Yeast Morphology Research Articles

Preliminary isolation and in vitro antiyeast activity of active fraction from crude extract of Gracilaria changii.

Objective:To isolate the active fraction from crude extract of Gracilaria changii and to determine its in vitro antifungal activity.Materials and Methods:The active fraction was isolated from the crude extract of G. changii by various purification procedures such as column chromatography, thin layer chromatography, bioauthograph etc. The in vitro antifungal activity (Candida albicans) of the active fraction (1.00, 0.50, and 0.25 mg/ml) was studied by disc diffusion method and the effect of the active fraction on the morphology of yeast was done by scanning electron microscope (SEM) studies.Results:An active fraction with remarkable antifungal activity was separated from the crude extract. The active fraction was effective as a fungicide against C. albicans and showed a dose-dependent antifungal activity. A Scanning Electron Microscope (SEM) study confirmed the fungicidal effect of G. changii active fraction on C. albicans, by changing the normal morphology of C. albicans.Conclusion:From G. changii crude extract, an active fraction with remarkable in vitro antifungal activity has been isolated.

Differentiation of Candida albicans from non-albicans yeast directly from blood cultures by Gram stain morphology

Clusters of pseudohyphae are commonly seen on Gram stain of blood cultures from patients with Candida albicans fungemia. Whether this morphologic feature is useful for differentiating C. albicans from other yeasts has not previously been systematically evaluated. Yeast morphology on Gram stain of blood cultures from consecutive patients with fungemia detected by the Bactec automated blood culture system was prospectively assessed and correlated with the final culture-based yeast identification. The distribution of yeast in 60 consecutive patients with fungemia included Candida spp. (C. albicans 43%, Candida glabrata 28%, Candida parapsilosis 8%, Candida krusei 7%, Candida tropicalis 5%, Candida dubliniensis 2%, and Candida lusitaniae 2%), Rhodotorula mucilaginosa 2%, and Cryptococcus neoformans 3%. Upon analysis of the first positive blood culture bottle per patient, the presence of clustered pseudohyphae on Gram stain had a sensitivity, specificity, positive predictive value, and negative predictive value of 85, 97, 96, and 89%, respectively, for C. albicans. The sensitivity and specificity of aerobic vs Myco/F blood culture bottles were 96 and 95% vs 25 and 100%, p < 0.001, respectively. Inter-rater agreement for ten separate observations among five reviewers was 100%. The presence of pseudohyphae clusters by Gram stain of blood cultures is useful in distinguishing C. albicans from non-albicans yeast. Additional studies are necessary to determine the clinical impact of these findings and their validity with other blood culture systems.

The Yeast Vacuole -A Scanning Electron Microscopy Study During High Gravity Wort Fermentations

The yeast vacuole has been shown to exhibit morphological responses to environmental conditions when exposed to worts of different gravity during fermentation. Marked effects of high gravity wort (20° Plato) on yeast morphology compared to more conventional wort gravity (12° Plato) were observed. High gravity worts caused vacuolar enlargement compared to conventional gravity wort. These results suggested that yeast cells experienced severe alterations with the vacuolar tonoplast when exposed to high osmotic pressure and elevated levels of ethanol.

Involvement of Candida albicans pyruvate dehydrogenase complex protein X (Pdx1) in filamentation

For 50 years, physiologic studies in Candida albicans have associated fermentation with filamentation and respiration with yeast morphology. Analysis of the mitochondrial proteome of a C. albicans NDH51 mutant, known to be defective in filamentation, identified increased expression of several proteins in the respiratory pathway. Most notable was a 15-fold increase in pyruvate dehydrogenase complex protein X (Pdx1), an essential component of the pyruvate dehydrogenase complex. In basal salts medium with ⩽100 mM glucose as carbon source, two independent pdx1 mutants displayed a filamentation defect identical to ndh51; reintegration of one PDX1 allele restored filamentation. Concentrations of glucose ⩽100 mM did not correct the filamentation defect. Expanding on previous work, these studies suggest that increased expression of proteins extraneous to the electron transport chain compensates for defects in the respiratory pathway to maintain yeast morphology. Mitochondrial proteomics can aid in the identification of C. albicans genes not previously implicated in filamentation.

