Telomerase RNA (TR) carries the template for synthesis of telomere DNA and provides a scaffold for telomerase assembly. Fungal TRs are long and have been compared to higher eukaryotes, where they show considerable diversity within phylogenetically close groups. TRs of several Saccharomycetaceae were recently identified, however, many of these remained uncharacterised in the template region. Here we show that this is mainly due to high variability in telomere sequence. We predicted the telomere sequences using Tandem Repeats Finder and then we identified corresponding putative template regions in TR candidates. Remarkably long telomere units and the corresponding putative TRs were found in Tetrapisispora species. Notably, variable lengths of the annealing sequence of the template region (1-10 nt) were found. Consequently, species with the same telomere sequence may not harbour identical TR templates. Thus, TR sequence alone can be used to predict a template region and telomere sequence, but not to determine these exactly. A conserved feature of telomere sequences, tracts of adjacent Gs, led us to test the propensity of individual telomere sequences to form G4. The results show highly diverse values of G4-propensity, indicating the lack of ubiquitous conservation of this feature across Saccharomycetaceae.
- MeSH
- benzothiazoly metabolismus MeSH
- fluorescence MeSH
- G-kvadruplexy MeSH
- genetická variace * MeSH
- genetické matrice * MeSH
- reprodukovatelnost výsledků MeSH
- RNA genetika MeSH
- Saccharomycetales genetika MeSH
- sekvence nukleotidů MeSH
- telomerasa genetika MeSH
- telomery genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Citrus black spot (CBS) and post-bloom fruit drop (PFD), caused by Phyllosticta citricarpa and Colletotrichum abscissum, respectively, are two important citrus diseases worldwide. CBS depreciates the market value and prevents exportation of citrus fruits to Europe. PFD under favorable climatic conditions can cause the abscission of flowers, thereby reducing citrus production by 80%. An ecofriendly alternative to control plant diseases is the use of endophytic microorganisms, or secondary metabolites produced by them. Strain LGMF1631, close related to Diaporthe cf. heveae 1, was isolated from the medicinal plant Stryphnodendron adstringens and showed significant antimicrobial activity, in a previous study. In view of the potential presented by strain LGMF1631, and the absence of chemical data for secondary metabolites produced by D. cf. heveae, we decided to characterize the compounds produced by strain LGMF1631. Based on ITS, TEF1, and TUB phylogenetic analysis, strain LGMF1631 was confirmed to belong to D. cf. heveae 1. Chemical assessment of the fungal strain LGMF1631 revealed one new seco-dihydroisocoumarin [cladosporin B (1)] along with six other related, already known dihydroisocoumarin derivatives and one monoterpene [(-)-(1S,2R,3S,4R)-p-menthane-1,2,3-triol (8)]. Among the isolated metabolites, compound 5 drastically reduced the growth of both phytopathogens in vitro and completely inhibited the development of CBS and PFD in citrus fruits and flowers. In addition, compound 5 did not show toxicity against human cancer cell lines or citrus leaves, at concentrations higher than used for the inhibition of the phytopathogens, suggesting the potential use of (-)-(3R,4R)-cis-4-hydroxy-5-methylmellein (5) to control citrus diseases.
- MeSH
- Ascomycota účinky léků fyziologie MeSH
- Citrus mikrobiologie MeSH
- Colletotrichum účinky léků fyziologie MeSH
- Fabaceae mikrobiologie MeSH
- fungicidy průmyslové chemie metabolismus farmakologie MeSH
- fylogeneze MeSH
- isokumariny chemie metabolismus farmakologie MeSH
- listy rostlin mikrobiologie MeSH
- nemoci rostlin mikrobiologie MeSH
- ovoce mikrobiologie MeSH
- Saccharomycetales chemie klasifikace genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
Schistosomiasis caused by parasitic blood flukes of the genus Schistosoma is a global health problem with over 200 million people infected. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease critical for digestion of host blood proteins as a source of nutrients. SmCB1 is a validated drug target, and inhibitors of SmCB1 represent promising anti-schistosomals. A comprehensive structural and functional characterization of SmCB1 provides a starting point for the rational design of selective and potent SmCB1 inhibitors. Here, we report optimized protocols for (1) the production of recombinant SmCB1 in the Pichia pastoris expression system and its purification, (2) the measurement of SmCB1 activity and inhibition in a kinetic fluorescence assay, and (3) the preparation and crystallization of SmCB1 in complex with a model vinyl sulfone inhibitor, and the determination of its crystal structure.
