Optimization of cellular uptake of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine for maximal photoinactivation of Candida albicans

Cellular uptake and photodynamic action of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc⁴⁺) was examined in Candida albicans. In vitro investigations showed that ZnPPc⁴⁺ was rapidly bound to C. albicans cells. The binding of phthalocyanine to cells was dependent on ZnPPc⁴⁺ concentrations (1–10 μM) and cells densities (10⁶–10⁸ cells mL^?1). A high amount of ZnPPc⁴⁺ retained in the cells after two washing steps, indicating a strong interaction between the photosensitizer and C. albicans. The uptake was temperature dependent, although the difference between 37 °C and 4 °C was about 10 %. Also, the amount of ZnPPc⁴⁺ bound to C. albicans was affected when the cells were incubated for a longer time with azide and 2,4-dinitrophenol (DNP) prior to treatment with ZnPPc⁴⁺. Cell survival after irradiation was dependent on the irradiation period, ZnPPc⁴⁺ concentration and cells density. Photoinactivation of C. albicans cells was elevated even after two washing steps. The strong dependence of uptake on cell density reveals the strength and avidity of the binding of ZnPPc⁴⁺ to C. albicans cells. The accumulation behaviour of ZnPPc⁴⁺ suggests that mainly an affinity-mediated binding mechanism can be involved. Therefore, ZnPPc⁴⁺ is an interesting phthalocyanine for photodynamic inactivation (PDI) of yeasts in liquid suspensions.     

A micromethod for the quantitative determination of the viability of Candida albicans hyphae

A micromethod for the quantitative determination of the viability of Candida albicans hypae was devised which takes advantage of the dimorphic nature of C. albicans which grows exclusively in the yeast form when incubated aerobically on Sabouraud dextrose agar at 30°C. When tested by thisd method, all viable, C. albicans hyphae were recognized as microcolonies consisting of one hypha surrounded by several yeast form progeny. In contrast to this, no yeast form progeny emerged from nonviable hypae. By counting appropriate total numbers (200–400) of microcolony-forming hypae and infertile hyphae, it was possible to determine the ratio of viable to nonviable cells in a given hyphal suspension. This micromethod may be used for quantitative assessment of the candidacidal effects of various antimycotic agents or phagocytes C. albicans hyphae whose viability could not have been determined by the conventional plating technique because of the species' high propensity to clump.     

Pseudomembranous Candidiasis in HIV/AIDS Patients in Cali, Colombia

Candida albicans is the most frequently isolated yeast from the oral cavity of HIV/AIDS individuals. The use of fluconazole has increased the number of resistant or less-sensitive Candida species different than C. albicans. The purpose of this study was to identify the Candida species producing pseudomembranous candidiasis in patients suffering from AIDS, their relationship with CD4⁺ counts and their sensitivity to fluconazole and itraconazole. We studied 71 patients at a hospital in the city of Cali. Samples of white plaque were seeded on CHROMagar Candida, yeast identification was done with API 20C Aux, and susceptibility testing was determined by E test. Ninety-three yeast isolates were obtained, 52 single and 41 mixed. C. albicans was the most isolated, followed by C. glabrata. An increased frequency of isolates and variety of Candida species occurred in patients with a CD4⁺ cell count ≤100 cells/mm³ without significant differences (p = 0.29). The susceptibility study showed that 8 (8.6 %) isolates were resistant to fluconazole and 11 (11.8 %) to itraconazole, while 6 (8.8 %) C. albicans were simultaneously resistant. No association was found between the isolates of C. albicans or Candida species different than C. albicans and the use of fluconazole (p = 0.21). The results of this study indicate that in the tested population, fluconazole continues to be the best treatment option for oropharyngeal candidiasis in patients suffering from AIDS (HIV/AIDS); however, susceptibility tests are necessary in patients who present therapeutic failure.     

Induction of germ-tube formation by Candida albicans in amino acid liquid synthetic medium at 25°C

Candida albicans (3153A) was found to exhibit extensive germ-tube and mycelial development at 25°C when transferred from amino acid synthetic medium at pH 6 to medium of pH 7. Significant germ-tube formation was detectable after approximately 8 h and in all experimental treatments, the peaks of maximal germination occurred at approximately 40–44 h. Such a transition was not only dependent on the initial pH of the medium but also on the glucose concentration and inoculum size. The optimum initial glucose concentration and inoculum size for maximal germ-tube development was 1.25% and 2×10⁶ cells ml^–1 respectively and above or below these values the extent of germ-tube formation was greatly reduced.     

