Systemic fungal diseases and antifungal resistance represent a serious problem in human medicine and con-tribute to increased patient mortality. The most common causes of these diseases are opportunistic yeasts of the genus Candida. C. albicansis considered to be the main pathogen, together with C. glabrata, C. tropicalis, C. par-apsilosis, and C.krusei. Azole antifungals predominate in the treatment of the systemic mycoses. For antifungal re-sistance in Candidaspp. some genes and their mutations are responsible, the genes ERG11, CDR1, CDR2and MDR1being considered the most important. The main target of azole antifungals is the process of ergosterol syn-thesis. Due to ergosterol crucial functions and its unique structural properties, the synthesis of ergosterol and its individual steps represent the target of most clinically available antifungals. The biofilm appears to be a signifi-cant virulence factor of the yeast Candidaspp. It allows hematogenous dissemination of cells, prevents the effect of antifungals on all cells during treatment and leads to a high level of antimicrobial resistance. The antifungal re-sistance in candidiasis often has a multifactorial origin, which must be considered in the treatment of systemic mycoses and in the development of new antifungals.
- MeSH
- antifungální látky chemie farmakologie terapeutické užití MeSH
- azoly farmakologie terapeutické užití MeSH
- biofilmy účinky léků MeSH
- Candida patogenita účinky léků MeSH
- ergosterol biosyntéza MeSH
- faktory virulence MeSH
- flukonazol farmakologie terapeutické užití MeSH
- fungální léková rezistence MeSH
- itrakonazol farmakologie terapeutické užití MeSH
- kandidóza * farmakoterapie mikrobiologie MeSH
- lidé MeSH
- vorikonazol farmakologie terapeutické užití MeSH
- Check Tag
- lidé MeSH
KlUpc2p, a transcription factor belonging to the fungal binuclear cluster family, is an important regulator of ergosterol biosynthesis and azole drug resistance in Kluyveromyces lactis. In this work, we show that the absence of KlUpc2p generates Rag- phenotype and modulates the K. lactis susceptibility to oxidants and calcofuor white. The KlUPC2 deletion leads to increased expression of KlMGA2 gene, encoding an important regulator of hypoxic and lipid biosynthetic genes in K. lactis and also KlHOG1 gene. The absence of KlUpc2p does not lead to statistically significant changes in glycerol, corroborating the expression of KlGPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase, that is similar in both the deletion mutant and the parental wild-type strain. Increased sensitivity of Klupc2 mutant cells to brefeldin A accompanied with significant increase in KlARF2 gene expression point to the involvement of KlUpc2p in intracellular signaling. Our observations highlight the connections between ergosterol and fatty acid metabolism to modulate membrane properties and point to the possible involvement of KlUpc2p in K. lactis oxidative stress response.
Candida albicans is an opportunistic pathogen accounting for the majority of cases of Candida infections. Currently, C. albicans are developing resistance towards different classes of antifungal drugs and this has become a global health burden that does not spare Lebanon. This study aims at determining point mutations in genes known to be involved in resistance acquisition and correlating resistance to virulence and ergosterol content in the azole resistant C. albicans isolate CA77 from Lebanon. This pilot study is the first of its kind to be implemented in Lebanon. We carried out whole genome sequencing of the azole resistant C. albicans isolate CA77 and examined 18 genes involved in antifungal resistance. To correlate genotype to phenotype, we evaluated the virulence potential of this isolate by injecting it into BALB/c mice and we quantified membrane ergosterol. Whole genome sequencing revealed that eight out of 18 genes involved in antifungal resistance were mutated in previously reported and novel residues. These genotypic changes were associated with an increase in ergosterol content but no discrepancy in virulence potential was observed between our isolate and the susceptible C. albicans control strain SC5314. This suggests that antifungal resistance and virulence potential in this antifungal resistant isolate are not correlated and that resistance is a result of an increase in membrane ergosterol content and the occurrence of point mutations in genes involved in the ergosterol biosynthesis pathway.
