

Characterization of the interactions between human high-molecular-mass kininogen and cell wall proteins of pathogenic yeasts Candida tropicalis.

Candida tropicalis is one of the most frequent causes of serious disseminated candidiasis in human patients infected by non-albicans Candida species, but still relatively little is known about its virulence mechanisms. In our current study, the interactions between the cell surface of this species and a multifunctional human protein - high-molecular-mass kininogen (HK), an important component of the plasma contact system involved in the development of the inflammatory state - were characterized at the molecular level. The quick release of biologically active kinins from candidal cell wall-adsorbed HK was presented and the HK-binding ability was assigned to several cell wall-associated proteins. The predicted hyphally regulated cell wall protein (Hyr) and some housekeeping enzymes exposed at the cell surface (known as "moonlighting proteins") were found to be the major HK binders. Accordingly, after purification of selected proteins, the dissociation constants of the complexes of HK with Hyr, enolase, and phosphoglycerate mutase were determined using surface plasmon resonance measurements, yielding the values of 2.20 × 10(-7) M, 1.42 × 10(-7) M, and 5.81 × 10(-7) M, respectively. Therefore, in this work, for the first time, the interactions between C. tropicalis cell wall proteins and HK were characterized in molecular terms. Our findings may be useful for designing more effective prevention and treatment approaches against infections caused by this dangerous fungal pathogen.

Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption.

The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-ß-glucan content. Among these factors, cell wall thickness and 1,3-ß-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-ß-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.

Enzymatic Production of Bioxylitol from Sawdust Hydrolysate: Screening of Process Parameters.

Xylose-rich sawdust hydrolysate can be an economic substrate for the enzymatic production of xylitol, a specialty product. It is important to identify the process factors influencing xylitol production. This research aimed to screen the parameters significantly affecting bioxylitol synthesis from wood sawdust by xylose reductase (XR). Enzymatic bioxylitol production was conducted to estimate the effect of different variables reaction time (2-18 h), temperature (20-70 °C), pH (4.0-9.0), NADPH (1.17-5.32 g/L), and enzyme concentration (2-6 %) on the yield of xylitol. Fractional factorial design was followed to identify the key process factors. The screening design identified that time, temperature, and pH are the most significant factors influencing bioxylitol production among the variables with the values of 12 h, 35 °C, and 7.0, respectively. These conditions led to a xylitol yield of 71 % (w/w). This is the first report on the statistical screening of process variables influencing enzyme-based bioxylitol production from lignocellulosic biomass.

[Peripheral blood cells luminol-dependent chemiluminescence at the different stages of atopic dermatitis].

UNLABELLED: Aim of this work was to record the luminol-dependent spontaneous and induced chemiluminescence at the different stages of atopic dermatitis. METHODS: Peripheral blood cells were obtained from adult patient with atopic dermatitis followed by the registration of luminol-dependent chemiluminescence on luminograph. Opsonized zymosan as well as yeasts Candida tropicalis have been used to induce the chemiluminescence. RESULTS: Spontaneous and induced chemiluminescence were slightly elevated at the mild atopic dermatitis but were decreased at the severe stage of disease. Statistically significant difference has been found between group with mild and severe atopic dermatitis, Skin contamination by yeasts Candida tropicalis causes the increased level of blood cells chemiluminescence at the first week of atopic relapse when the disease was mild. Severe stage of atopic dermatitis was coupled with statistically significant inhibition of both, spontaneous and induced chemiluminescence. CONCLUSIONS: Luminol-dependent chemiluminescence of peripheral blood cells from adult atopic dermatitis patients may be stimulated at the mild stage and suppressed at severe stage of atopic dermatitis.

Identification and assessment of the effects of yeast decarboxylases expressed in Escherichia coli for producing higher alcohols.