The effect of temperature of concanavalin A-mediated agglutination of cells with rigid receptors

The agglutination of a yeast, Candida albicans, by concanavalin A has been described. The agglutination was cell-number dependent. Prolonged incubation (60 min) was needed to reach maximum agglutination at 37° C. The rate but not the extent of agglutination was temperature dependent. The dimeric forms of concanavalin A, obtained either at low pH or after succinylation, agglutinated the yeast cells as well as the tetramer. Temperature changes affected the agglutination of yeast cells by dimers and by tetramers to the same extent.     

Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. ¹H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or “closed chain” structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.     

Two subpopulations of α1-adrenergic receptors with high and low affinity for agonists: Short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including β-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in α₁-adrenergic receptors induced by agonists. α₁-receptors were studied on DDT₁ MF-2 smooth muscle cells (DDT₁-MF-2 cells) by specific [³H]prazosin binding. In competition binding on membranes and on intact cells at 4°C or at 37°C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37°C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [³H]prazosin binding inhibited by 20 μM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [³H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4°C, but only 30% at 37°C. On DDT₁-MF-2 cells preequilibrated with [³H]prazosin at 4°C, and then shifted to 37°C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4°C it is the native form of α₁-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37°C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 μM norepinephrine. The nature of this low-affinity form was further investigated. On DDT₁-MF-2 cells preincubated with the agonist and then extensively washed at 4°C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [³H]prazosin at 4°C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4°C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37°C may be due to the agonist-induced sequestration of α₁-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.     

Growth of the yeast Kluyveromyces marxianus CBS 6556 on different sugar combinations as sole carbon and energy source

The yeast Kluyveromyces marxianus has been pointed out as a promising microorganism for a variety of industrial bioprocesses. Although genetic tools have been developed for this yeast and different potential applications have been investigated, quantitative physiological studies have rarely been reported. Here, we report and discuss the growth, substrate consumption, metabolite formation, and respiratory parameters of K. marxianus CBS 6556 during aerobic batch bioreactor cultivations, using a defined medium with different sugars as sole carbon and energy source, at 30 and 37 °C. Cultivations were carried out both on single sugars and on binary sugar mixtures. Carbon balances closed within 95 to 101 % in all experiments. Biomass and CO₂ were the main products of cell metabolism, whereas by-products were always present in very low proportion (<3 % of the carbon consumed), as long as full aerobiosis was guaranteed. On all sugars tested as sole carbon and energy source (glucose, fructose, sucrose, lactose, and galactose), the maximum specific growth rate remained between 0.39 and 0.49 h^?1, except for galactose at 37 °C, which only supported growth at 0.31 h^?1. Different growth behaviors were observed on the binary sugar mixtures investigated (glucose and lactose, glucose and galactose, lactose and galactose, glucose and fructose, galactose and fructose, fructose and lactose), and the observations were in agreement with previously published data on the sugar transport systems in K. marxianus. We conclude that K. marxianus CBS 6556 does not present any special nutritional requirements; grows well in the range of 30 to 37 °C on different sugars; is capable of growing on sugar mixtures in a shorter period of time than Saccharomyces cerevisiae, which is interesting from an industrial point of view; and deviates tiny amounts of carbon towards metabolite formation, as long as full aerobiosis is maintained.     

The dynamics of carbohydrate metabolism in Candida albicans

The total cellular concentrations of the intermediary metabolites and the carbohydrate end products were determined for starved Candida albicans yeast cells and cells forming germ tubes during a 60-min incubation in imidazole-HCl buffer in the absence and presence of 2.5 mM glucose, 2.5 mM glutamine, and 0.2% serum at 37°C. These cells were also incubated in the presence of tracer [U-¹⁴C]glucose and the specific radioactivities of the metabolites and end products determined. The labeling data indicated (1) a minimum of two metabolically independent pools of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate, and uridine diphosphoglucose; (2) compartmentation of the pathways of catabolism and anabolism; (3) channeling of the exogenous tracer glucose into the anabolic pathway compartments of the starved cells; and (4) a significant rate of turnover of cell wall carbohydrates in cells incubated under nongrowth conditions and rapid turnover of these pools in germ tube forming cells. The labeling data will be used to construct kinetic models of carbohydrate metabolism in C. albicans.     

