Anticancer drugs as inducers of thermotolerance in yeast

Yeast cell viability was evaluated microscopically following exposure to heat shock for 30 min at 53°C. The cells were previously grown in the presence of potential stressors (anticancer drugs;e.g., 5-fluorouracil, methotrexate, cisplatin, bleomycin, mitomycin-C and camptothecin-11). The induction of thermotolerance was documented by significantly increased viability after heat shock. This effect, which was reversed by cycloheximide, was comparable to that observed following exposure to a mild heat stress. These data demonstrate that pretreatment with sub-toxic concentrations of some of the clinically used antineoplastic agents conferres thermotolerance to yeast, possibly through the synthesis of protein components.     

Reversible arrest of Chinese hamster V79 cells in G2 by dibutytyl AMP.

Mouse L cells 929 were cloned in supplemented Eagle's minimal medium enriched with lactalbumin and yeast extract and buffered with HEPES. Multiplication was followed photographically in single clones from the 8-cell stage through 6–7 days. Addition of the folic acid analogue methotrexate (amethopterin) in 5 × 10^?6 M concentration slowed growth only after two cell generations; 10^?4 M uridine had no effect on growth except when combined with methotrexate. The two agents together blocked cell division quickly and symptoms of thymine-less death developed in few days. The cells could be rescued before 48 h by removal of the inhibitors, or by addition of folic acid or thymidine. The combination of methotrexate with uridine blocks DNA synthesis in Tetrahymena by inhibition of thymidylate synthesis and of thymidine uptake from the complex medium. Apparently the same mechanisms operate in L cells grown in a complex medium containing thymidine.     

Proline enhances <Emphasis Type="Italic">Torulopsis glabrata</Emphasis> growth during hyperosmotic stress

This study investigated that the importing of compatible solute proline could enhance the growth of the yeast Torulopsis glabrata under hyperosmotic stress. Osmolarity progressively increased from 860 to 2,603 mOsmol/kg by accumulation of sodium pyruvate in the culture broth, leading to a significant decrease in cell growth. When 1.0 g/L of proline as a compatible solute was added to the culture medium, it was imported and enhanced cell growth by 59.0% at 2,603 mOsmol/kg. By addition of proline during pyruvate production, the concentration, productivity, and yield of pyruvate increased 22.1, 38.4, and 14.3%, respectively. These results suggested that T. glabrata can import proline as an osmoprotectant against high osmotic stress, thus enhance pyruvate productivity. The improvement of yeast growth and viability under hyperosmotic stress by the addition of proline provided an alternative approach to enhance the organic acids production by yeast.     

Ketoconazole resistance in Torulopsis glabrata

The majority of clinical isolates of T. glabrata has been shown highly sensitive to ketoconazole, when tested with an agar dilution method, and resistant, when using a broth dilution method. The fact was accounted for the high degree of mutation associated with the haploid state present in this species. Experimental T. glabrata infection in immunocompetent and immunocompromised mice appears not respond to the oral administration of the drug. The results agree with that of broth dilution tests and not with those produced by the agar methods. It seems that agar dilution sensitivity tests are inadequate for yeast species with an high rate of mutation, as T. glabrata.     

The Candida glabrata sterol scavenging mechanism,mediated by the ATP‐binding cassette transporter Aus1p,is regulated by iron limitation

During disseminated infection by the opportunistic pathogen Candida glabrata, uptake of sterols such as serum cholesterol may play a significant role during pathogenesis. The ATP‐binding cassette transporter Aus1p is thought to function as a sterol importer and in this study, we show that uptake of exogenous sterols occurred under anaerobic conditions in wild‐type cells of C. glabrata but not in AUS1‐deleted mutant (aus1Δ) cells. In aerobic cultures, growth inhibition by fluconazole was prevented in the presence of serum, and AUS1 expression was upregulated. Uptake of sterol by azole treated cells required the presence of serum, and sterol alone did not reverse FLC inhibition of growth. However, if iron availability in the growth medium was limited by addition of the iron chelators ferrozine or apo‐transferrin, growth of wild‐type cells, but not aus1Δ cells, was rescued. In a mouse model of disseminated infection, the C. glabrata aus1Δ strain caused a significantly decreased kidney fungal burden than the wild‐type strain or a strain in which AUS1 was restored. We conclude that sterol uptake in C. glabrata can occur in iron poor environment of host tissues and thus may contribute to C. glabrata pathogenesis.     

