Name: production and visualization of bacterial spheroplasts and protoplasts to characterize antimicrobial peptide localization
Uploaded: 2018-08-11T15:00:00
Description: Watch this Scientific Journal Video about Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization at JoVE.com

وكان دعم البحوث "المعهد الوطني للحساسية" و "الأمراض المعدية" (المعاهد الوطنية للصحة-نييد) جائزة R15AI079685.

1-حل إعداد
ملاحظة: إعداد الحلول هو موضح في الخطوات 1.1-1.9 و 1.8-1.11 بغية إنتاج جدارها كولاي وبروتوبلاستس ميجاتيريوم (ب) ، على التوالي.
<ol><li>تحضير 1 م تريس-Cl، الأس الهيدروجيني 7.8 بتذويب 10.34 ز HCl تريس وز 4.17 أوه تريس في 50 مل من dH2س في قارورة 125 مل. تعقيم بعملية التصفية من خل?...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+antimicrobial+peptides+and+their+mechanisms+of+action.">Diversity of antimicrobial peptides and their mechanisms of action.</a> Biochim Biophys Acta. 1462, 11-28 (1999).</li><li>Drin, G., Rousselle, C., Scherrmann, J. -. M., Rees, A. R., Temsamani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+Delivery+to+the+Brain+via+Adsorptive-Mediated+Endocytosis:+Advances+With+SynB+Vectors.">Peptide Delivery to the Brain via Adsorptive-Mediated Endocytosis: Advances With SynB Vectors.</a> AAPS PharmSciTech. 4 (4), 61-67 (2002).</li><li>Splith, K., Neundorf, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+with+cell-penetrating+peptide+properties+and+vice+versa.">Antimicrobial peptides with cell-penetrating peptide properties and vice versa.</a> European Biophysics Journal. 40 (4), 387-397 (2011).</li><li>Bustillo, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+analysis+of+hipposin,+a+histone-derived+antimicrobial+peptide+consisting+of+membrane+translocating+and+membrane+permeabilizing+fragments.">Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments.</a> Biochim Biophys Acta. 1838 (9), 2228-2233 (2014).</li><li>Koo, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+relations+of+parasin+I,+a+histone+H2A-derived+antimicrobial+peptide.">Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide.</a> Peptides. 29 (7), 1102-1108 (2008).</li><li>Libardo, M. D., Cervantes, J. L., Salazar, J. C., Angeles-Boza, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+bioactivity+of+antimicrobial+peptides+by+addition+of+amino-terminal+copper+and+nickel+(ATCUN)+binding+motifs.">Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs.</a> ChemMedChem. 9 (8), 1892-1901 (2014).</li><li>Park, C. B., Kim, H. S., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Action+of+the+Antimicrobial+Peptide+Buforin+II:+Buforin+II+Kills+Microorganisms+by+Penetrating+the+Cell+Membrane+and+Inhibiting+Cellular+Functions.">Mechanism of Action of the Antimicrobial Peptide Buforin II: Buforin II Kills Microorganisms by Penetrating the Cell Membrane and Inhibiting Cellular Functions.</a> Biochemical and Biophysical Research Communications. , 253-257 (1998).</li><li>Park, C. B., Yi, K. -. S., Matsuzaki, K., Kim, M. S., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+analysis+of+buforin+II,+a+histone+H2A-derived+antimicrobial+peptide:+The+proline+hinge+is+responsible+for+the+cell-penetrating+ability+of+buforin+II.">Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (15), 8245-8250 (2000).</li><li>Pavia, K. E., Spinella, S. A., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+histone-derived+antimicrobial+peptides+use+different+antimicrobial+mechanisms.">Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms.</a> Biochim Biophys Acta. 1818 (3), 869-876 (2012).</li><li>Wei, L., LaBouyer, M. A., Darling, L. E., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Spheroplasts+as+a+Model+for+Visualizing+Membrane+Translocation+of+Antimicrobial+Peptides.">Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.</a> Antimicrobial Agents and Chemotherapy. 60 (10), 6350-6352 (2016).</li><li>Chassy, B. M., Giuffrida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+Lysis+of+Gram-Positive,+Asporogenous+Bacteria+with+Lysozyme.">Method for the Lysis of Gram-Positive, Asporogenous Bacteria with Lysozyme.</a> Appl. Environ. Microbiol. 39 (1), 153-158 (1980).</li><li>Fitz-James, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+and+Chemical+Studies+of+the+Browth+of+Protoplasts+of+Bacillus+megaterium.">Cytological and Chemical Studies of the Browth of Protoplasts of Bacillus megaterium.</a> J. Biophysic. and Biochem. Cytol. 4 (3), 257-266 (1958).