Effect of farnesol on Candida dubliniensis morphogenesis

Cell-cell signalling in Candida albicans is a known phenomenon and farnesol was identified as a quorum sensing molecule determining the yeast morphology. The aim of this work was to verify if farnesol had a similar effect on Candida dubliniensis, highlighting the effect of farnesol on Candida spp. morphogenesis. Two different strains of C. dubliniensis and one of C. albicans were grown both in RPMI 1640 and in serum in the presence of absence of farnesol. At 150 micromol l(-1) farnesol the growth rate of both Candida species was not affected. On the contrary, farnesol inhibited hyphae and pseudohyphae formation in C. dubliniensis. Farnesol seems to mediate cell morphology in both Candida species. The effect of farnesol on C. dubliniensis morphology was not reported previously.

Antimicrobial effects of liquid anesthetic isoflurane on Candida albicans

Candida albicans is a dimorphic fungus that can grow in yeast morphology or hyphal form depending on the surrounding environment. This ubiquitous fungus is present in skin and mucus membranes as a potential pathogen that under opportunistic conditions causes a series of systemic and superficial infections known as candidiasis, moniliasis or simply candidiasis. There has been a steady increase in the prevalence of candidiasis that is expressed in more virulent forms of infection. Although candidiasis is commonly manifested as mucocutaneous disease, life-threatening systemic invasion by this fungus can occur in every part of the body. The severity of candidal infections is associated with its morphological shift such that the hyphal morphology of the fungus is most invasive. Of importance, aberrant multiplication of Candida yeast is also associated with the pathogenesis of certain mucosal diseases. In this study, we assessed the anti-candidal activity of the volatile anesthetic isoflurane in liquid form in comparison with the anti-fungal agent amphotericin B in an in vitro culture system. Exposure of C. albicans to isoflurane (0.3% volume/volume and above) inhibited multiplication of yeast as well as formation of hyphae. These data suggest development of potential topical application of isoflurane for controlling a series of cutaneous and genital infections associated with this fungus. Elucidiation of the mechanism by which isoflurane effects fungal growth could offer therapeutic potential for certain systemic fungal infections.

Evaluation of image processing programs for accurate measurement of budding and fission yeast morphology

To study the cellular functions of gene products, various yeast morphological mutants have been investigated. To describe yeast morphology objectively, we have developed image processing programs for budding and fission yeast. The programs, named CalMorph for budding yeast and F-CalMorph for fission yeast, directly process microscopic images and generate quantitative data about yeast cell shape, nuclear shape and location, and actin distribution. Using CalMorph, we can easily and quickly obtain various quantitative data reproducibly. To study the utility and reliability of CalMorph, we evaluated its data in three ways: (1) The programs extracted three-dimensional bud information from two-dimensional digital images with a low error rate (<1%). (2) The absolute values of the diameters of manufactured fluorescent beads calculated with CalMorph were very close to those given in the manufacturer's data sheet. (3) The programs generated reproducible data consistent with that obtained by hand. Based on these results, we determined that CalMorph could monitor yeast morphological changes accompanied by the progression of the cell cycle. We discuss the potential of the CalMorph series as a novel tool for the analysis of yeast cell morphology.

A Novel Arabidopsis Gene Causes Bax-like Lethality in Saccharomyces cerevisiae

Overexpression of the mammalian proapoptotic protein Bax induces cell death in plant and yeast cells. The Bax inihibitor-1 (BI-1) gene rescues yeast and plant from Bax-mediated lethality. Using the Arabidopsis BI-1 (AtBI-1) gene controlled by the GAL1 promoter as a cell death suppressor in yeast, Cdf1 (cell growth defect factor-1) was isolated from Arabidopsis cDNA library. Overexpression of Cdf1 caused cell death in yeast, whereas such an effect was suppressed by co-expression of AtBI-1. The Cdf1 protein fused with a green fluorescent protein was localized in the mitochondria and resulted in the loss of mitochondrial membrane potential in yeast. The Bax-resistant mutant BRM1 demonstrated tolerance against Cdf1-mediated lethality, whereas the Deltaatp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.