Two non-exclusive strategies employed to protect Torulopsis glabrata against hyperosmotic stress

Several recent reports described an apoptosis-like programmed cell death (PCD) process in yeast in response to different environmental challenges. In this study, hyperosmotic stress caused by high NaCl concentration in culture medium induced cell death in the haploid yeast Torulopsis glabrata. Propidium iodide (PI) and PI/rhodamine-123 (Rh123) dual staining with flow cytometry showed that high salinity decreased intact cells by 16.5 %, increased necrotic cells by nearly twofold, and altered fermentative parameters appreciably. Morphological and biochemical indicators of apoptosis were apparent, specifically a decrease in mitochondrial membrane potential (?Ψ_m), translocation of phosphatidylserine (PS) from the inner to the outer side of the plasma membrane, generation of reactive oxygen species (ROS), and involvement of caspase all while plasma membrane integrity was maintained. Additionally, it was found that overexpression of YCA1 drastically stimulated cell death, indicating that activation of metacaspase might lead to cell death. However, T. glabrata growth under hyperosmotic stress was enhanced when FIS1, HOG1, and GPD2 were overexpressed, or when exogenous proline or glutathione (GSH) were added into the cultures, both of which could repress caspase-3 activity. Thus, in these concrete cases of overexpression of anti-apoptotic or anti-necrotic factors and pharmacological manipulations, it decreased T. glabrata cell death that might help to achieve higher fermentative efficiency.     

Iron-depletion promotes mitophagy to maintain mitochondrial integrity in pathogenic yeast Candida glabrata

Candida glabrata, a haploid budding yeast, is the cause of severe systemic infections in immune-compromised hosts. The amount of free iron supplied to C. glabrata cells during systemic infections is severely limited by iron-chelating proteins such as transferrin. Thus, the iron-deficiency response in C. glabrata cells is thought to play important roles in their survival inside the host's body. In this study, we found that mitophagy was induced under iron-depleted conditions, and that the disruption of a gene homologous to ATG32, which is responsible for mitophagy in Saccharomyces cerevisiae, blocked mitophagy in C. glabrata. The mitophagic activity in C. glabrata cells was not detected on short-period exposure to nitrogen-starved conditions, which is a mitophagy-inducing condition used in S. cerevisiae. The mitophagy-deficient atg32Δ mutant of C. glabrata also exhibited decreased longevity under iron-deficient conditions. The mitochondrial membrane potential in Cgatg32Δ cells was significantly lower than that in wild-type cells under iron-depleted conditions. In a mouse model of disseminated infection, the Cgatg32Δ strain resulted in significantly decreased kidney and spleen fungal burdens compared with the wild-type strain. These results indicate that mitophagy in C. glabrata occurs in an iron-poor host tissue environment, and it may contribute to the longevity of cells, mitochondrial quality control, and pathogenesis.     

Induction of morphological alterations by antineoplastic agents in yeast

Saccharomyces cerevisiae was used as an alternative experimental model in order to investigate the effects of antineoplastic agents on eukaryotic cells. After being exposed to the most common clinically used antineoplastic agents, yeast cells were examined under the light microscope. Folate and pyrimidine antagonists, platinum derivatives, mitomycin C, actinomycin D and bleomycin induced alterations in yeast cellular morphology, which were not observed following treatment with drugs belonging to any category other than the antineoplastics, leading to the suggestion that these alterations could potentially be used as an experimental tool in pre-screening for new chemotherapeutic leads.     

Growth and macromolecular content of the dimorphic fungus Aureobasidium pullulans and the effect of hydroxyurea and other inhibitors

The growth kinetics and the macromolecular content of the yeast and ethanol-induced hyphal forms of Aureobasidium pullulans were studied. During the morphological transition from yeasts to hyphae, both the protein and RNA content decreased significantly, the mycelial form containing only 76% of the amount of protein in the yeasts, and 38% of the RNA. The DNA was the only component tested whose level increased during the transition. Among several compounds inhibiting macromolecular synthesis, only hydroxyurea showed a remarkable effect on the morphology of A. pullulans, inducing the mycelial morphology. The macromolecular composition of hydroxyurea-treated cultures changed with time in a way similar to that of the ethanol-Tween 80-ammonia medium, and to that of carbon-starved cultures, without ethanol or glucose.     

