Biolistischen Transformation einer Leuchtstoff Tagged-Gens in den Opportunistic Pilzpathogenentwicklung<em> Cryptococcus neoformans</em
Summary
Biolistische Transformation ein Verfahren verwendet werden, um eine stabile Integration der DNA in das Genom der opportunistisches Pathogen Cryptococcus neoformans durch homologe Rekombination zu erzeugen. Wir biolistische Transformation eines Konstrukts, das den kodierenden Gens Acetatkinase zur Fluoreszenzmarkierung mCherry in C. neoformans fusioniert hat demonstrieren.
Abstract
Die Basidiomyceten Cryptococcus neoformans, eine invasive opportunistische Erreger des zentralen Nervensystems, ist die häufigste Ursache für Pilzmeningitis weltweiten was zu mehr als 625.000 Todesfälle pro Jahr weltweit. Obwohl Elektroporation zur Transformation von Plasmiden in Cryptococcus entwickelt, liefert nur biolistischen Anlieferung ein wirksames Mittel, um eine lineare DNA, die in das Genom durch homologe Rekombination integriert werden kann, zu transformieren.
Acetat wurde gezeigt, ein Hauptfermentationsprodukt während cryptococcal Infektion sein, aber die Bedeutung dieser Tatsache wird noch nicht bekannt. Eine bakterielle Wegs der Enzyme Xylulose-5-Phosphat / Fructose-6-phosphat Phosphoketolase (XFP) und Acetatkinase (Ack) zusammengesetzt ist, eines von drei möglichen Pfade für Acetatproduktion in C. neoformans. Hier zeigen wir die biolistische Transformation eines Konstrukts,was hat die Ack kodierende Gen in die fluoreszierende Markierung verschmolzen mCherry, in C. neoformans. Wir bestätigen dann die Integration des ACK -mCherry Fusion in die ACK-Locus.
Introduction
Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis resulting in more than 625,000 deaths per year worldwide 1. Acetate has been shown to be a major fermentation product during cryptococcal infection 2,3,4, and genes encoding enzymes from three putative acetate-producing pathways have been shown to be upregulated during infection 5. This suggests that acetate production and transport may be a necessary and required part of the pathogenic process; however, the significance of this is not yet understood. One possible pathway for acetate production is the xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) – acetate kinase (Ack), a pathway previously thought to be present only in bacteria but recently identified in both euascomycete as well as basidiomycete fungi, including C. neoformans 6.
To determine the localization of these enzymes of this pathway in the cell, a construct carrying a neomycin resistance gene downstream of an ACK gene fusion to the fluorescent tag mCherry (ACK:mCherry:Neo) will be introduced into C. neoformans using the well-established method of biolistic transformation 7,8. Although electroporation is an efficient method for transformation of plasmids that will be maintained as episomes into Cryptococcus 9, it is not useful in creating stable homologous transformants 8. Only biolistic delivery using a gene gun provides an effective means to transform linear DNAs that will be integrated into the genome by homologous recombination. For example, Edman et al. showed that of the transformants resulting from electroporation of a plasmid-borne URA5 selectable marker into C. neoformansura5 mutants, just 0.001 to 0.1% of transformants were stable 9. Chang et al. achieved just a 0.25% stable transformation efficiency using electroporation to reconstitute capsule production in an acapsular mutant 10. Unlike electroporation, biolistic transformation has been shown to result in stable transformation efficiency of 2-50% depending on the gene that is being altered 7,8,11.
This visual experiment will provide a step-by-step demonstration of biolistic transformation of the linear ACK:mCherry:Neo DNA construct into C. neoformans, and will describe how to confirm its proper integration via homologous recombination into the ack locus. The protocol demonstrated here is a modification of the method developed in the Perfect laboratory 8.
Protocol
Representative Results
Discussion
Utilizing this protocol, biolistic transformation can be accomplished in which linear DNA is integrated into a desired locus in the Cryptococcus neoformans genome by homologous recombination. Certain steps in the protocol can have a dramatic effect on the effectiveness/efficiency of the transformation. For a successful transformation, it is imperative that the DNA utilized in the shoot has a concentration of at least 1 µg. However, the volume of DNA added to the gold beads can be increased in the chance the…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Diese Arbeit wurde durch Auszeichnungen von der National Science Foundation (Preis # 0.920.274) und dem South Carolina Experiment Station Projekt SC-1.700.340 unterstützt. Dieses Papier isTechnical Beitrag Nr 6283 der Clemson University Experiment Station. Die Autoren danken Dr. Lukasz Kozubowski für seine hilfreiche Ratschläge bei der Entwicklung dieser letzten Protokoll und Dr. Cheryl Ingram-Smith, Katie Glenn, und Grace Kisirkoi für die kritische Durchsicht des Manuskripts.