Effect of hyperbaric stress on yeast morphology: study by automated image analysis.

The effects of hyperbaric stress on the morphology of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 MPa and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide), covering aerobic and anaerobic conditions. A method using automatic image analysis for classification of S. cerevisiae cells based on their morphology was developed and applied to experimental data. Information on cell size distribution and bud formation throughout the cell cycle is reported. The results show that the effect of pressure on cell activity strongly depends on the nature of the gas used for pressurization. While nitrogen and air to a maximum of 0.6 MPa of pressure were innocuous to yeast, oxygen and carbon dioxide pressure caused cell inactivation, which was confirmed by the reduction of bud cells with time. Moreover, a decrease in the average cell size was found for cells exposed for 7.5 h to 0.6 MPa CO2.

Development of image processing program for yeast cell morphology.

Every living organism has its own species-specific morphology. Despite the relatively simple ellipsoidal shape of budding yeast cells, the global regulation of yeast morphology remains unclear. In the past, each mutated gene from many mutants with abnormal morphology had to be classified manually. To investigate the morphological characteristics of yeast in detail, we developed a novel image-processing program that extracts quantitative data from microscope images automatically. This program extracts data on cells that are often used by yeast morphology researchers, such as cell size, roundness, bud neck position angle, and bud growth direction, and fits an ellipse to the cell outline. We evaluated the ability of the program to extract quantitative parameters. The results suggest that our image-processing program can play a central objective role in yeast morphology studies.

The protein kinase kin1, the fission yeast orthologue of mammalian MARK/PAR-1, localises to new cell ends after mitosis and is important for bipolar growth

The kin1 protein kinase of the fission yeast Schizosaccharomyces pombe is a member of the PAR-1/MARK (partitioning-defective 1/microtubule-associated protein/microtubule affinity-regulating kinase) family important in eukaryotic cell polarity and cytoskeletal dynamics. We show here that kin1 plays a role in establishing the characteristic rod-shaped morphology of fission yeast. Cells in which kin1 was deleted are viable but are impaired in growth, and are rounded at one end or both ends. They are monopolar because after mitosis they fail to activate bipolar growth, and are delayed in cytokinesis, resulting in a high proportion of septated cells often with multiple septa. This phenotype can be partially rescued by heterologous expression of human MARKs, which restore bipolar growth in most cells, but do not correct the delay in cytokinesis. Using chromosomal epitope tagging, we show that kin1p localises to the cell ends, except during mitosis when it disappears from cell ends. After mitosis, kin1p first reappears at the new cell end. Overexpression of kin1 results in a loss of polarity, with partially or fully rounded cells. From these results we suggest that kin1 is required to direct the growth machinery to the cell ends.

Maintenance and Protection of Yeast Morphology by Contact with Wine Polyphenols During Simulation of Wine Aging on Lees

ABSTRACTScanning electron microscopy was used to monitor yeast cell morphology during simulated wine aging. Although yeast cells in lees in the absence of wine polyphenols rapidly reach a flat shape after the end of alcoholic fermentation, they keep a spherical (and almost intact) shape in the presence of wine polyphenols. We confirmed these visual observations by calculating the dynamic viscosity of the corresponding lees. Because yeast autolysis is not affected by the contact with wine polyphenols, such maintenance of yeast morphology maybe the consequence of protection of some constitutive parts of yeast cell walls by wine polyphenols toward the action of hydrolytic enzymes.

The Effects of Osmotic Pressure and Ethanol on Yeast Viability and Morphology

The selection of a brewing yeast strain with the required fermentation and recycling characteristics is critical. The yeast strain will influence the rate and extent of fermentation, the flavour characteristics and the overall quality and stability of the finished beer, and consequently, the economic viability of the brewery. Since high gravity worts can have a deleterious effect on yeast fermentation performance, it is imperative that the strain selected be suitable for this environment, which includes a capacity to withstand high osmotic pressures and elevated ethanol levels. Under controlled in vitro osmotic and ethanol induced stresses, there was a decline in mean cell volume in both lager and ale yeast strains. Whilst significant reductions in viability were observed in the lager strains, the ale strains studied were not affected. Cell surface investigations revealed shrinkage of the yeast cells and crenation of the outside envelope under both stresses, although exposure to ethanol had a more marked effect on the yeast cell surface than sorbitol-induced elevated osmotic pressure.