AIMS: To contribute to the improvement of methods for the regulation and production of higher alcohols using micro-organisms, we assessed the yields achieved using 10 decarboxylase genes from three different yeast species (Saccharomyces cerevisiae, Candida tropicalis and Pichia pastoris) by cloning them into vectors and overexpressing them in Escherichia coli hosts of different genotypes. Genes that produced the greatest yields in higher alcohol production were further assessed for the catalytic effects of the decarboxylase enzymes in the different E. coli hosts. METHODS AND RESULTS: A major metabolic pathway is structured via overexpressing a series of five genes, to detect the effect of decarboxylase on the production of higher alcohols. Results suggested that these genes can facilitate production of specific types of higher alcohols by diverse types of E. coli. We also showed that they play direct roles in the metabolic pathways that lead to production of higher alcohols in E. coli. The gene ARO10 from S. cerevisiae produced the highest yields for producing isobutanol and isopentanol in the host JM109. Significant differences were found in the types of higher alcohols and yields produced within the same host, for the genes PAD1, GAD1, SPE1 from S. cerevisiae. Similar results were observed for the genes ODC1 and gadB from Candida tropicalis and P. pastoris, respectively. CONCLUSIONS: Investigation of these genes for identification of the key enzymatic steps or regulatory pathways involved in the Ehrlich metabolic network to produce higher alcohols is paramount for producing biofuels. The selected genes are promising targets for the development of improved production strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first published assessment of the effects of decarboxylases from different yeast species that were expressed in E. coli, for the production of higher alcohols. Our results provide guidance for future studies about the use of yeast enzymes for transforming or constructing a new metabolic pathway utilizing E. coli for the production of target higher alcohols.

Determination of biofilm production by Candida tropicalis isolated from hospitalized patients and its relation to cellular surface hydrophobicity, plastic adherence and filamentation ability.

Candida tropicalis is an emerging virulent species. The aim of this study is to determine the biofilm-forming ability of 29 strains of C. tropicalis isolated from inpatients, and to examine its relation with other virulence factors such as cellular surface hydrophobicity (CSH), immediate (15 min, IA) and late (24 h, LA) plastic adherence and filamentation ability. The study was performed in parallel using two incubation temperatures - 37 and 22 °C - to determine the effect of growth temperature variations on these pathogenic attributes of C. tropicalis. Biofilm formation (BF) was measured by optical density (OD) and by XTT reduction (XTT); Slime index (SI), which includes growth as a correction factor in BF, was calculated in both methods. All strains were hydrophobic and adherent - at 15 min and 24 h - at both temperatures, with higher values for 22 °C; the adhered basal yeast layer appears to be necessary to achieve subsequent development of biofilm. Filamentation ability varied from 76.2% of strains at 37 °C to 26.6% at 22 °C. All C. tropicalis strains were biofilm producers, with similar results obtained using OD determination and XTT measurement to evaluation methods; SI is useful when good growth is not presented. BF at 37 °C was similar at 24 h and 96 h incubation; conversely, at 22 °C, the highest number of biofilm-producing strains was detected at 96 h. CSH is an important pathogenic factor which is involved in adherence, is influenced by the filamentation of yeast, and plays a critical role in BF.

Evaluation of corncob hemicellulosic hydrolysate for xylitol production by adapted strain of Candida tropicalis.

A maximum xylose extraction of 21.98 g/L was obtained in hydrolysate with a solid to liquid ratio of 1:8 (w/v) at 1% H(2)SO(4) and treated for 30 min. The optimized and treated corncob hemicellulosic hydrolysate medium supplemented with (g/L) yeast extract 5.0, KH(2)PO(4) 2.0, MgSO(4)·7H(2)O 0.3 and methanol 10 mL whose pH was adjusted to 4.5 acts as production medium. Under this condition; the adapted strain of C. tropicalis resulted in 1.22-fold increase in xylitol yield and 1.70-fold enhancement in volumetric productivity was obtained as compared to parent strain of C. tropicalis. On concentrating the hydrolysate under vacuum using rotavapor proves to be efficient in terms of improved xylitol yield and productivity over microwave assisted concentration using adapted strain of C. tropicalis. The immobilized cells of C. tropicalis resulted in more than 70% efficiency up to third cycle. The xylitol production could be scaled up to 10 L fermentor.