Redirection of the NADH oxidation pathway in Torulopsis glabrata leads to an enhanced pyruvate production

This study aimed at increasing the pyruvate productivity of a multi-vitamin auxotrophic yeast Torulopsis glabrata by redirecting NADH oxidation from adenosine triphosphate (ATP)-production pathway (oxidative phosphorylation pathway) to non-ATP production pathway (fermentative pathway). Two respiratory-deficient mutants, RD-17 and RD-18, were screened and selected after ethidium bromide (EtBr) mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, cytochrome aa ₃ and b in electron transfer chain (ETC) of RD-18 and cytochrome b in RD-17 were disrupted. As a consequence, the activities of key ETC enzymes of the mutant RD-18, including F₀F₁-ATP synthase, complex I, complex I + III, complex II + III, and complex IV, decreased by 22.2, 41.6, 53.1, 23.6, and 84.7%, respectively. With the deficiency of cytochromes in ETC, a large amount of excessive cytosolic NADH was accumulated, which hampered the further increase of the glycolytic flux. An exogenous electron acceptor, acetaldehyde, was added to the strain RD-18 culture to oxidize the excessive NADH. Compared with the parent strain, the concentration of pyruvate and the glucose consumption rate of strain RD-18 were increased by 26.5 and 17.6%, respectively, upon addition of 2.1 mM of acetaldehyde. The strategy for increasing the glycolytic flux in T. glabrata by redirecting the NADH oxidation pathway may provide an alternative approach to enhance the glycolytic flux in yeast.     

Pitfalls in the measurement of membrane potential in yeast cells using tetraphenylphosphonium

The uptake of the lipophilic cation tetraphenylphosphonium (Ph₄P⁺) by Saccharomyces cerevisiae was measured using yeast grown on glucose and harvested either at the logarithmic or at the stationary phase of growth. When yeast was collected at the stationary phase, Ph₄P⁺ uptake proceeded steadily during several hours until an equilibrium was reached. When yeast was collected in the logarithmic phase of growth, a biphasic uptake was observed. The second phase of uptake began when the glucose of the incubation medium had been exhausted. From experiments in the presence of cycloheximide or chloramphenicol it is concluded that the second phase of Ph₄P⁺ uptake is dependent on the synthesis of some protein(s) repressed by glucose but unrelated with the existence of functional mitochondria. The addition of compounds which collapse the membrane potential provokes an efflux from the yeast cells of the Ph₄P⁺ accumulated both during the first phase and the second phase of uptake. It is concluded that accumulation of Ph₄P⁺ in yeast cells is a complex process and that Ph₄P⁺ cannot be used to give a quantitative measure of the yeast plasma membrane potential.     

Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12

Summary The regulatory regions for the rpsU-dnaG-rpoD macromolecular synthesis operon have been fused to a structural gene whose product is readily assayed (the Cm^r structural gene coding for chloramphenicol acetyl transferase, CAT). The promoters (P₁, P₂, P₃, P_a, P_b, P_hs) for the macromolecular synthesis operon have different strengths as shown by their relative abilities to drive expression of the CAT gene. Promoter occlusion by P₁ can be demonstrated within this operon. Regions 5kb upstream have a profound effect on operon gene expression. There is a thermoinducible promoter located within the dnaG structural gene. One of the macromolecular synthesis operon promoters is under lexA control. Although the operon structure allows coordinate expression of rpsU, dnaG and rpoD these additional features suggest that expression of individual genes can be independently regulated in response to altered growth conditions.Abbreviations Ap^r ampicillin resistance - CAT chloramphenicol acetyl transferase - Cm^r chloramphenicol resistance - kb kilobase pair - orf open reading frame - P promoter - T terminator - Tc^r tetracycline resistance     

Selective Inhibition by Pantoyl Lactone and Butyl Alcohol of the Initiation of DNA Replication in E. coli

We studied action mechanisms of pantoyl lactone and butyl alcohol on the macromolecular synthesis of E. coli. Protein synthesis was not significantly suppressed by these agents. DNA synthesis was more remarkably affected than RNA synthesis by them. Synchronous cultures of E. coli were subsequently used to investigate the inhibition of DNA replication with these agents. It was consequently shown that these agents inhibit the initiation of a new cycle of DNA replication in this organism but permit the completion of DNA replication initiated before addition of these agents to the medium.     

Evaluation of the Commercial Rapid Trehalose Test (GLABRATA RTT) for the Point of Isolation Identification of <Emphasis Type="Italic">Candida glabrata</Emphasis> Isolates in Primary Cultures