- MeSH
- aktivace enzymů MeSH
- elektroporace MeSH
- exprese genu MeSH
- genetické vektory metabolismus MeSH
- glykosylace MeSH
- kathepsin B antagonisté a inhibitory chemie izolace a purifikace metabolismus MeSH
- kinetika MeSH
- krystalizace MeSH
- mutace genetika MeSH
- rekombinantní proteiny izolace a purifikace metabolismus MeSH
- Saccharomycetales genetika MeSH
- Schistosoma mansoni enzymologie MeSH
- transformace genetická MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The yeast Magnusiomyces capitatus is an opportunistic human pathogen causing rare yet severe infections, especially in patients with hematological malignancies. Here, we report the 20.2 megabase genome sequence of an environmental strain of this species as well as the genome sequences of eight additional isolates from human and animal sources providing an insight into intraspecies variation. The distribution of single-nucleotide variants is indicative of genetic recombination events, supporting evidence for sexual reproduction in this heterothallic yeast. Using RNAseq-aided annotation, we identified genes for 6518 proteins including several expanded families such as kexin proteases and Hsp70 molecular chaperones. Several of these families are potentially associated with the ability of M. capitatus to infect and colonize humans. For the purpose of comparative analysis, we also determined the genome sequence of a closely related yeast, Magnusiomyces ingens. The genome sequences of M. capitatus and M. ingens exhibit many distinct features and represent a basis for further comparative and functional studies.
- MeSH
- anotace sekvence MeSH
- antifungální látky farmakologie MeSH
- faktory virulence MeSH
- fenotyp MeSH
- fylogeneze MeSH
- genom fungální * MeSH
- genomika * metody MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- multigenová rodina MeSH
- mykózy mikrobiologie MeSH
- oportunní infekce mikrobiologie MeSH
- rekombinace genetická MeSH
- Saccharomycetales klasifikace genetika růst a vývoj patogenita MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
There are only a few antifungal drugs used systemically in treatment, and invasive fungal infections that are resistant to these drugs are an emerging problem in health care. In this study, we performed a high-copy-number genomic DNA (gDNA) library screening to find and characterize genes that reduce susceptibility to amphotericin B, caspofungin, and voriconazole in Saccharomyces cerevisiae We identified the PDR16 and PMP3 genes for amphotericin B, the RMD9 and SWH1 genes for caspofungin, and the MRS3 and TRI1 genes for voriconazole. The deletion mutants for PDR16 and PMP3 were drug susceptible, but the other mutants had no apparent susceptibility. Quantitative-PCR analyses suggested that the corresponding drugs upregulated expression of the PDR16, PMP3, SWH1, and MRS3 genes. To further characterize these genes, we also profiled the global expression patterns of the cells after treatment with the antifungals and determined the genes and paths that were up- or downregulated. We also cloned Candida albicans homologs of the PDR16, PMP3, MRS3, and TRI1 genes and expressed them in S. cerevisiae Heterologous expression of Candida homologs also provided reduced drug susceptibility to the budding yeast cells. Our analyses suggest the involvement of new genes in antifungal drug resistance.
- MeSH
- amfotericin B farmakologie MeSH
- antifungální látky farmakologie MeSH
- Candida albicans účinky léků genetika metabolismus MeSH
- fungální léková rezistence genetika MeSH
- kaspofungin farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae účinky léků genetika metabolismus MeSH
- Saccharomycetales účinky léků genetika metabolismus MeSH
- vorikonazol farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Candida krusei is a pathogenic yeast species that is phylogenetically outside both of the well-studied yeast groups, whole genome duplication and CUG. Like all other yeast species, it needs to accumulate high amounts of potassium cations, which are needed for proliferation and many other cell functions. A search in the sequenced genomes of nine C. krusei strains revealed the existence of two highly conserved genes encoding putative potassium uptake systems. Both of them belong to the TRK family, whose members have been found in all the sequenced genomes of species from the Saccharomycetales subclade. Analysis and comparison of the two C. krusei Trk sequences revealed all the typical features of yeast Trk proteins but also an unusual extension of the CkTrk2 hydrophilic N-terminus. The expression of both putative CkTRK genes in Saccharomyces cerevisiae lacking its own potassium importers showed that only CkTrk1 is able to complement the absence of S. cerevisiae's own transporters and provide cells with a sufficient amount of potassium. Interestingly, a portion of the CkTrk1 molecules were localized to the vacuolar membrane. The presence of CkTrk2 had no evident phenotype, due to the fact that this protein was not correctly targeted to the S. cerevisiae plasma membrane. Thus, CkTrk2 is the first studied yeast Trk protein to date that was not properly recognized and targeted to the plasma membrane upon heterologous expression in S. cerevisiae.
