Epoch - Poster Final

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<ul><li><p>8/12/2019 Epoch - Poster Final</p><p> 1/1</p><p>ISOLATION, PURIFICATION, AND CHARACTERIZATION</p><p>OF THE MYCOBACTERIOPHAGE EPOCHGUSTAVO MIRALDA, DEMING, K.E., HUGHES, L.E. AND R.C. BENJAMIN</p><p>Universi ty of Nor th Texas</p><p>The novel mycobacteriophageEpochwas isolated fr om an environmental samplecollected under a tree in a residential front lawn through a process known as enrichment,</p><p>usingMycobacterium smegmatis as the host bacteria. The initial plate possessed plaques of</p><p>three different morphologies and Epoch was isolated as a unique phage following several</p><p>rounds of plaque purification. The mycobacteriophage was then characterized using several</p><p>different methods. These included plaque morphology, restriction digestion, gel</p><p>electrophoresis and electron microscopy. Based upon the results of these studiesEpochhas</p><p>been given a preliminary assignment to mycobacteriophage group A5. This study was</p><p>conducted as part of the Howard Hughes-sponsored National Genomics Research Initiative.</p><p>The focus of this initiative is the isolation of a l arge numbers of unique mycobacteriophage</p><p>from the environment and an assessment of the diversity of characteristics, both physical and</p><p>at the genomic level, of mycobacteriophages worldwide. Long term goals of the project are</p><p>expected to include benefits to phage scientists as well as other researchers in the fields of</p><p>healthcare, environmental/ecological applications, as well as biomedicine.</p><p>Epochhas the characteristic features of the siphoviridae virus family, which includea isometric head/capsid and a flexible noncontractile tail. As of early 2011, over</p><p>90% of known mycobacteriophages are categorized in the siphoviridae family. The</p><p>genome ofEpoch, estimated to be 52 kilobases in length, likely contains at least 70</p><p>genes and this group has a guanine/cytosine content of at least 52%. Plaques</p><p>produced in the early protocols of serial dilutions did not demonstrate the same</p><p>characteristics as shown in Figure 2. As noted previousl y, the plaque type we</p><p>classified as A was chosen for purification from the original enrichment plate.</p><p>Interestingly, the size of the plaques produced by this phage increased as the phage</p><p>purification process progressed. What began as a uniformly turbid plaque attained</p><p>the characteristics of the halo plaque morphology (lytic inner circle, turbid outer</p><p>ring) by the high titer lysate stage of purification. Thus, althoughEpoch</p><p>demonstrated a traditional lysogenic life cycle at early stages, it appeared to be</p><p>more lytic when phage were present at high numbers on the plate. It is thought</p><p>that this change may have occurred due to t he under favorable conditions for the</p><p>mycobacteriophages or host cells that would be found at the centers of large plaques</p><p>under web plate conditions.</p><p>The calculated DNA yield forEpochis on par with the results obtained with other</p><p>mycobacteriophages within the A cluster. Phages within the A cluster range from agenome size between 47 to 54 kilobases and this is again consistent with our</p><p>findings for Epoch. Other characteristics of t he A cluster include a GC percentage</p><p>between 59 and 65, as well as a range of total genes from 75 to 104. Based upon the</p><p>restriction analysis ofEpoch, it was concluded that the results best fit those of the</p><p>A2 subcluster. This includes a lack ofEcoRI andHindIII sites as well as the</p><p>presence of numerous cutting sites for ClaI. Within the A2 subcluster, the phage L5</p><p>has characteristics very similar to Epoch, including a genome length of 52,297 base</p><p>pairs, a similar plaque morphology, a noncontractile tail and a isometric head.</p><p>Genome sequencing ofEpochat some future date would allow for a better</p><p>understanding of its lysogenic/lytic cycle, as well as its characteristic genes. It is not</p><p>known whether the observed change in plaque morphology that occurred during</p><p>phage purification/isolation was the result of actual phage physiology characteristic</p><p>ofEpochor a mutation that occurred in the phage line during purification. This</p><p>question cannot be readily answered without the full sequencing of Epoch.