Yeast expression system
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BACKGROUND: Yeast infections are often connected with formation of biofilms that are extremely difficult to eradicate. An excellent model system for deciphering multifactorial determinants of yeast biofilm development is the colony biofilm, composed of surface ("aerial") and invasive ("root") cells. While surface cells have been partially analyzed before, we know little about invasive root cells. In particular, information on the metabolic, chemical and morphogenetic properties of invasive versus surface cells is lacking. In this study, we used a new strategy to isolate invasive cells from agar and extracellular matrix, and employed it to perform genome wide expression profiling and biochemical analyses of surface and invasive cells. RESULTS: RNA sequencing revealed expression differences in 1245 genes with high statistical significance, indicating large genetically regulated metabolic differences between surface and invasive cells. Functional annotation analyses implicated genes involved in stress defense, peroxisomal fatty acid β-oxidation, autophagy, protein degradation, storage compound metabolism and meiosis as being important in surface cells. In contrast, numerous genes with functions in nutrient transport and diverse synthetic metabolic reactions, including genes involved in ribosome biogenesis, biosynthesis and translation, were found to be important in invasive cells. Variation in gene expression correlated significantly with cell-type specific processes such as autophagy and storage compound accumulation as identified by microscopic and biochemical analyses. Expression profiling also provided indications of cell-specific regulations. Subsequent knockout strain analyses identified Gip2p, a regulatory subunit of type 1 protein phosphatase Glc7p, to be essential for glycogen accumulation in surface cells. CONCLUSIONS: This is the first study reporting genome wide differences between surface and invasive cells of yeast colony biofilms. New findings show that surface and invasive cells display very different physiology, adapting to different conditions in different colony areas and contributing to development and survival of the colony biofilm as a whole. Notably, surface and invasive cells of colony biofilms differ significantly from upper and lower cells of smooth colonies adapted to plentiful laboratory conditions.
V posledních desetiletích vzrostl význam kvasinek rodu Candida jako původců nozokomiálních infekcí. Jedním z významných faktorů virulence, které se na vzniku a rozvoji těchto infekcí podílejí, je tvorba biofilmu. Tento faktor virulence kvasinkám umožňuje kolonizovat nativní povrchy i umělé implantáty. Nejčastějším zdrojem infekce je sám pacient, zvi. jeho gastrointestinální trakt a kůže. Jako vektor exogenních kvasinkových infekcí se uplatňují především ruce personálu a kontaminované zdravotnické pomůcky. O adhezi kvasinek na povrch implantátu rozhodují vlastnosti povrchů implantátu i kvasinky. Následuje prohferace, tvorba mikrokolonií a produkce extracelulární matrix. Velký význam pro konečnou strukturu biofilmu má též tvorba hyf a pseudohyf. Celý proces tvorby biofilmu je řízen řadou regulačních systémů, mezi kterými hraje klíčovou úlohu systém quorum sensing. Stejně jako přisedlé bakteriální kultury, také kandidy rostoucí ve formě biofilmu jsou vysoce rezistentní k antimikrobiální terapii. Rezistence kvasinkových biofilmů k antimykotikům je komplexní proces, na kterém se podílí více faktorů. Jde především o zvýšenou expresi genů (např. genů kódujících tzv. multidrug efflux pumps), o omezenou penetraci látek přes extracelulární matrix, o zpomalený růst buněk a o změnu mikroprostředí v hlubších vrstvách biofilmu. Koncentrace antimykotik schopných efektivně zasáhnout buňky v biofilmu přesahují až o několik řádů, hodnoty konvenčně stanovených MIC. Z důvodů této vysoké rezistence biofilmu pak léčba biofilmových infekcí pomocí antimykotik často selhává. Proto by měl být, pokud je to možné, kolonizovaný implantát odstraněn. Při konzervativní terapii je pak potřeba volit antimykotika s prokázaným efektem na biofilm (např. caspofungin). Nejefektivnějším opatřením v boji proti biofilmovým infekcím je prevence, která se týká především dodržováním aseptických postupů při manipulaci s implantáty a jejich správné ošetřování.