Analyses of Candida Cdc13 orthologues revealed a novel OB fold dimer arrangement, dimerization-assisted DNA binding, and substantial structural differences between Cdc13 and RPA70.

The budding yeast Cdc13-Stn1-Ten1 complex is crucial for telomere protection and has been proposed to resemble the RPA complex structurally and functionally. The Cdc13 homologues in Candida species are unusually small and lack two conserved domains previously implicated in telomere regulation, thus raising interesting questions concerning the mechanisms and evolution of these proteins. In this report, we show that the unusually small Cdc13 homologue in Candida albicans is indeed a regulator of telomere lengths and that it associates with telomere DNA in vivo. We demonstrated high-affinity telomere DNA binding by C. tropicalis Cdc13 (CtCdc13) and found that dimerization of this protein through its OB4 domain is important for high-affinity DNA binding. Interestingly, CtCdc13-DNA complex formation appears to involve primarily recognition of multiple copies of a six-nucleotide element (GGATGT) that is shared by many Candida telomere repeats. We also determined the crystal structure of the OB4 domain of C. glabrata Cdc13, which revealed a novel mechanism of OB fold dimerization. The structure also exhibits marked differences to the C-terminal OB fold of RPA70, thus arguing against a close evolutionary kinship between these two proteins. Our findings provide new insights on the mechanisms and evolution of a critical telomere end binding protein.

Candida tropicalis biofilms: artificial urine, urinary catheters and flow model.

Adhesion to medical devices and biofilm formation are considered important virulence factors of Candida tropicalis. This work aimed to use artificial urine (AU) and urinary catheters, under flow conditions, for studying C. tropicalis biofilms. Adhesion and biofilm formation on silicone and latex urinary catheters were quantified by crystal violet staining and determination of colony forming units. Candida surface hydrophobicity was also evaluated, as well as the biofilms' matrix content in terms of proteins and carbohydrates. Candida tropicalis was able to adhere and to form biofilms along the entire length of the catheters under flow conditions. It was found that the isolate U69 adhered significantly more to both types of catheters than did the reference strain. However, U69 biofilms contained significantly less cultivable cells and higher biofilm biomass than those of the reference strain. Detachment of cells from biofilms on latex catheter was lower compared to silicone catheter. This model using AU appeared to be suitable for studies mimicking the real body conditions. Additionally, C. tropicalis was in fact able to colonize urinary catheters in the presence of AU and to detach from these catheters, demonstrating their capacity to colonize distal sites.

Effect of monorhamnolipid on the degradation of n-hexadecane by Candida tropicalis and the association with cell surface properties.

The effect of monorhamnolipid (monoRL) on the degradation of n-hexadecane by Candida tropicalis was investigated in this study. The concentration of hexadecane, cell growth, cell surface hydrophobicity (CSH), cell surface zeta potential (CSZP), and FT-IR spectra of cellular envelope were tested to determine the mechanisms. MonoRL at the initial concentrations of 11.4, 19, and 38 mg/l improved the degradation of hexadecane, and 19 mg/l was the best concentration. However, 114 mg/l monoRL suppressed the biodegradation probably because of the reduced bioavailability of hexadecane caused by the micelles. The presence of monoRL changed the cell surface properties, which was demonstrated by the increased CSH, the increased CSZP, and the changed FT-IR spectra of cellular envelope at 680 and 620 cm(-1). The changes of cell surface properties may be a reason for the enhanced biodegradation of hexadecane by the yeast. The results indicate the potential application of monoRL in the bioremediation of hydrocarbons.

Two squalene synthase inhibitors, E5700 and ER-119884, interfere with cellular proliferation and induce ultrastructural and lipid profile alterations in a Candida tropicalis strain resistant to fluconazole, itraconazole, and amphotericin B.