- MeSH
- Candida klasifikace genetika růst a vývoj metabolismus MeSH
- draslík metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- fylogeneze MeSH
- genetická variace MeSH
- genom fungální genetika MeSH
- iontový transport MeSH
- proteiny přenášející kationty genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae klasifikace genetika růst a vývoj metabolismus MeSH
- Saccharomycetales klasifikace genetika MeSH
- testy genetické komplementace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Saprochaete and Geotrichum spp. are rare emerging fungi causing invasive fungal diseases in immunosuppressed patients and scarce evidence is available for treatment decisions. Among 505 cases of rare IFD from the FungiScope(™) registry, we identified 23 cases of invasive infections caused by these fungi reported from 10 countries over a 12-year period. All cases were adults and previous chemotherapy with associated neutropenia was the most common co-morbidity. Fungaemia was confirmed in 14 (61%) cases and deep organ involvement included lungs, liver, spleen, central nervous system and kidneys. Fungi were S. capitata (n=14), S. clavata (n=5), G. candidum (n=2) and Geotrichum spp. (n=2). Susceptibility was tested in 16 (70%) isolates. All S. capitata and S. clavata isolates with the exception of one S. capitata (MIC 4 mg/L) isolate had MICs>32 mg/L for caspofungin. For micafungin and anidulafungin, MICs varied between 0.25 and >32 mg/L. One case was diagnosed postmortem, 22 patients received targeted treatment, with voriconazole as the most frequent first line drug. Overall mortality was 65% (n=15). Initial echinocandin treatment was associated with worse outcome at day 30 when compared to treatment with other antifungals (amphotericin B ± flucytosine, voriconazole, fluconazole and itraconazole) (P=.036). Echinocandins are not an option for these infections.
- MeSH
- amfotericin B farmakologie terapeutické užití MeSH
- antifungální látky farmakologie terapeutické užití MeSH
- dospělí MeSH
- echinokandiny farmakologie terapeutické užití MeSH
- flukonazol farmakologie terapeutické užití MeSH
- fungemie diagnóza farmakoterapie mikrobiologie MeSH
- geotrichóza farmakoterapie mikrobiologie mortalita MeSH
- Geotrichum klasifikace účinky léků genetika izolace a purifikace MeSH
- hostitel s imunodeficiencí MeSH
- invazivní mykotické infekce farmakoterapie mikrobiologie mortalita MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipopeptidy farmakologie terapeutické užití MeSH
- mikrobiální testy citlivosti MeSH
- mladiství MeSH
- mladý dospělý MeSH
- neutropenie komplikace farmakoterapie mikrobiologie MeSH
- registrace * MeSH
- Saccharomycetales klasifikace účinky léků genetika izolace a purifikace MeSH
- senioři MeSH
- vorikonazol farmakologie terapeutické užití MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
The budding yeast (Saccharomyces cerevisiae) can serve as a unique experimental system for functional studies of heterologous genes, allowing not only complementation of readily available yeast mutations but also generation of overexpression phenotypes and in some cases also rescue of such phenotypes. Here we summarize the main considerations that have to be taken into account when using the yeast expression system for investigating the function of plant genes participating in cell morphogenesis; outline the strategies of experiment planning, yeast strain selection (or construction), and expression vector choice; and provide detailed protocols for yeast transformation, transformant selection, and phenotype evaluation.
- MeSH
- exprese genu MeSH
- fenotyp MeSH
- genová knihovna MeSH
- plazmidy genetika MeSH
- rostlinné buňky metabolismus MeSH
- rostlinné geny * MeSH
- Saccharomycetales genetika MeSH
- testy genetické komplementace MeSH
- transformace genetická MeSH
- vývoj rostlin fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