Vicia villosa B4 lectin inhibits nucleotide pyrophosphatase activity toward UDP-GalNAc specifically

Plant seed lectins play a defense role against plant-eating animals. Here, GalNAc-specific Vicia villosa B₄ lectin was found to inhibit hydrolysis of UDP-GalNAc by animal nucleotide pyrophosphatases, which are suggested to regulate local levels of nucleotide sugars in cells. Inhibition was marked at low concentrations of UDP-GalNAc, and was reversed largely by the addition of GalNAc to the reaction mixture. In contrast, lectin inhibited enzymatic hydrolysis of other nucleotide sugars, such as UDP-Gal and UDP-GlcNAc, only to a small extent, and GalNAc did not affect such an inhibition. The binding constant of the lectin for UDP-GalNAc was as high as 2.8×10⁵ M^?1 at 4°C, whereas that for GalNAcα-1-phosphate was 1.3×10⁵ M^?1. These findings indicate that lectin inhibition of pyrophosphatase activity toward low concentrations of UDP-GalNAc arises mainly from competition between lectin and enzyme molecules for UDP-GalNAc. This type of inhibition was also observed to a lesser extent with GalNAc-specific Wistaria floribunda lectin, but not apparently with GalNAc-specific soybean or Dolichos biflorus lectin. Thus, V. villosa B₄ lectin shows unique binding specificity for UDP-GalNAc and has the capacity to modulate UDP-GalNAc metabolism in animal cells.     

Purification and characterization of the cold-active killer toxin from the psychrotolerant yeast Mrakia frigida isolated from sea sediments in Antarctica

The psychrotolerant yeast Mrakia frigida 2E00797 isolated from sea sediments in Antarctica was found to be able to produce killer toxin against Metschnikowia bicuspidata, Candida tropicalis and Candida albicans. In the present study, the killer toxin was purified and characterized. The molecular weight of the purified killer toxin was estimated to be 55.6 kDa and the purified killer toxin shared 35.1% sequence homology with a protein kinase. The purified killer toxin's optimal temperature and pH for killing activity were 16 °C and 4.5, respectively, and it was stable in the temperature range from 10 to 25 °C at pH 4.5. The toxin's highest killing activity was observed in the presence of 3.0 g/100 ml NaCl. The purified killer toxin was able to actively kill whole cells of M. bicuspidata but could not kill the protoplast of the sensitive yeast. Of the eight yeast species tested in this study, the killer toxin was able to kill C. tropicalis and C. albicans in addition to M. bicuspidata.     

Role of Force-Sensitive Amyloid-Like Interactions in Fungal Catch Bonding and Biofilms

The Candida albicans Als adhesin Als5p has an amyloid-forming sequence that is required for aggregation and formation of model biofilms on polystyrene. Because amyloid formation can be triggered by force, we investigated whether laminar flow could activate amyloid formation and increase binding to surfaces. Shearing Saccharomyces cerevisiae cells expressing Als5p or C. albicans at 0.8 dyne/cm² increased the quantity and strength of cell-to-surface and cell-to-cell binding compared to that at 0.02 dyne/cm². Thioflavin T fluorescence showed that the laminar flow also induced adhesin aggregation into surface amyloid nanodomains in Als5p-expressing cells. Inhibitory concentrations of the amyloid dyes thioflavin S and Congo red or a sequence-specific anti-amyloid peptide decreased binding and biofilm formation under flow. Shear-induced binding also led to formation of robust biofilms. There was less shear-activated increase in adhesion, thioflavin fluorescence, and biofilm formation in cells expressing the amyloid-impaired V326N-substituted Als5p. Similarly, S. cerevisiae cells expressing Flo1p or Flo11p flocculins also showed shear-dependent binding, amyloid formation, biofilm formation, and inhibition by anti-amyloid compounds. Together, these results show that laminar flow activated amyloid formation and led to enhanced adhesion of yeast cells to surfaces and to biofilm formation.     

Species-specific activation of Cu/Zn SOD by its CCS copper chaperone in the pathogenic yeast Candida albicans