</li><li>Martinac, B., Buechner, M., Delcour, A. H., Adler, J., Kung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-sensitive+ion+channel+in+Escherichia+coli.">Pressure-sensitive ion channel in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 2297-2301 (1986).</li><li>Martinac, B., Rohde, P. R., Cranfield, C. G., Nomura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+electrophysiology+for+the+study+of+bacterial+ion+channels+in+giant+spheroplasts+of+E.+coli.">Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.</a> Methods Mol Biol. 966, 367-380 (2013).</li><li>Nadeau, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Experimental Biophysics: Biological Methods for Physical Scientists. , (2016).</li><li>Sun, Y., Sun, T. L., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+Escherichia+coli+spheroplast+membranes.">Physical properties of Escherichia coli spheroplast membranes.</a> Biophysical Journal. 107 (9), 2082-2090 (2014).</li><li>Branco, P., Viana, T., Albergaria, H., Arneborg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+(AMPs)+produced+by+Saccharomyces+cerevisiae+induce+alterations+in+the+intracellular+pH,+membrane+permeability+and+culturability+of+Hanseniaspora+guilliermondii+cells.">Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells.</a> Int J Food Microbiol. 205, 112-118 (2015).</li><li>Kobayashi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Translocation+Mechanism+of+the+Antimicrobial+Peptide+Buforin+2.">Membrane Translocation Mechanism of the Antimicrobial Peptide Buforin 2.</a> Biochemistry. 43 (49), 15610-15616 (2004).</li><li>Spinella, S. A., Nelson, R. B., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+peptide+translocation+into+large+unilamellar+vesicles.">Measuring peptide translocation into large unilamellar vesicles.</a> J Vis Exp. (59), e3571 (2012).</li><li>van der Kraan, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrampin:+a+novel+antimicrobial+peptide+in+the+N1-domain+of+bovine+lactoferrin.">Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin.</a> Peptides. 25 (2), 177-183 (2004).</li><li>Sun, Y., Sun, T. L., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+electrophysiology+for+the+study+of+bacterial+ion+channels+in+giant+spheroplasts+of+E.+coli.">Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.</a> Biophys J. 111 (1), 132-139 (2016).</li><li>Xie, Y., Fleming, E., Chen, J. L., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+proline+position+on+the+antimicrobial+mechanism+of+buforin+II.">Effect of proline position on the antimicrobial mechanism of buforin II.</a> Peptides. 32 (4), 677-682 (2011).</li><li>Kobayashi, S., Takeshima, K., Park, C. B., Kim, S. C., Matsuzaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+Novel+Antimicrobial+Peptide+Buforin+2+with+Lipid+Bilayers:+Proline+as+a+Translocation+Promoting+Factor.">Interactions of the Novel Antimicrobial Peptide Buforin 2 with Lipid Bilayers: Proline as a Translocation Promoting Factor.</a> Biochem. 39 (29), 8648-8654 (2000).</li><li>Decad, G. M., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+Membrane+of+Gram-Negative+Bacteria+XII.+Molecular-Sieving+Function+of+Cell+Wall.">Outer Membrane of Gram-Negative Bacteria XII. Molecular-Sieving Function of Cell Wall.</a> J. Bacteriol. 128 (1), 325-336 (1976).</li><li>Choi, H., Yang, Z., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell,+real-time+detection+of+oxidative+stress+induced+in+Escherichia+coli+by+the+antimicrobial+peptide+CM15.">Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15.</a> Proc Natl Acad Sci U S A. 112 (3), E303-E310 (2015).</li><li>Yang, Z., Choi, H., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melittin-Induced+Permeabilization,+Re-sealing,+and+Re-permeabilization+of+E.+coli+Membranes.">Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes.</a> Biophys J. 114 (2), 368-379 (2018).</li><li>Ruthe, H. J., Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+bacterial+spheroplasts+by+electric+fields.">Fusion of bacterial spheroplasts by electric fields.</a> Biochim. Biophys. Acta. 819 (1), (1985).</li></ol>

البروتوكولات المقدمة هنا جعله قابلاً للباحثين للحصول على أحجام العينة أكبر من الصور البكتيرية أكثر سرعة لأن البكتريا الموسع، كروية أسهل بكثير تحديد وتوجيه، وصورة. هذه القدرة المحسنة لجمع البيانات قيمة من نواح عدة. أولاً، أنه يمكن تحليل كمي أكثر منهجية لأنماط التعريب الببتيد. بينما يمكن ?...