- MeSH
- azoly farmakologie MeSH
- bodová mutace MeSH
- Candida albicans chemie účinky léků genetika patogenita MeSH
- ergosterol analýza MeSH
- fenotyp MeSH
- fungální léková rezistence genetika MeSH
- genotyp MeSH
- lidé MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- pilotní projekty MeSH
- sekvenování celého genomu * MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Libanon MeSH
Naturally occurring antimicrobial peptides and their synthetic analogues are promising candidates for new antifungal drugs. We focused on three groups of peptides isolated from the venom of bees and their synthetic analogues (lasioglossins, halictines and hylanines), which all rapidly permeabilised the plasma membrane. We compared peptides' potency against six pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei and C. dubliniensis) and the non-pathogenic model yeast Saccharomyces cerevisiae. Their activity was independent of the presence of the multidrug-resistant pumps of C. glabrata but was influenced by the lipid composition of cell plasma membranes. Although the direct interaction of the peptides with ergosterol was negligible in comparison with amphotericin B, the diminished ergosterol content after terbinafine pretreatment resulted in an increased resistance of C. glabrata to the peptides. The tested peptides strongly interacted with phosphatidylglycerol, phosphatidic acid and cardiolipin and partly with phosphatidylinositol and phosphatidylethanolamine. The interactions between predominantly anionic phospholipids and cationic peptides indicated a mainly electrostatic binding of peptides to the membranes. The results obtained also pointed to a considerable role of the components of lipid rafts (composed from sphingolipids and ergosterol) in the interaction of yeast cells with the peptides.
- MeSH
- antifungální látky farmakologie MeSH
- buněčná membrána účinky léků MeSH
- Candida účinky léků MeSH
- ergosterol metabolismus MeSH
- fungální léková rezistence účinky léků MeSH
- hmyz metabolismus MeSH
- membránové lipidy metabolismus MeSH
- mikrobiální testy citlivosti metody MeSH
- peptidy farmakologie MeSH
- Saccharomyces cerevisiae účinky léků MeSH
- včelí jedy farmakologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Farnesol (FAR) has already demonstrated an inhibitory effect on Candida albicans biofilm. The aim of this work was to determine the effectiveness of externally added FAR in combination with fluconazole (FLC) on Candida albicans biofilm and on regulation of the ergosterol genes ERG20, ERG9, and ERG11. The effectiveness of compounds was determined by MTT assay and evaluated by the minimal inhibitory concentrations reducing a sessile biofilm to 50% activity (0.5 μg/mL and 200 μmol/L for FLC and FAR, respectively). These concentrations as well as 30 and 100 μmol/L FAR were selected for a study of the effectiveness of the FAR/FLC combination. The reduction in biofilm robustness mainly caused by the presence of 200 μmol/L FAR-alone or in combination with FLC-was accompanied by a significant inhibition of the yeast-to-hyphae transition that was observed by light microscopy and CLSM. Results from qRT-PCR indicated that while 30 μmol/L FAR only slightly regulated the expression of all 3 genes in the 48-h biofilm, the presence of 200 μmol/L FAR downregulated all the tested genes. However, the addition of 0.5 μg/mL of FLC to the samples with 200 μmol/L FAR restored the downregulation of the ERG20 and ERG11 genes to the control level. Moreover, the gene ERG9 was slightly upregulated. In summary, FAR acted via multiple effects on the C. albicans biofilm, but only a higher concentration of FAR proved to be effective.