In recent years, the role of Candida yeasts as causative agents of nosocomial infections has increased. One of the important virulence factors contributing to the development of such infections is biofilm production. This virulence factor enables yeast to colonize both native surfaces and artificial implants. The most common sources of infection are patients themselves, in particular the gastromtestinal tract and skin. The vectors of exogenous yeast infections are predominantly the hands of the health personnel and contaminated medical instruments. The adhesion of yeasts to the implant surfaces is determined both by implant surface and yeast characteristics. This is followed by proliferation and production of microcolonies and extracellular matrix. The final biofilm structure is also influenced by the production of hyphae and pseudohyphae. The entire process of biofilm production is controlled by numerous regulatory systems, with the key role being played by the quorum sensing system. Like the adhered bacterial cultures, Candidas growing in the form of a biofilm are highly resistant to antimicrobial therapy. Resistance of yeast biofilms to antifungals is a complex process with multiple contributing factors. These are especially increased gene expression (e.g. genes encoding the so called multidrug efflux pumps), limited penetration of substances through the extracellular matrix, inhibited cell growth and altered microenvironment in deeper biofilm layers. The concentrations of antifungals able to effectively affect the biofilm cells exceed, by several orders of magnitude, the values of conventionally determined MICs. High biofilm resistance results in ineffective antifungal therapy of biofilm infections. Therefore, if possible, the colonized implant should be removed. Conservative therapy should involve antifungals with a proven effect on the biofilm (e.g. caspofungin). The most effective measure in fighting biofilm infections is prevention, especially adhering to aseptic techniques when manipulating with implants and their correct maintenance.
- MeSH
- antifungální látky MeSH
- biofilmy růst a vývoj účinky léků MeSH
- buněčná adheze MeSH
- Candida patogenita MeSH
- finanční podpora výzkumu jako téma MeSH
- financování organizované MeSH
- fungální léková rezistence MeSH
- infekce spojené s protézou farmakoterapie mikrobiologie prevence a kontrola MeSH
- infekce spojené se zdravotní péčí farmakoterapie mikrobiologie MeSH
- kvasinky patogenita MeSH
Pediocin PA-1 is a bacteriocin that shows strongly anti-microbial activity against some Gram-positive pathogens such as Listeria monocytogenes, Staphylococcus aureus, and Enterococcus faecalis. With the broad inhibitory spectrum as well as high-temperature stability, pediocin has a potential application in the food preservation and pharmaceutical industry. Pediocin has been studied to express in many heterologous expression systems such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris as a free peptide. Here we showed in this study a new strategy by using yeast surface display system to produce the anchored pediocin PA-1 on the cell surface of Saccharomyces cerevisiae, which could be used directly as a pediocin resource. We had successfully constructed a recombinant S. cerevisiae W303 strain that could express pediocin PA-1 on the cell surface. The pediocin-expressing yeast could inhibit the growth of Shigella boydii and Shigella flexneri, which have never been reported before for pediocin activity. Besides, the pediocin expression level of the recombinant S. cerevisiae strain was also evaluated in three different media: synthetic defined (SD), basic medium (BM), and fermentation medium (FM). BM medium was shown to give the highest production yield of the recombinant yeast (4.75 ± 0.75 g dry cell weight per 1 L of culture) with the ratio number of the pediocin-expressing cells of 93.46 ± 2.45%. Taken together, the results clearly showed that pediocin can be displayed on yeast cell surface as anchored protein. The application of yeast cell surface system enables a new door of pediocin application on either food or feed industries. Graphical abstract.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacteria účinky léků MeSH
- bakteriociny MeSH
- fermentace MeSH
- genetické vektory MeSH
- mikrobiální testy citlivosti MeSH
- pediociny genetika metabolismus farmakologie MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Yeast biofilms are complex multicellular structures, in which the cells are well protected against drugs and other treatments and thus highly resistant to antifungal therapies. Colony biofilms represent an ideal system for studying molecular mechanisms and regulations involved in development and internal organization of biofilm structure as well as those that are involved in fungal domestication. We have identified here antagonistic functional interactions between transcriptional regulators Cyc8p and Tup1p that modulate the life-style of natural S. cerevisiae strains between biofilm and domesticated mode. Herein, strains with different levels of Cyc8p and Tup1p regulators were constructed, analyzed for processes involved in colony biofilm development and used in the identification of modes of regulation of Flo11p, a key adhesin in biofilm formation. Our data show that Tup1p and Cyc8p regulate biofilm formation in the opposite manner, being positive and negative regulators of colony complexity, cell-cell interaction and adhesion to surfaces. Notably, in-depth analysis of regulation of expression of Flo11p adhesin revealed that Cyc8p itself is the key repressor of FLO11 expression, whereas Tup1p counteracts Cyc8p's repressive function and, in addition, counters Flo11p degradation by an extracellular protease. Interestingly, the opposing actions of Tup1p and Cyc8p concern processes crucial to the biofilm mode of yeast multicellularity, whereas other multicellular processes such as cell flocculation are co-repressed by both regulators. This study provides insight into the mechanisms regulating complexity of the biofilm lifestyle of yeast grown on semisolid surfaces.