Candida albicans is a pathogenic yeast of important public health relevance. Virulence of C. albicans requires a copper and zinc containing superoxide dismutase (SOD1), but the biology of C. albicans SOD1 is poorly understood. To this end, C. albicans SOD1 activation was examined in baker’s yeast (Saccharomyces cerevisiae), a eukaryotic expression system that has proven fruitful for the study of SOD1 enzymes from invertebrates, plants, and mammals. In spite of the 80 % similarity between S. cerevisiae and C. albicans SOD1 molecules, C. albicans SOD1 is not active in S. cerevisiae. The SOD1 appears incapable of productive interactions with the copper chaperone for SOD1 (CCS1) of S. cerevisiae. C. albicans SOD1 contains a proline at position 144 predicted to dictate dependence on CCS1. By mutation of this proline, C. albicans SOD1 gained activity in S. cerevisiae, and this activity was independent of CCS1. We identified a putative CCS1 gene in C. albicans and created heterozygous and homozygous gene deletions at this locus. Loss of CCS1 resulted in loss of SOD1 activity, consistent with its role as a copper chaperone. C. albicans CCS1 also restored activity to C. albicans SOD1 expressed in S. cerevisiae. C. albicans CCS1 is well adapted for activating its partner SOD1 from C. albicans, but not SOD1 from S. cerevisiae. In spite of the high degree of homology between the SOD1 and CCS1 molecules in these two fungal species, there exists a species-specific barrier in CCS–SOD interactions which may reflect the vastly different lifestyles of the pathogenic versus the noninfectious yeast.     

Gamma-aminobutyric acid (GABA) increases in vitro germ-tube formation and phospholipase B1 mRNA expression in Candida albicans

Candida albicans is a commensal yeast in humans that disseminates in immunocompromised persons. Its spreading is modulated by melanin, hormones, or some neurotransmitters, among other factors. The neurotransmitter gamma-aminobutyric acid (GABA) is used by bacteria, plants, and fungi as a carbon and nitrogen source. In this article, the in vitro effect of different doses of GABA on germ-tube formation and expression of phospholipase B1 (PLB1) mRNA in two Candida albicans strains was investigated. Results demonstrated that GABA increases both germ-tube formation and PLB1 mRNA expression in the two Candida strains in a dose-dependent manner, which suggests that GABA promotes the growth of C. albicans.     

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis

The protein kinase p34^cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34^CDC2 protein kinase from chicken. The response of the chicken p34^CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34^CDC2 protein divide at reduced size at 31°?C. Cells are temperature sensitive at 35.5°?C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34^CDC2 are cold sensitive. At 19°?C to 25°?C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34^CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest.     

Estudios de la viabilidad y la vitalidad frente al congelado de la levadura probiótica Saccharomyces boulardii: efecto del preacondicionamiento fisiológico

The aim of this study was to evaluate the vitality and viability of the probiotic yeast Saccharomyces boulardii after freezing/thawing and the physiological preconditioning effect on these properties. The results indicate that the specific growth rate (0.3/h^?1) and biomass (2-3 × 10⁸ cells/ml) of S. boulardii obtained in flasks shaken at 28 °C and at 37 °C were similar. Batch cultures of the yeast in bioreactors using glucose or sugar-cane molasses as carbon sources, reached yields of 0.28 g biomass/g sugar consumed, after 10 h incubation at 28 °C; the same results were obtained in fed batch fermentations. On the other hand, in batch cultures, the vitality of cells recovered during the exponential growth phase was greater than the vitality of cells from the stationary phase of growth. Vitality of cells from fed-batch fermentations was similar to that of stationary growing cells from batch fermentations. Survival to freezing at –20 °C and subsequent thawing of cells from batch cultures was 0.31% for cells in exponential phase of growth and 11.5% for cells in stationary phase. Pre-treatment of this yeast in media with water activity (a_w) 0.98 increased the survival to freezing of S. boulardii cells stored at –20 °C for 2 months by 10 fold. Exposure of the yeast to media of reduced a_w and/or freezing/thawing process negatively affected cell vitality. It was concluded that stress conditions studied herein decrease vitality of S. boulardii. Besides, the yeast strain studied presented good tolerance to bile salts even at low pH values.     

Calcium-Dependent and Calcium-Independent Adhesive Mechanisms Are Present During Initial Binding Events of Neural Retina Cells

The hypothesis that intercellular adhesion can be subdivided into two separable phenomena, an initial recognition event and a subsequent stabilization, is supported by the use of a new cell binding assay that provides a quantitative measure of intercellular binding strengths. Radioactive single cells are brought into contact with cell monolayers at 4°C in sealed compartments. The compartments are inverted and a centrifugal force is then applied tending to dislodge the probe cells from the monolayers. By varying the speed of centrifugation, the force maintaining associations between embryonic chick neural retina cells was determined to be on the order of 10^?5 dynes after incubation at 4°C. Brief incubations at 37°C resulted in significant strengthening of the intercellular bond. Using this cell binding assay, neural retina cells were shown to exhibit both a Ca⁺⁺-independent and a Ca⁺⁺-dependent mechanism in their initial binding to one another.