- MeSH
- antifungální látky farmakologie MeSH
- biofilmy účinky léků růst a vývoj MeSH
- Candida albicans účinky léků růst a vývoj MeSH
- ergosterol genetika metabolismus MeSH
- farnesol farmakologie MeSH
- flukonazol farmakologie MeSH
- geny hub genetika MeSH
- hyfy účinky léků MeSH
- metabolické sítě a dráhy účinky léků MeSH
- mikrobiální testy citlivosti MeSH
- regulace genové exprese u hub účinky léků MeSH
- synergismus léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Publikační typ
- časopisecké články MeSH
The total yield of ergosterol produced by the fermentation of the yeast Saccharomyces cerevisiae depends on the final amount of yeast biomass and the ergosterol content in the cells. At the same time ergosterol purity-defined as percentage of ergosterol in the total sterols in the yeast-is equally important for efficient downstream processing. This study investigated the development of both the ergosterol content and ergosterol purity in different physiological (metabolic) states of the microorganism S. cerevisiae with the aim of reaching maximal ergosterol productivity. To expose the yeast culture to different physiological states during fermentation an on-line inference of the current physiological state of the culture was used. The results achieved made it possible to design a new production strategy, which consists of two preferable metabolic states, oxidative-fermentative growth on glucose followed by oxidative growth on glucose and ethanol simultaneously. Experimental application of this strategy achieved a value of the total efficiency of ergosterol production (defined as product of ergosterol yield coefficient and volumetric productivity), 103.84 × 10-6g L-1h-1, more than three times higher than with standard baker's yeast fed-batch cultivations, which attained in average 32.14 × 10-6g L-1h-1. At the same time the final content of ergosterol in dry biomass was 2.43%, with a purity 86%. These results make the product obtained by the proposed control strategy suitable for effective down-stream processing. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:838-848, 2017.
Comparison of the genomes of free-living Bodo saltans and those of parasitic trypanosomatids reveals that the transition from a free-living to a parasitic life style has resulted in the loss of approximately 50% of protein-coding genes. Despite this dramatic reduction in genome size, B. saltans and trypanosomatids still share a significant number of common metabolic traits: glycosomes; a unique set of the pyrimidine biosynthetic pathway genes; an ATP-PFK which is homologous to the bacterial PPi -PFKs rather than to the canonical eukaryotic ATP-PFKs; an alternative oxidase; three phosphoglycerate kinases and two GAPDH isoenzymes; a pyruvate kinase regulated by fructose-2,6-bisphosphate; trypanothione as a substitute for glutathione; synthesis of fatty acids via a unique set of elongase enzymes; and a mitochondrial acetate:succinate coenzyme A transferase. B. saltans has lost the capacity to synthesize ubiquinone. Among genes that are present in B. saltans and lost in all trypanosomatids are those involved in the degradation of mureine, tryptophan and lysine. Novel acquisitions of trypanosomatids are components of pentose sugar metabolism, pteridine reductase and bromodomain-factor proteins. In addition, only the subfamily Leishmaniinae has acquired a gene for catalase and the capacity to convert diaminopimelic acid to lysine.
- MeSH
- aminokyseliny metabolismus MeSH
- Bacteria genetika metabolismus MeSH
- dolichol metabolismus MeSH
- ergosterol biosyntéza MeSH
- Eukaryota genetika metabolismus MeSH
- fosfolipidy metabolismus MeSH
- glukoneogeneze MeSH
- glykolýza MeSH
- Kinetoplastida enzymologie genetika metabolismus MeSH
- koenzymy metabolismus MeSH
- kyselina listová metabolismus MeSH
- kyselina mevalonová metabolismus MeSH
- metabolismus lipidů MeSH
- metabolismus sacharidů MeSH
- mikrotělíska metabolismus MeSH
- mitochondrie enzymologie metabolismus MeSH
- močovina metabolismus MeSH
- oxidoreduktasy metabolismus MeSH
- pentózofosfátový cyklus MeSH
- peroxizomy metabolismus MeSH
- polyaminy metabolismus MeSH
- prenylace proteinů MeSH
- protozoální geny genetika MeSH
- protozoální proteiny genetika MeSH
- puriny biosyntéza metabolismus MeSH
- pyrimidiny biosyntéza metabolismus MeSH
- reaktivní formy kyslíku MeSH
- Trypanosomatina enzymologie genetika metabolismus MeSH
- ubichinon metabolismus MeSH
- vitaminy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
We investigated fungal growth and community composition in buried meshbags, amended with apatite, biotite or hornblende, in Norway spruce (Picea abies) forests of varying nutrient status. Norway spruce needles and soil collected from forests overlying serpentinite had low levels of potassium and phosphorus, those from granite had low levels of magnesium, whereas those from amphibolite had comparably high levels of these nutrients. We assayed the fungal colonization of meshbags by measuring ergosterol content and fungal community with 454 sequencing of the internal transcribed spacer region. In addition, we measured fine root density. Fungal biomass was increased by apatite amendment across all plots and particularly on the K- and P-deficient serpentinite plots, whereas hornblende and biotite had no effect on fungal biomass on any plots. Fungal community (total fungal and ectomycorrhizal) composition was affected strongly by sampling location and soil depth, whereas mineral amendments had no effect on community composition. Fine root biomass was significantly correlated with fungal biomass. Ectomycorrhizal communities may respond to increased host-tree phosphorus demand by increased colonization of phosphorus-containing minerals, but this does not appear to translate to a shift in ectomycorrhizal community composition. This growth response to nutrient demand does not appear to exist for potassium or magnesium limitation.