- MeSH
- biofilmy * MeSH
- buněčná adheze fyziologie MeSH
- jaderné proteiny genetika metabolismus MeSH
- membránové glykoproteiny genetika metabolismus MeSH
- mezibuněčná komunikace fyziologie MeSH
- regulace genové exprese u hub * MeSH
- represorové proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
During development of yeast colonies, various cell subpopulations form, which differ in their properties and specifically localize within the structure. Three branches of mitochondrial retrograde (RTG) signaling play a role in colony development and differentiation, each of them activating the production of specific markers in different cell types. Here, aiming to identify proteins and processes controlled by the RTG pathway, we analyzed proteomes of individual cell subpopulations from colonies of strains, mutated in genes of the RTG pathway. Resulting data, along with microscopic analyses revealed that the RTG pathway predominantly regulates processes in U cells, long-lived cells with unique properties, which are localized in upper colony regions. Rtg proteins therein activate processes leading to amino acid biosynthesis, including transport of metabolic intermediates between compartments, but also repress expression of mitochondrial ribosome components, thus possibly contributing to reduced mitochondrial translation in U cells. The results reveal the RTG pathway's role in activating metabolic processes, important in U cell adaptation to altered nutritional conditions. They also point to the important role of Rtg regulators in repressing mitochondrial activity in U cells.
- MeSH
- aminokyseliny metabolismus MeSH
- analýza jednotlivých buněk MeSH
- biosyntetické dráhy genetika MeSH
- chromatografie kapalinová MeSH
- intracelulární signální peptidy a proteiny genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- proteom genetika metabolismus MeSH
- proteomika MeSH
- regulace genové exprese u hub genetika MeSH
- represorové proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- signální transdukce genetika MeSH
- tandemová hmotnostní spektrometrie MeSH
- transkripční faktory BHLH-Zip genetika metabolismus MeSH
- Publikační typ
- časopisecké články 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
Transcription factors are prominent regulators of gene expression that execute responses to various intracellular and extracellular stimuli. Recombinant transcription reporter systems can be conveniently used to study the DNA binding preferences and regulatory activity of a transcription factor under a range of conditions. Several reporter genes have been used to study transcription regulation in the fission yeast Schizosaccharomyces pombe. Each of these reporters has distinct advantages, such as high sensitivity or ease of use, and limitations, such as prohibitive costs or use of hazardous substances. To combine the strengths and mitigate the weaknesses of individual reporter genes, we have created pREPORT, a flexible multi-readout transcription reporter vector for fission yeast that employs an enhanced GFP-lacZ fusion and a customizable minimal promoter. With pREPORT, gene expression driven by the transcription factor of interest can be quantified in a number of ways, both in live cells and in vitro, using a single reporter construct.
- MeSH
- beta-galaktosidasa analýza MeSH
- genetické vektory MeSH
- regulace genové exprese u hub * MeSH
- reportérové geny * MeSH
- Schizosaccharomyces pombe - proteiny genetika metabolismus MeSH
- Schizosaccharomyces genetika MeSH
- stanovení celkové genové exprese metody MeSH
- transkripční faktory genetika metabolismus MeSH
- zelené fluorescenční proteiny analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Expresní systém kvasinky Pichia pastoris se ve velkém měřítku používá na produkci rekombinantních proteinů již několik desetiletí. Pichia pastoris v sobě spojuje výhody eukaryotních i prokaryotních expresních systému. V rámci této práce byly v bioreaktoru produkovány mutantní formy proteinu cryptogeinu. Bylo vyprodukováno a purifikováno pět proteinů: X24, L41F, V84F, V84F/L41F a K13V s průměrnými výtěžky 40-60 mg proteinu na litr bazálního media. Buněčné hustoty na konci fermentace dosahovali 250-400 g/l.