وقد اكتسبت الببتيدات المضادة للميكروبات (الامبير) الاهتمام بسبب إمكانية استخدامها كبدائل للمضادات الحيوية التقليدية1،2،3،،من45. الامبير تقتل البكتيريا أما ترانسلوكاتينج عبر غشاء الخلية، والتفاعل مع المكونات داخل الخلايا مثل الأحماض النووية أو بيرميبيليزينج الغشاء مما تسبب في تسرب محتويات الخلية6. بالإضافة إلى استخدامها كالمضادات الحيوية، ترانسلوكاتينج الامبير قد تكون مكيفة لطلبات تسليم المخدرات نظراً لأنها غير ديسروبتيفيلي يمكن عبر7،غشاء الخلية كتيمة8. ولذلك، نحن، تسعى إلى فهم آليات أمبير الأساسية العمل على إرساء الأسس لاستخدامها في تصميم الأدوية.
[كنفوكل] الفحص المجهري يوفر وسيلة لتقييم أنماط الترجمة من الامبير المسمى فلوريسسينتلي في الخلايا البكتيرية تقديم رؤى في إليه العمل9،10،،من1112، 13 , 14-بوصفها غشاء البكتيريا، واحد يمكن تحديد إذا يموضع ببتيد مسمى فلوريسسينتلي للغشاء أو الفضاء داخل الخلية للخلية البكتيرية. ومع ذلك، يقتصر هذا الأسلوب الشكل صغيرة الحجم وقضيب من البكتيريا، والتي يمكن أن تجعل التصوير صعبة بسبب حدود القرار مجاهر الخفيفة التقليدية والاتجاه المتغير من البكتيريا على شريحة15.
وهدف طريقة عرض تمكين التصور تعزيز أنماط التعريب الببتيد المسمى فلوريسسينتلي باستخدام الفحص المجهري [كنفوكل]. هو تعزيز التصور بتحويل صغيرة، رقيقة، على شكل قضيب الغرام الإشريكيّة القولونية (كولاي) وإيجابية Bacillus megaterium (ميجاتيريوم) البكتيريا إلى أشكال كروية الموسع، ويشار إليه بوصفه جدارها (لسلالات الغرام) وبروتوبلاستس (لسلالات إيجابية)16،17،،من1819،،من2021. جدارها وبروتوبلاستس أسهل للصورة بسبب زيادة حجمها وشكلها متماثل، مما يجعل التوجه للبكتيريا على شريحة غير ذي صلة التصوير. وباﻹضافة إلى ذلك، فإننا نقدم مقاربة منهجية لتحليل البيانات مجهرية [كنفوكل] كمياً لوصف الامبير الغشاء أما محلياً أو ترانسلوكاتينج. تطبيق هذه الأساليب يجعل من السهل التمييز بين المسمى فلوريسسينتلي أنماط التعريب الببتيد. يمكن استخدام البروتوكولات المقدمة هنا لتقييم إضفاء الطابع المحلي على مجموعة متنوعة من العوامل النشطة غشاء خلاف المكاتب الإقليمية للمرأة، بما في ذلك اختراق الخلية الببتيدات.
واحد ميزة واضحة لهذا الأسلوب هو أن فإنه يوفر نظرة ثاقبة إليه العمل للمكاتب الإقليمية للمرأة على مستوى خلية واحدة، التي قد تكشف التغايرية خلية إلى15، بدلاً من فحوصات الفلورية الأخرى استخداماً لتحديد آليات العمل للمكاتب الإقليمية للمرأة، التي توفر معظم التقديرات9،،من2223،24،25فقط. استخدام جدارها وبروتوبلاستس من أجل تقييم إدخال خلية أمبير هي مفيدة خاصة26 لأنها ذات الصلة أكثر فسيولوجيا15 من النماذج الأخرى المستخدمة لتقييم إدخال خلية، مثل الدهن حويصلات24.

