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.

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.

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.

Molecular cloning and characterization of NAD(+)-dependent xylitol dehydrogenase from Candida tropicalis ATCC 20913.

Induction of xylitol dehydrogenase of Candida tropicalis ATCC 20913 by various carbon sources was investigated. The enzyme activity was induced when the yeast was grown on l-arabinose and d-xylose. A novel gene encoding the enzyme was cloned and characterized. The 1,095-bp coding sequence of the gene encodes a polypeptide of 364 amino acids, with a molecular mass of 39.4 kDa. Sequence analysis of the putative protein showed it to be a member of the zinc-containing alcohol dehydrogenase family and to have homology to xylitol dehydrogenase genes from other yeasts and fungi. The recombinant xylitol dehydrogenase expressed in Escherichia coli oxidized polyols such as xylitol and d-sorbitol and reduced ketoses such as d-xylulose and d-fructose. It required exclusively NAD or NADH as a cofactor.

Comparación de diferentes métodos para la identificación de especies del género Candida / Comparison of different methods for species identification of genus Candida

Las diferentes especies del género Candida producen una variedad de enfermedades, desde infecciones mucocutáneas leves a formas diseminadas graves. Tradicionalmente, la taxonomía de las levaduras se ha llevado a cabo en base a estudios morfológicos y fisiológicos, pero éstos dependen de las condiciones de cultivo de las cepas, por lo que se han observado diversas dificultades. Por tal motivo, recientemente, se han probado técnicas de biología molecular. El objetivo de este trabajo es correlacionar los estudios taxonómicos de las especies correspondientes a las principales patógenas: C. albicans, C. krusei, C. parapsilosis, C. tropicalis y C. glabrata, realizados por técnicas fenotípicas tradicionales, métodos comerciales y por PCR fingerprinting. Al comparar las técnicas que identifican Candida albicans, agar harina de maíz y formación de tubos germinativos, estadísticamente se observa que no existen diferencias significativas entre ambos métodos (valor de la estadística X2 = 0,5 p = 0,4795). Comparando los métodos que discriminan especies de Candida: pruebas fisiológicas, CHROMagar, API20C y PCR fingerprinting se observó que no existen diferencias significativas en las proporciones de resultados que identifican cualquier Candida entre las pruebas fisiológicas, API20C y PCR fingerprinting. La proporción de resultados definitivos es mayor a la obtenida usando el método CHROMagar (p< 0,001).

Different species of genus Candida can cause a wide range of pathologies, since mucocutaneous trivial infections to disseminated serious forms. Traditionally, taxonomy of yeast has been performed taking into account morphologic and physiologic studies, but they depend on the culture conditions of strains, what cause certain difficulties. Thus, recently, molecular biology methods have been tried. The aim of this work is to correlate taxonomic studies of most important pathogenic species -C. albicans, C. krusei, C. parapsilosis, C. tropicalis and C. glabrata- all of them performed by phenotypic traditional methods, commercial ones, and by a molecular method, PCR fingerprinting. Comparing useful methods for C. albicans identification, corn flour agar and germinative tube formation, no statistical differences between them are observed (X2 =0.5, p=0.4795). By comparison between methods to discriminate different Candida species, physiological tests, CHROMagar, API 20C and PCR fingerprinting we observed no significative differences in proportion of accurate results, in test that can identify any Candida species, such as physiological assays, API 20C and PCR fingerprinting. The proportion of unequivocal results is greater than the obtained performing the CHROMagar culture method (p< 0.001).

Microbial adhesion to poly(ethylene oxide) brushes: influence of polymer chain length and temperature.