Expression system Pichia pastoris has been used for decades to production of recombinant proteins in large scale. Pichia pastoris combines the advantages of eukaryotic and prokaryotic expression systems. In this work mutant forms of protein cryptogein were produced in the fermenter. Five proteins: X24, L41F, V84F, V84F/L41F and K13V were produced in the bioreactor and purified with average yields of 40-60 mg protein per liter of basal media. Cell density was reached 250-400 g/l at the end of fermentation.
- Klíčová slova
- růstová křivka, fermentor,
- MeSH
- bioreaktory MeSH
- fermentace * fyziologie genetika MeSH
- fungální proteiny * biosyntéza genetika MeSH
- kultivační média * MeSH
- mikrobiologické techniky MeSH
- Pichia genetika metabolismus růst a vývoj MeSH
- proteomika * metody MeSH
- rekombinantní proteiny * biosyntéza genetika MeSH
- Publikační typ
- práce podpořená grantem MeSH
Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions via changes in nuclear gene expression. The most comprehensive picture of components and regulation of retrograde signaling has been obtained in Saccharomyces cerevisiae, where retrograde-target gene expression is regulated by RTG genes. In this chapter, we describe methods to measure mitochondrial retrograde pathway activation at the level of mRNA and protein products in yeast model systems, including cell suspensions and microcolonies. In particular, we will focus on three major procedures: mRNA levels of RTG-target genes, such as those encoding for peroxisomal citrate synthase (CIT2), aconitase, and NAD+-specific isocitrate dehydrogenase subunit 1 by real-time PCR; expression analysis of CIT2-gene protein product (Cit2p-GFP) by Western blot and fluorescence microscopy; the phosphorylation status of transcriptional factor Rtg1/3p which controls RTG-target gene transcription.
- MeSH
- akonitáthydratasa genetika metabolismus MeSH
- buněčné jádro genetika metabolismus MeSH
- citrátsynthasa genetika metabolismus MeSH
- fosforylace MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- isocitrátdehydrogenasa genetika metabolismus MeSH
- mitochondrie metabolismus patologie MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- signální transdukce MeSH
- transkripční faktory BHLH-Zip metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cadmium has no known physiological function in the body; however, its adverse effects are associated with cancer and many types of organ system damage. Although much has been shown about Cd toxicity, the underlying mechanisms of its responses to the organism remain unclear. In this study, the role of Tor1, a catalytic subunit of the target of rapamycin complex 2 (TORC2), in Cd-mediated effects on cell proliferation, the antioxidant system, morphology, and ionome balance was investigated in the eukaryotic model organism Schizosaccharomyces pombe. Surprisingly, spectrophotometric and biochemical analyses revealed that the growth rate conditions and antioxidant defense mechanisms are considerably better in cells lacking the Tor1 signaling. The malondialdehyde (MDA) content of Tor1-deficient cells upon Cd treatment represents approximately half of the wild-type content. The microscopic determination of the cell morphological parameters indicates the role for Tor1 in cell shape maintenance. The ion content, determined by inductively coupled plasma optical emission spectroscopy (ICP-OES), showed that the Cd uptake potency was markedly lower in Tor1-depleted compared to wild-type cells. Conclusively, we show that the cadmium-mediated cell impairments in the fission yeast significantly depend on the Tor1 signaling. Additionally, the data presented here suggest the yet-undefined role of Tor1 in the transport of ions.
- MeSH
- homeostáza účinky léků MeSH
- ionty metabolismus MeSH
- kadmium aplikace a dávkování toxicita MeSH
- malondialdehyd metabolismus MeSH
- oxidační stres účinky léků MeSH
- proteinkinasy genetika metabolismus MeSH
- regulace genové exprese u hub MeSH
- Schizosaccharomyces pombe - proteiny genetika metabolismus MeSH
- Schizosaccharomyces cytologie účinky léků fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Publikační typ
- časopisecké články MeSH