</p><p>Below is a short summary of the methods used in this study. These are taken from</p><p>The HHMI National Genomics Research Initiative Laboratory Manual.</p><p>The isolation of the novel phageEpochfrom the environment was performed using an</p><p>enrichment protocol and a soil sample collected from a residential lawn in Irving, Texas. The</p><p>enrichment process consisted of soil inoculation of a M. smegmatisculture, filter-sterilization</p><p>of the cultured lysate, serial dilutions and plaque purification.</p><p>A spot test was performed to verify putative plaques and a selected plaque was purified</p><p>by a series of phage-titer assays. A filter-sterilized sample medium titer lysate was created</p><p>from a web plate flooded with phage buffer. The final high titer lysate of the phageEpoch</p><p>was obtained using the medium titer lysate to create 10 web plates that were again flooded</p><p>with phage buffer to collect phage. Final phage yields were determined using standard phage</p><p>dilution/assay techniques.</p><p>Epoch was then analysed by electron microscopy and its capsid diameter and tail length</p><p>were determined. Phage genomic DNA was prepared for restriction digestion and gelelectrophoresis. The genome size of epoch was estimated from the resultant restriction data</p><p>and a comparison of the patterns obtained with each enzyme was used to make a preliminary</p><p>group assignment for the phage.</p><p>1. Mc Grath S and van Sinderen D (editors). (2007).Bacteriophage: Genetics and Molecular Biology(1st ed.).</p><p>Caister Academic Press.</p><p>Wommack, K. E.; Colwell, R. R. (2000). "Virioplankton: Viruses in Aquatic Ecosystems".Microbiology and</p><p>Molecular Biology Reviews64 (1): 69114.</p><p>2. Gabashvili, I.; Khan, S.; Hayes, S.; Serwer, P. (1997). "Polymorphism of bacteriophage T7". Journal of Molecular</p><p>Biology273 (3): 658.</p><p>Wright, A.; Hawkins, C.; Anggrd, E.; Harper, D. (2009). "A controlled clinical trial of a therapeutic bacteriophage</p><p>preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a preliminary report of</p><p>efficacy".Clinical Otolaryngology34 (4): 349357.</p><p>Collman, J. P. 2001.Naturally Dangerous: Surprising facts about food, health, and the Environment.Sausalito, CA:</p><p>University Science Books. Pg. 92.</p><p>3. Fiers, W.; Contreras, R.; Duerinck, F.; Haegeman, G.; Iserentant, D.; Merregaert, J.; Min Jou, W.; Molemans, F. et</p><p>al. (1976). "Complete nucleotide sequence of bacteriophage MS2 RNA: pri mary and secondary structure of the</p><p>replicase gene".Nature260 (5551): 5007.</p><p>Rhoads DD, Wolcott RD, Kuskowski MA, Wolcott BM, Ward LS, Sulakvelidze A (2009). Bacteriophage therapy of</p><p>venous leg ulcers in humans: results of a phase I safety trial. J Wound Care. 2009 Jun;18(6):237-8, 240-3.</p><p>4. Graham F. HATFULL 12 - M ycobacteriophages :Pathogenesis and Applications, pages 238-255 (in Waldor, M.K.,</p><p>D.I. Friedman, and S.L. Adhya, Phages: Their role in bacterial pathogenesis and biotechnology, 2005, University of</p><p>Michigan; Sankar L. Adhya, National Institutes of Health: ASM Press).</p><p>5. Murphy, Clare. "BBC NEWS | Health | 'Red Army' Virus to Combat MRSA."BBC News - Home. BBC News, 13</p><p>Aug. 2007. Web. 03 Dec. 2011. .</p><p>6. "The Bacteriophage Ecology Group - Phage Ecology and Evolutionary Biology."Home - The Ohio State Universityat Mansfield. The Ohio State University at Mansfield. Web. 03 Dec. 2011. .</p><p>7. "Phage Biology and Phage Therapy | Home of the Evergreen International Phage Biology</p><p>Meeting."Blogs.evergreen.edu | at The Evergreen State College. Evergreen. Web. 03 Dec. 2011.</p><p>.</p><p>The first r ound of screening yielded plaques of three different morphologies. Plaque A was roughly 1.4mm in diameter and was not visible unless it was held in direct</p><p>light due to its turbidity. This was chosen for the plaque streak protocol.