Rapid and cost-effective identification of Candida species using multipoint inoculation of CHROMagar Candida media, cycloheximide sensitivity and carbohydrate assimilation tests

<h2>Summary</h2><h3>Aims</h3> Increasing numbers of nosocomial infections with <i>Candida</i> species are caused by species other than <i>Candida albicans.</i> Definitive identification of <i>Candida</i> spp. usually utilises one commercial biochemical identification strip per isolate. As an alternative, CHROMagar Candida (CHROMagar, France) can be used to differentiate some species but lacks accuracy for some species, and is expensive when used as a primary isolation medium. The aim of this study was to evaluate a multipoint inoculation method for batch identification of <i>Candida</i> isolates, by generating a unique profile using CHROMagar Candida, a cycloheximide-containing medium and media containing seven in-house prepared carbohydrates. <h3>Methods</h3> Initially, 53 prospectively obtained clinical isolates, 52 stored clinical isolates and 15 reference laboratory-derived strains were tested using the multipoint inoculation method and compared with results obtained using the ID32C (BioMerieux, France) plus a germ tube test and yeast morphology agar. Nine <i>Candida</i> spp. were represented. Subsequently, 400 <i>Candida</i> isolates obtained from clinical specimens were prospectively tested using this method. <h3>Results</h3> All <i>Candida glabrata</i> (37), <i>Candida parapsilosis</i> (27), <i>Candida albicans</i> (26), <i>Candida tropicalis</i> (11), <i>Candida lusitaniae</i> (three), <i>Candida kefyr</i> (three) and <i>Candida famata</i> (two) were correctly identified. Of 26 <i>C. albicans</i> isolates, 25 gave a green colour on CHROMagar Candida and one gave a less characteristic white colour. One of five <i>Candida guilliermondii</i> failed to assimilate cellobiose. All six <i>Candida krusei</i> isolates consistently produced a unique unexpected profile which was then incorporated into the database and the method was integrated into the routine laboratory. Of 400 consecutive prospectively examined isolates, 390 (97.5%) were identifiable using this system; 53.7% were due to Candida species other than <i>C albicans.</i> Seven isolates (1.8%) were not identifiable using this system as they did not produce any of the expected profiles. <h3>Conclusions</h3> Utilising the described method and batch testing allowed considerable cost and labour savings for the laboratory identification of <i>Candida</i> species and determination of local <i>Candida</i> epidemiology. The estimated material and labour costs for a run of 30 yeasts in a batch is AUD$0.97. By comparison, the cost of a commercial ID32C is AUD$8.52 per isolate.

The mold-specific MS8 gene is required for normal hypha formation in the dimorphic pathogenic fungus Histoplasma capsulatum.

The dimorphic fungus Histoplasma capsulatum is the etiologic agent of one of the most common systemic mycoses of humans, histoplasmosis. In the environment, H. capsulatum grows in a differentiated mold form and shifts to an undifferentiated yeast form after mold fragments or spores are inhaled. This mold-to-yeast shift is required for disease. Little is known about the molecular biology of dimorphism in Histoplasma, and most studies have been directed toward yeast-specific genes. While it is important to examine the role of genes upregulated in the yeast morphotype, genes which are silenced in the yeast (i.e., mold-specific genes) may also play a critical role in dimorphism. To begin to examine this hypothesis, we report here the first misexpression and knockout analysis of a mold-specific gene in Histoplasma. The strongly expressed MS8 gene encodes a predicted 21-kDa protein extremely rich in glycine and glutamine. Forced expression of MS8 driven by the TEF1 promoter in yeast did not alter the yeast morphology at 37 degrees C or mold formation at 25 degrees C. Yeast expressing MS8 did exhibit clumping in liquid medium and formed "sticky" colonies on agar plates. Allelic replacement of MS8 was accomplished by a positive-negative selection procedure. ms8 knockout mutants formed apparently normal yeast at 37 degrees C but gave rise to aberrant mycelia at 25 degrees C. The mold colonies of the knockouts were less than half as large as normal, had a granular surface, produced a dark-red pigment, and formed short hyphae which were 40% wider with a distinctive twisted "zig-zag" shape.

