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.

Nail Raman spectroscopy: A promising method for the diagnosis of onychomycosis. An ex vivo pilot study.

Trichophyton rubrum and Candida species comprise the majority of onychomycosis pathogens. The aim of this study was to evaluate Raman spectroscopy for the differentiation between healthy and either T. rubrum or Candida infected nails. Raman measurements were performed on clippings (N = 52) infected either by T. rubrum (N = 12) or Candida species (N = 14; C. parapsilosis (sensu lato): N = 11, C. glabrata: N = 1, C. albicans: N = 2) with healthy nails (N = 26) used as controls. Systematic spectral differences were observed in the 500-520 cm-1 band region, attributable to a diverting imprint of the disulfide stretching of cystine and cysteine residues among samples. Particularly, Candida infected nails demonstrated a shoulder at 519 cm-1, corresponding to the signal of the less stable gauche-gauche-trans conformation of the disulfide bond. Two additional bands at 619 and 648 cm-1, corresponding to the C-S stretching vibration, were more evident in the T. rubrum infected nails. Finally, a Raman band at 1550 cm-1, attributable to amide II and tryptophan (Trp) content, was undetectable in Candida infected nails. Using principal component analysis (PCA), efficient differentiation of healthy, T. rubrum and Candida species infected nails was achieved. Soft independent modeling of class analogy (SIMCA) and partial least squares-discriminant analysis (PLS-DA) were further applied to generate diagnostic algorithms for the classification of Raman spectra. Both techniques succeeded in modeling clinical nail samples in three groups according to their mycological categories. Raman spectroscopy is a promising method for the differentiation of healthy vs. diseased nails, including efficient differentiation between onychomycosis caused by T. rubrum and Candida species.

Enhanced pyruvate production in Candida glabrata by overexpressing the CgAMD1 gene to improve acid tolerance.

OBJECTIVES: To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism. RESULTS: The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 (H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l. CONCLUSION: CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.

UNLABELLED: Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear. Here we describe structural, biochemical, and in vivo analyses of FKBP12s from the pathogenic fungi Candida albicans, Candida glabrata, and Aspergillus fumigatus Strikingly, multiple apo A. fumigatus and C. albicans FKBP12 crystal structures revealed a symmetric, intermolecular interaction involving the deep insertion of an active-site loop proline into the active-site pocket of an adjacent subunit. Such interactions have not been observed in previous FKBP structures. This finding indicates the possibility that this is a self-substrate interaction unique to the A. fumigatus and C. albicans fungal proteins that contain this central proline. Structures obtained with the proline in the cis and trans states provide more data in support of self-catalysis. Moreover, cysteine cross-linking experiments captured the interacting dimer, supporting the idea that it forms in solution. Finally, genetic studies exploring the impact of mutations altering the central proline and an adjacent residue provide evidence that any dimeric state formed in vivo, where FKBP12 concentrations are low, is transient. Taken together, these findings suggest a unique mechanism of self-substrate regulation by fungal FKBP12s, lending further novel understanding of this protein for future drug-targeting efforts. IMPORTANCE: FKBP12 is a cis-trans peptidyl-prolyl isomerase that plays key roles in cellular protein homeostasis. FKBP12s also bind the immunosuppressive drug FK506 to inhibit the phosphatase calcineurin (CaN). CaN is required for virulence of A. fumigatus, C. albicans, C. glabrata, and other deadly fungal pathogens, marking FKBP12 and CaN as potential broad-spectrum drug targets. Here we describe structures of fungal FKBP12s. Multiple apo A. fumigatus and C. albicans FKBP12 structures reveal the insertion of a proline, conspicuously conserved in these proteins, into the active sites of adjacent molecules. This suggests that these proteins might serve as their own substrates. Cysteine disulfide trapping experiments provide support for this self-interaction and hence possible intermolecular catalysis by these enzymes.

Synthesis and carbonic anhydrase inhibitory properties of sulfamides structurally related to dopamine.

A series of novel sulfamides incorporating the dopamine scaffold were synthesized. Reaction of amines and tert-butyl-alcohol/benzyl alcohol in the presence of chlorosulfonyl isocyanate (CSI) afforded sulfamoyl carbamates, which were converted to the title compounds by treatment with trifluoroacetic acid or by palladium-catalyzed hydrogenolysis. Inhibition of six α-carbonic anhydrases (CAs, EC 4.2.1.1), that is, CA I, CA II, CA VA, CA IX, CA XII and CA XIV, and two ß-CAs from Candida glabrata (CgCA) and Mycobacterium tuberculosis (Rv3588) with these sulfamides was investigated. All CA isozymes were inhibited in the low micromolar to nanomolar range by the dopamine sulfamide analogues. K(i)s were in the range of 0.061-1.822 µM for CA I, 1.47-2.94 nM for CA II, 2.25-3.34 µM for CA VA, 0.041-0.37 µM for CA IX, 0.021-1.52 µM for CA XII, 0.007-0.219 µM for CA XIV, 0.35-5.31 µM for CgCA and 0.465-4.29 µM for Rv3588. The synthesized sulfamides may lead to inhibitors targeting medicinally relevant CA isoforms with potential applications as antiepileptic, antiobesity antitumor agents or anti-infective.

Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata.

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal "FG" repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

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).

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.