- MeSH
- biomasa MeSH
- draslík farmakologie MeSH
- ergosterol metabolismus MeSH
- fosfor farmakologie MeSH
- hořčík farmakologie MeSH
- houby účinky léků růst a vývoj MeSH
- lesy * MeSH
- listy rostlin chemie účinky léků MeSH
- minerály metabolismus MeSH
- multivariační analýza MeSH
- půdní mikrobiologie * MeSH
- smrk účinky léků fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- cholekalciferol biosyntéza metabolismus MeSH
- dehydrocholesteroly metabolismus účinky záření MeSH
- endokrinní disruptory * metabolismus MeSH
- ergokalciferoly chemická syntéza metabolismus MeSH
- ergosterol biosyntéza metabolismus účinky záření MeSH
- Hominidae metabolismus MeSH
- kalcifediol metabolismus účinky záření MeSH
- kalcitriol metabolismus účinky záření MeSH
- kůže * účinky záření MeSH
- lidé MeSH
- nedostatek vitaminu D MeSH
- rostliny metabolismus MeSH
- ryby metabolismus MeSH
- savci metabolismus MeSH
- sinice MeSH
- statistika jako téma MeSH
- vitamin D * metabolismus terapeutické užití MeSH
- Check Tag
- lidé MeSH
We investigated the impact of the deletions of genes from the final steps in the biosynthesis of ergosterol (ERG6, ERG2, ERG3, ERG5, ERG4) on the physiological function of the Saccharomyces cerevisiae plasma membrane by a combination of biological tests and the diS-C3(3) fluorescence assay. Most of the erg mutants were more sensitive than the wild type to salt stress or cationic drugs, their susceptibilities were proportional to the hyperpolarization of their plasma membranes. The different sterol composition of the plasma membrane played an important role in the short-term and long-term processes that accompanied the exposure of erg strains to a hyperosmotic stress (effect on cell size, pH homeostasis and survival of yeasts), as well as in the resistance of cells to antifungal drugs. The pleiotropic drug-sensitive phenotypes of erg strains were, to a large extent, a result of the reduced efficiency of the Pdr5 efflux pump, which was shown to be more sensitive to the sterol content of the plasma membrane than Snq2p. In summary, the erg4Δ and erg6Δ mutants exhibited the most compromised phenotypes. As Erg6p is not involved in the cholesterol biosynthetic pathway, it may become a target for a new generation of antifungal drugs.
- MeSH
- ABC transportéry genetika metabolismus MeSH
- antifungální látky farmakologie MeSH
- biosyntetické dráhy genetika MeSH
- buněčná membrána chemie fyziologie MeSH
- ergosterol biosyntéza chemie MeSH
- flukonazol farmakologie MeSH
- fluorescenční mikroskopie MeSH
- koncentrace vodíkových iontů MeSH
- membránové potenciály fyziologie MeSH
- methyltransferasy genetika metabolismus MeSH
- mnohočetná fungální léková rezistence účinky léků genetika fyziologie MeSH
- molekulární struktura MeSH
- mutace MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae chemie genetika fyziologie MeSH
- tolerance k soli genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