Three quinuclidine-based squalene synthase (SQS) inhibitors (BPQ-OH, E5700, and ER-119884) were evaluated against five Candida tropicalis strains with different susceptibility profiles to fluconazole (FLC), itraconazole (ITC), terbinafine (TRB), and amphotericin B (AMB). Although the quinuclidine derivatives were inactive against most C. tropicalis strains tested at concentrations up to 16 µg/ml, E5700 and ER-119884 showed antifungal activity against C. tropicalis ATCC 28707, a strain resistant to FLC, ITC, and AMB, with IC(50) and IC(90) values (i.e., the minimum inhibitory concentrations of the drugs determined as the lowest drug concentrations leading to a 50 and 90% of reduction in turbidity at 492 nm, respectively, after 48 h of incubation) of 1 and 4 µg/ml, respectively. Analysis of free sterols showed that non-treated C. tropicalis ATCC 28707 cells contained only 14-methylated sterols and that treatment with E5700 or ER-119884 led to a marked reduction of squalene content and the complete disappearance of the endogenous sterols. The fatty acid and phospholipid profiles in C. tropicalis ATCC 28707 cells grown in the presence of E5700 and ER-119884 were also markedly altered, with a large increase in the content of linolenic acid (C18:3), associated with a reduction in the content of linoleic (C18:2) and oleic (C18:1) acids. Treatment of C. tropicalis ATCC 28707 with E5700 or ER-119884 IC(50) values induced several ultrastructural alterations, including a marked increase in the thickness of the cell wall and the appearance of a large number of electron-dense vacuoles. In conclusion, our results indicated that E5700 and ER-119884 inhibited the growth and altered the lipid prolife and the ultrastructure of a multiple drug-resistant C. tropicalis strain. Therefore, such compounds could act as leads for the development of new treatment options against multidrug resistant Candida species.

Unique profiles of changes in cell membrane fluidity during ethanol-induced yeast-to-pseudohyphal transition in Candida tropicalis.

A dimorphic transition from the yeast form to filamentous one in Candida tropicalis pK233 is triggered by the addition of ethanol into the glucose semi-defined liquid medium and the process of filamentation accompanies temporal depolarization of yeast cells. The transition is completely prevented by further supplementation of myo-inositol at the start of cultivation. The addition of ethanol caused an increase in membrane fluidity during the process of depolarization, and then fluidity was gradually lowered to the level equivalent with that of the stationary-phase yeast cells in accordance with filamentation. The increase in membrane fluidity of ethanol-induced cells appeared parallel with reduction in the content of membrane phosphatidylinositol, which was rich in saturated palmitic acid. Introduction of exogenous myo-inositol or 1 M sorbitol into the ethanol-supplemented culture at the start of cultivation restored yeast growth and the reduction of membrane fluidity occurred, coupled with the recovery of the phosphatidylinositol content.

Structure and role of sialic acids on the surface of Aspergillus fumigatus conidiospores.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes a life-threatening invasive fungal disease (invasive aspergillosis, IA) in immunocompromised individuals. The first step of pathogenesis is thought to be the attachment of conidia to proteins in lung tissue. Previous studies in our laboratory have shown that conidia adhere to basal lamina proteins via negatively charged sugars on their surface, presumably sialic acids. Sialic acids are a family of more than 50 substituted derivatives of a nine-carbon monosaccharide, neuraminic acid. The purpose of this study was 2-fold: (1) to determine the structure of sialic acids and the glycan acceptor on A. fumigatus oligosaccharides and (2) to determine the effect on the removal of sialic acids from conidia on conidial binding to the extracellular matrix protein fibronectin and phagocytosis of conidia by cultured macrophages and type 2 pneumocytes. Surface sialic acids were removed using Micromonospora viridifaciens sialidase or using acetic acid, mild acid hydrolysis. Lectin binding studies revealed that the majority of conidial sialic acids are alpha2,6-linked to a galactose residue. High-pressure liquid chromatography of derivatized sialic acids released from conidia revealed that unsubstituted N-acetylneuraminic acid is the predominant sialic acid on the surface of conidia. Enzymatic removal of sialic acid significantly decreased the binding of conidia to fibronectin by greater than 65% when compared with sham-treated controls. In addition, removal of sialic acids decreased conidial uptake by cultured murine macrophages and Type 2 pneumocytes by 33% and 53%, respectively. Hence, sialylated molecules on A. fumigatus conidia are ligands for both professional and nonprofessional phagocytes.