Feasibility of copper uptake by the yeast Pichia guilliermondii isolated from sewage sludge

Copper is one of the most abundant toxic heavy metals in municipal wastewaters and, in consequence, in sewage sludge and compost. The ability of a strain of the yeast Pichia guilliermondii, which was isolated from sewage sludge, to eliminate copper has been evaluated, using both viable and nonviable biomass. It has been found that raising concentrations of copper affected both morphology and physiological parameters of the viable yeast, and it is thought that a process of bioaccumulation may be involved in its copper uptake. The growth rate of nonadapted cells decreased with increasing concentrations of copper, mainly due to a decrease in the biomass yield. The cells could be adapted by training with increasing copper concentrations up to 317.7 mg/l. This adaptation was an all-or-nothing process: once cells had adapted, the biomass yield, metabolic flux and consequent growth rate were constant and independent of the external copper concentration. Also, it was determined that up to 20 mg of copper per gram of viable adapted biomass could accumulate from the medium (i.e., double the amount when using nonadapted viable biomass). Finally, adsorption data on nonviable cells were found to be well modeled by the Langmuir isotherm ( q max=9.09 mg/g of biomass).

Protein phosphatase 2A on track for nutrient-induced signalling in yeast.

Early studies identified two bona fide protein phosphatase 2A (PP2A)-encoding genes in Saccharomyces cerevisiae, designated PPH21 and PPH22. In addition, three PP2A-related phosphatases, encoded by PPH3, SIT4 and PPG1, have been identified. All share as much as 86% sequence similarity at the amino acid level. This review will focus primarily on Pph21 and Pph22, but some aspects of Sit4 regulation will also be discussed. Whereas a role for PP2A in yeast morphology and cell cycle has been readily recognized, uncovering its function in yeast signal transduction is a more recent breakthrough. Via their interaction with phosphorylated Tap42, PP2A and Sit4 play a pivotal role in target of rapamycin (TOR) signalling. PPH22 overexpression mimics overactive cAMP-PKA (protein kinase A) signalling and PP2A and Sit4 might represent ceramide signalling targets. The methylation of its catalytic subunit stabilizes the heterotrimeric form of PP2A and might counteract TOR signalling. We will show how these new elements could lead us to understand the role and regulation of PP2A in nutrient-induced signalling in baker's yeast.

Glutathione metabolism and dimorphism inAureobasidium pullulans

Yeast<-->mycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors. In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions. In accordance with this hypothesis, the specific GSH levels found in A. pullulans yeast cells were always significantly higher than those in mycelia. One the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology. The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols. Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g. glutathione reductase (GR), gamma-glutamyltranspeptidase (gamma GT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels. It is noteworthy that very similar specific gamma GT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology. On the other hand, the specific gamma GT and G6PD activities of A. pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences.

Glutathione metabolism and dimorphism in Aureobasidium pullulans

Yeast⟷mycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors. In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions. In accordance with this hypothesis, the specific GSH levels found in A. pullulans yeast cells were always significantly higher than those in mycelia. On the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology. The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols. Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g. glutathione reductase (GR),γ-glutamyltranspeptidase (γGT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels. It is noteworthy that very similar specific γGT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology. On the other hand, the specific γGT and G6PD activities of A. pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences.

Glutathione metabolism and dimorphism in Aureobasidium pullulans.

Yeast<-->mycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors. In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions. In accordance with this hypothesis, the specific GSH levels found in A. pullulans yeast cells were always significantly higher than those in mycelia. One the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology. The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols. Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g. glutathione reductase (GR), gamma-glutamyltranspeptidase (gamma GT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels. It is noteworthy that very similar specific gamma GT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology. On the other hand, the specific gamma GT and G6PD activities of A. pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences.