अवसरवादी फफूंद पैथोजन में एक फ्लोरोसेंट टैग की गईं जीन की biolistic परिवर्तन<em> क्रिप्टोकोकस neoformans</em
Summary
Biolistic परिवर्तन मुताबिक़ पुनर्संयोजन के माध्यम से अवसरवादी रोगज़नक़ क्रिप्टोकोकस neoformans के जीनोम में डीएनए के स्थिर एकीकरण उत्पन्न करने के लिए प्रयोग किया जाता है एक विधि है। हम सी neoformans में फ्लोरोसेंट टैग mCherry के लिए जुड़े जीन एन्कोडिंग एसीटेट काइनेज है, जो एक निर्माण, के biolistic परिवर्तन प्रदर्शन करेंगे।
Abstract
basidiomycete क्रिप्टोकोकस neoformans, केंद्रीय तंत्रिका तंत्र के लिए एक आक्रामक अवसरवादी रोगज़नक़, दुनिया भर में दुनिया भर में प्रति वर्ष से अधिक 625.000 मौतों का कारण फंगल दिमागी बुखार के सबसे लगातार कारण है। Electroporation के क्रिप्टोकोकस में plasmids के परिवर्तन के लिए विकसित किया गया है, केवल biolistic डिलीवरी मुताबिक़ पुनर्संयोजन द्वारा जीनोम में एकीकृत किया जा सकता है कि रैखिक डीएनए को बदलने के लिए एक प्रभावी साधन प्रदान करता है।
एसीटेट क्रिप्टोकोकल संक्रमण के दौरान एक प्रमुख किण्वन उत्पाद होना दिखाया गया है, लेकिन इस बात का महत्व अभी तक ज्ञात नहीं है। एंजाइमों xylulose-5-फॉस्फेट / फ्रुक्टोज-6-फॉस्फेट phosphoketolase (Xfp) और एसीटेट काइनेज (ACK) से बना एक बैक्टीरियल मार्ग सी में एसीटेट उत्पादन के लिए तीन संभावित रास्ते में से एक है neoformans। यहाँ, हम एक निर्माण के biolistic परिवर्तन का प्रदर्शन,Ack एन्कोडिंग जीन, mCherry फ्लोरोसेंट टैग के लिए जुड़े हुए सी में है जो neoformans। हम तो एसीके ठिकाना में एसीके -mCherry संलयन के एकीकरण की पुष्टि करें।
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
यह काम राष्ट्रीय विज्ञान फाउंडेशन (पुरस्कार # 0,920,274) और दक्षिण कैरोलिना प्रयोग स्टेशन परियोजना अनुसूचित जाति-1,700,340 से पुरस्कार द्वारा समर्थित किया गया। Clemson विश्वविद्यालय प्रयोग स्टेशन के इस पत्र isTechnical योगदान नं 6283। लेखकों के लिए इस अंतिम प्रोटोकॉल और पांडुलिपि के अपने महत्वपूर्ण पढ़ने के लिए डॉ चेरिल इनग्राम स्मिथ, केटी ग्लेन, और अनुग्रह Kisirkoi के विकास में उनके लिए मददगार सलाह के लिए डा लुकाज़ Kozubowski धन्यवाद।
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. Genetics. 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).