Glass surfaces were modified by end-grafting poly(ethylene oxide) (PEO) chains having molecular weights of 526, 2000, or 9800 Da. Characterization using water contact angles, ellipsometry, and X-ray photoelectron spectroscopy confirmed the presence of the PEO brushes on the surface with estimated lengths in water of 2.8-, 7.5-, and 23.7-nm, respectively. Adhesion of two bacterial (Staphylococcus epidermidis and Pseudomonas aeruginosa) and two yeast (Candida albicans and Candida tropicalis) strains to these brushes was studied and compared to their adhesion to bare glass. For the bacterium P. aeruginosa and the yeast C. tropicalis, adhesion to the 2.8-nm brush was comparable to their adhesion on bare glass, whereas adhesion to the 7.5- and 23.7-nm brushes was greatly reduced. For S. epidermidis, adhesion was only slightly higher to the 2.8-nm brush than that to the longer brushes. Adhesion of the yeast C. albicans to the PEO brushes was lower than that to glass, but no differences in adhesion were found between the three brush lengths. After passage of an air bubble, nearly all microorganisms adhering to a brush were removed, irrespective of brush length, whereas retention of the adhering organisms on glass was much higher. No significant differences were found in adhesion nor retention between experiments conducted at 20 and those conducted at 37 degrees C.

A novel technique to evaluate the adhesion of Candida species to gingival epithelial cells.

We developed an in vitro ATP assay technique to extract cellular and fungal ATP separately, which allowed to evaluate quantitatively the adhesion of the yeasts to monolayers of human gingival epithelial cells. Thirteen isolates of Candida spp. representing three species (i.e. Candida albicans, C. tropicalis and C. glabrata) were used in the present study. When the adherent capacity of the Candida species was compared, C. albicans exhibited highest capacity of adherence to gingival epithelial cells, followed by C. tropicalis, and C. glabrata was the lowest [analysis of variance (ANOVA), P < 0.01]. The germ tubes of C. albicans exhibited significantly higher adherence capacity than their blastoconidia cells (ANOVA, P < 0.01), which was not observed with a C. albicans isolate, defect of germ tube formation. Our results suggested that the adherence of C. albicans is promoted by germ tube formation and may play an important role in the pathogenesis of the fungus.

A combination of alpha-tocopherol, vitamin C and N-acetyl cysteine increases unsaturated fatty acid levels in hydrogen peroxide-induced Candida tropicalis (ATCC 13803).

This research was aimed at evaluating the antioxidant effects of combinations of alpha lipoic acid (LA), vitamin C (VC), N-acetyl cysteine (NAC) and alpha-tocopherol (TOC) on lipid level and fatty acid composition of C. tropicalis (ATCC 13803) against hydrogen peroxide toxicity. According to the experimental results, the cell density of C. tropicalis increased significantly in NAC+LA+H2O2, NAC+TOC+ H2O2 and NAC+VC+H2O2 groups (p<0.001) at the end of 48 and 72 h incubation times. The total lipid level in H2O2 and H2O2 + antioxidant-supplemented groups was lower than that of the control group. In the fatty acid composition of C. tropicalis, the palmitic acid level was raised in the NAC group (p<0.05), whereas its level was reduced in the other supplemented groups. While the oleic acid level increased in NAC+TOC+H2O2 and NAC+VC+H2O2 (p<0.001) groups, its level slightly decreased in the H2O2 group. The linolenic acid level was low in all the supplemented groups, but linoleic acid and total mono-unsaturated fatty acid (MUFA) levels were high in these groups compared with the control group. Total polyunsaturated fatty acid level (PUFA) decreased in NAC and H2O2 groups (p<0.01), but its level increased in NAC+LA+H2O2 and NAC+TOC+H2O2 groups (respectively, p<0.01, p<0.001). Total saturated fatty acid level decreased significantly in NAC+TOC+H2O2, NAC+H2O2 and NAC+VC+H2O2 (p<0.001) groups (p<0.01), whereas total unsaturated fatty acid level increased in NAC, NAC+H2O2, NAC+LA+H2O2, NAC+TOC+H2O2 and NAC+VC+H2O2 groups. In conclusion, our data showed that the levels of total unsaturated fatty acid, MUFA and PUFA were raised with the combinations of NAC and TOC, LA and VC in C. tropicalis cells subjected to hydrogen peroxide toxicity.