Materials
| Product | Company | Catalog # | Website |
| 0.6 μm gold beads | Bio-Rad | 165-2262 | http://www.bio-rad.com |
| Spermadine-free base | Sigma- Aldrich | S0266 | https://www.sigmaaldrich.com |
| G418 – Sulfate (Neomycin) | Gold Biotechnology | G-418-10 | www.goldbio.com |
| Hygromycin | Gold Biotechnology | H-270-1 | www.goldbio.com |
| 1350 psi Rupture Discs | Bio-Rad | 165-2330 | http://www.bio-rad.com |
| Stopping Screens | Bio-Rad | 165-2336 | http://www.bio-rad.com |
| Macrocarriers discs | Bio-Rad | 165-2335 | http://www.bio-rad.com |
| YPD Broth | Becton Dickinson & Co. | 242820 | www.bd.com |
| Agar | Becton Dickinson & Co. | 214530 | www.bd.com |
| Sorbitol | Fisher Scientific | BP439 | http://www.fishersci.com |
| PDS-1000/He System | Bio-Rad | 165-2257 | http://www.bio-rad.com |
| Microscope | Zeiss | Axio | http://www.zeiss.com/microscopy |
| KOD One Step PCR Kit | EMD Millipore | 71086-4 | http://www.emdmillipore.com |
| One Step RT-PCR Kit | Qiagen | 210212 | www.qiagen.com |
| Wizard Genomic DNA Purification Kit | Promega | A1120 | www.promega.com |
| RNeasy Mini Kit | Qiagen | 74104 | www.qiagen.com |
| Mini Beadbeater – 1 | BioSpecs | 3110BX | http://www.biospec.com |
| Microfuge 18 Centrifuge | Beckman Coulter | F241.5P | www.beckmancoulter.com |
| Microplate Spectrophotometer | BioTek | EPOCH | www.biotek.com |
References
- Price, M. S., et al. Cryptococcusneoformans requires a functional glycolytic pathway for disease but not persistence in the host. MBio. 2, e00103-e00111 (2011).
- Bubb, W. A., et al. Heteronuclear NMR studies of metabolites produced by Cryptococcusneoformans in culture media: identification of possible virulence factors. Magn Reson Med. 42, 442-453 (1999).
- Himmelreich, U., et al. Identification of metabolites of importance in the pathogenesis of pulmonary cryptococcoma using nuclear magnetic resonance spectroscopy. Microbes Infect. 5, 285-290 (2003).
- Wright, L., et al. Metabolites released by Cryptococcusneoformans var. neoformans and var. gattii differentially affect human neutrophil function. Microbes Infect. 4, 1427-1438 (2002).
- Hu, G., Cheng, P. Y., Sham, A., Perfect, J. R., Kronstad, J. W. Metabolic adaptation in Cryptococcusneoformans during early murine pulmonary infection. Mol Microbiol. 69, 1456-1475 (2008).
- Ingram-Smith, C., Martin, S. R., Smith, K. S. Acetate kinase: not just a bacterial enzyme. Trends Microbiol. 14, 249-253 (2006).
- Davidson, R. C., et al. Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genet Biol: FG & B. 29, 38-48 (2000).
- Toffaletti, D. L., Rude, T. H., Johnston, S. A., Durack, D. T., Perfect, J. R. Gene transfer in Cryptococcusneoformans by use of biolistic delivery of DNA. J. Bacteriol. 175, 1405-1411 (1993).
- Edman, J. C., Kwon-Chung, K. J. Isolation of the URA5 gene from Cryptococcusneoformans var. neoformans and its use as a selective marker for transformation. Mol Cell Biol. 10, 4538-4544 (1990).
- Chang, Y. C., Kwon-Chung, K. J. Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol. 14, 4912-4919 (1994).
- Del Poeta, M., et al. Topoisomerase I is essential in Cryptococcusneoformans: role In pathobiology and as an antifungal target. 유전학. 152, 167-178 (1999).
- McClelland, C. M., Chang, Y. C., Kwon-Chung, K. J. High frequency transformation of Cryptococcusneoformans and Cryptococcusgattii by Agrobacteriumtumefaciens. Fungal Genet Biol:FG & B. 42, 904-913 (2005).
- Gan, W. B., Grutzendler, J., Wong, W. T., Wong, R. O., Lichtman, J. W. Multicolor ‘DiOlistic’ labeling of the nervous system using lipophilic dye combinations. Neuron. 27, 219-225 (2000).
- Nicola, A. M., Frases, S., Casadevall, A. Lipophilic dye staining of Cryptococcusneoformans extracellular vesicles and capsule. Eukaryot Cell. 8, 1373-1380 (2009).