Candidaemias account for 10–20% of nosocomial bloodstream infections depending on the study. Whilst Candida albicans remains the most frequently isolated species, Candida glabrata may be responsible for as many as 10–25% of all candidaemias. Moreover, C. glabrata is generally less susceptible to the azole antifungals than the majority of other pathogenic yeast species. Thus, a rapid test for the specific identification of isolates of C. glabrata would be useful for patient management if it could be performed at point of isolation, on primary cultures grown on standard mycological media directly from patient specimens. Under certain conditions, C. glabrata rapidly hydrolyses trehalose into glucose. The GLABRATA RTT kit allows detection of the preformed enzyme responsible for this action. This study has assessed GLABRATA RTT as an identification tool specifically at point of isolation. Sixty test isolates were evaluated: 39 clinical isolates of C. glabrata identified at the UK Mycology Reference Laboratory, examples of the recently described genetic relatives of C. glabrata, Candida nivariensis (n = 6) and Candida bracarensis (n = 1), and a selection of other common pathogenic yeast species (n = 14). The test provided results within 30 min. Although 77% (30/39) of confirmed C. glabrata isolates were correctly identified by GLABRATA RTT (positive trehalase test), 23% (9/39) of isolates gave negative or equivocal results. All other yeast species gave negative results. The performance of GLABRATA RTT in this study is compared to previous evaluations of the test which employed isolates pre-cultured on specialised media and to other existing conventional identification methodologies.     

An unexpected response of Torulopsis glabrata fusion products to x-irradiation

Intra-species fusion products of Saccharomyces cerevisiae, Saccharomyces unisporus and Torulopsis glabrata have been isolated following polyethylene glycol-induced fusion of protoplasts and selection for prototrophic colonies. Staining with lomofungin showed that all fusion products were uninucleate. Measurement of DNA content mostly gave values between haploid and diploid levels indicating that the majority of fusion products were aneuploid. Nevertheless fusion products of S. cerevisiae and S. unisporus were, as expected, more resistant to X-irradiation than their haploid parents. By contrast, the X-ray doze—response curve of all T. glabrata fusion products was indistinguishable from their progenitors despite the fact that mitotic segregants could be recovered amongst the survivors to X-rays. A possible explanation for the behaviour towards X-rays of T. glabrata fusion products is that this species lacks a DNA repair pathway involving recombination between homologous chromosomes. We conclude from this study that the shape of the X-ray dose—response curve should not be taken to indicate the ploidy of new yeast isolates without supporting data.     

An essential role for phosphatidylinositol 3‐kinase in the inhibition of phagosomal maturation,intracellular survival and virulence in Candida glabrata

The yeast class III phosphoinositide 3‐kinase (PI3K) that catalyses production of the lipid signalling molecule, phosphatidylinositol‐3‐phosphate, is primarily implicated in vesicle‐mediated transport and autophagy. In this study, we identified, through a genetic screen, the Candida glabrata CgVPS15 gene, an orthologue of the Saccharomyces cerevisiae PI3K regulatory subunit‐encoding open reading frame (ORF) to be required for impairment of phagosomal maturation in human macrophages. We also disrupted catalytic subunit of the C. glabrata PI3K complex, CgVps34, and found it to be pivotal to arrest mature phagolysosome biogenesis. Further, deletion of either CgVPS15 or CgVPS34 rendered C. glabrata cells hyperadherent to epithelial cells and susceptible to the antimicrobial arsenal of primary murine and cultured human macrophages and diverse stresses. Despite no growth retardation at 37°C, Cgvps15Δ and Cgvps34Δ mutants were severely virulence attenuated in mice. We demonstrate that trafficking and/or processing of the vacuolar lumenal hydrolase, carboxypeptidase Y, and the major adhesin, Epa1, rely on PI3K regulatory mechanisms in C. glabrata. By disrupting autophagy‐related PI3K complex genes, we show that C. glabrata PI3K‐impeded phagolysosomal acidification is primarily owing to its role in cellular trafficking events. Altogether, our findings underscore the essentiality of PI3K signalling in modulation of host immune response, intracellular survival and virulence in C. glabrata.     

Evolutionary divergence in the fungal response to fluconazole revealed by soft clustering

Background  

Fungal infections are an emerging health risk, especially those involving yeast that are resistant to antifungal agents. To understand the range of mechanisms by which yeasts can respond to anti-fungals, we compared gene expression patterns across three evolutionarily distant species - Saccharomyces cerevisiae, Candida glabrata and Kluyveromyces lactis - over time following fluconazole exposure.     

Pyruvate production in Candida glabrata: manipulation and optimization of physiological function

Candida glabrata, a multi-vitamin auxotrophic yeast, can accumulate a large amount of pyruvate extracellularly using glucose as the carbon source, a characteristic that has facilitated the cost-effective biotechnological production of pyruvate on an industrial scale. In this review, we describe the current advances in further improving the performance of C. glabrata for efficient pyruvate production, which includes: optimization of the vitamin and dissolved oxygen concentrations, regulation of intracellular cofactor levels and improvement of the environmental robustness of C. glabrata. We also discuss the current efforts using systems biology to understand the metabolism of C. glabrata. Finally, perspectives on engineering and exploiting C. glabrata as a cell factory for efficiently producing various chemicals and materials are discussed.