</p><p> Plaque B had the largest diameter on average of 2.0mm and its plaque morphology was very</p><p>cloudy, often grouped in large clumps of other plaques. This was chosen for plaque</p><p>purification but not studied further beyond that point.</p><p> Plaque C was roughly about 0.7mm in diameter and showed minor turbidity in comparison</p><p>to the other plaques. This type of plaque was not chosen for further study.</p><p>Sample A, chosen for the phage purification/isolation, contained a 10-1dilution and 19 pfu</p><p>from 5 lof medium titer lysate. Plaques in average had a diameter of 2.5 mm and an area of</p><p>5.0 mm. The titer of the phage lysate was 3.8 x 104pfu/ml. The total number of pfu needed to</p><p>create a lysed web pattern was estimated to be 1140 pfu, corresponding to phage lysate</p><p>volume of 3.0 x 10-3ml. The final medium titer lysate was determined to have 1.69 x</p><p>1010pfu/ml. The medium titer lysate was used to produce a ten plate high titer lysate from</p><p>which genomic DNA was prepared and which was used as the phage source for electron</p><p>microscopy. The titer of the high titer lysate was 1.56 x 1013pfu/ml.</p><p>Electron microscopy of uranyl acetate s tainedEpoch reveal ed a siphoveri dae phage. The</p><p>tail length was determined to be 125 nm and its capsid diameter was 61 nm. (Figure 1)</p><p>The purification of phage genomic DNA yielded a total DNA volume of 86 lwith a 54.5ng/ul DNA concentration. Total DNA recovered was thus 4.7 g.</p><p>Gel electrophoresis of restriction digestions ofEpochDNA, specifically digestions with</p><p>BamHI and ClaI, gave an estimated genome size of 52,000 bp. (Figure 3)</p><p>Although the focus of basic research on mycobacteriophages is fairly recent, it hasalready yielded findings highly beneficial to scientists in a range of disciplines. One of the</p><p>useful properties of mycobacteriophages revolve around their bactericidal nature, which,</p><p>unlike antibiotics, only targets specific bacteria. The virus-host bacteria interaction is</p><p>unaffected by multi-drug-resistance of the target cells, which allows healthcare professionals</p><p>to use these phages to control specific disease-causing strains of bacteria without impacting</p><p>normal human bacterial flora. Since the 1930s, bacteriophages have been used as an</p><p>alternative to antibiotics when combating gangrene, leprosy and E. coli infections. These</p><p>phage regiments have also been completed without any known detrimental side-effects.</p><p>Although few western countries have adopted the use of mycobacteriophages in the field of</p><p>health, researchers at the Eliava Institute in Georgia have developed practical antibacterial</p><p>uses for the phages. Recently in the United States, however, the Food and Drug</p><p>Administration approved the spraying of meats with phages in order to combatLysteria</p><p>monocytogenes without the risk of promoting drug-resistant strains of the pathogens. Overall,</p><p>the use of mycobacteriophages in medicine has benefitted individuals, but the research</p><p>required for broader application of the technique has been restricted because of its limited use</p><p>in western countries to this time. With additional research and new applications, the use of</p><p>mycobacteriophages will develop into a more mainstream alternative to the use of antibiotics,</p><p>which will be much more beneficial to target infectious diseases.</p><p>ABSTRACT</p><p>INTRODUCTION</p><p>MATERIALS AND METHODS</p><p>RESULTS CONCLUSIONS</p><p>REFERENCES</p><p>Figure 1. Electron microscopy image of</p><p>Epoch. The tail length is 125.35nm and</p><p>the capsid diameter is 61.29nm.</p><p>Figure 2. Plaque morphology ofEpoch. The</p><p>plaques demonstrate halo-like</p><p>characteristics with lytic inner circles and</p><p>turbid outer rings. The average plaque</p><p>diameter was roughly 2.8mm. After 24</p><p>hours at 37C.</p><p>Figure 3. Gel electrophoresis of restriction digestions of Epoch. The estimated genome size averaged</p><p>from two restriction digestions (BamHI and ClaI)is estimated to be 52,000 base pairs.Epochis most</p><p>similar to the A cluster due to its lack of EcoRI andHindIII sites, which is a characteristics the A2</p><p>subcluster.</p></li></ul>