<table>
	<tbody>
		<tr>
			<td>Trizma hydrocloride (Tris HCl)</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base (Tris OH)</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma</td>
			<td>106361</td>
			<td>Used Sigma 106361 in original protocol development; 106361 discontinued with ED2SS as replacement</td>
		</tr>
		<tr>
			<td>Cephalexin hydrate</td>
			<td>Sigma</td>
			<td>C4895</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Trypticase soy broth</td>
			<td>Fisher Scientific</td>
			<td>B11768</td>
			<td></td>
		</tr>
		<tr>
			<td>BF2 P11A FITC</td>
			<td>NeoScientific</td>
			<td>Custom ordered</td>
			<td></td>
		</tr>
		<tr>
			<td>di-8-ANEPPS</td>
			<td>Biotium</td>
			<td>61012</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>34869</td>
			<td>Used Sigma D8779 in original protocol development; D8779 discontinued with 34869 as replacement</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrodisc 25 mm Syringe Filter w/ 0.2 &#956;m HT Tuffryn Membrane</td>
			<td>Pall Corporation</td>
			<td>4192</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SP5 II</td>
			<td>For image acquisition</td>
		</tr>
		<tr>
			<td>Leica Application Suite, Advanced Fluorescence</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>For image processing</td>
		</tr>
	</tbody>
</table>

production and visualization of bacterial spheroplasts and protoplasts to characterize antimicrobial peptide localization

تحول دون استخدام الفحص المجهري [كنفوكل] كطريقة لتقييم أنماط التعريب الببتيد داخل البكتيريا عادة بحدود القرار مجاهر الخفيفة التقليدية. كما لا يمكن تعزيز القرار مجهر معين بسهولة، نقدم البروتوكولات لتحويل صغيرة على شكل قضيب الغرام الإشريكيّة القولونية (كولاي) وإيجابية Bacillus megaterium (ميجاتيريوم) ودعا إلى أشكال كروية المصورة بسهولة أكبر، وجدارها أو بروتوبلاستس. ويسمح هذا التحول المراقبين لتحديد سرعة ووضوح سواء الببتيدات تقديم أنفسهم إلى الغشاء الجرثومي (أي، ترجمة غشاء) أو عبر الغشاء بدخول الخلية (أي، ترانسلوكاتينج). مع هذا النهج، نقدم أيضا أسلوب منهجي لتوصيف الببتيدات كغشاء إضفاء الطابع المحلي أو ترانسلوكاتينج. بينما يمكن استخدام هذا الأسلوب لمجموعة متنوعة من الببتيدات غشاء نشطة والسلالات البكتيرية، نظهر فائدة هذا البروتوكول عن طريق مراقبة التفاعل بين بوفورين الثاني P11A (BF2 P11A)، ببتيد مضادات الميكروبات (أمبير)، مع كولاي جدارها وبروتوبلاستس ميجاتيريوم (ب) .

طريق توسيع البكتيريا وجعلها كروية، يمكننا أن نميز بسهولة سواء الببتيدات تعريب للغشاء الجرثومي أو سهولة ترانسلوكاتي عبر الغشاء الجرثومي. حدود القرار مجاهر الخفيفة التقليدية تجعل من صعوبة التمييز بين ما إذا كانت الإشارات الببتيد تنشأ من الغشاء أو مساحة داخل الخلية في ال?...

Watch this Scientific Journal Video about Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization at JoVE.com

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

نقدم هنا بروتوكولا لإنتاج الغرام الإشريكيّة القولونية (كولاي) جدارها وإيجابية Bacillus megaterium (باء-ميجاتيريوم) بروتوبلاستس تصور واضح وتميز سرعة التفاعلات البكتيريا الببتيد. يوفر هذا أسلوب منهجي لتعريف غشاء إضفاء الطابع المحلي وترانسلوكاتينج الببتيدات.

الإنتاج والتصور جدارها البكتيرية وبروتوبلاستس لتميز التعريب الببتيد مضادات الميكروبات

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

This method can help answer key questions in the antimicrobial peptide field about whether peptides localize to or trans locate across the cell membrane. Using spheroplasts and protoplasts allows the visualization and analysis of peptide localization in a physiologically relevant model system without the resolution challenges encountered with bacterial cell imaging. Though this method can provide insights into antimicrobial peptides, it can also be applied to other membrane active agents such as cell penetrating peptides.Gram-negative E.coli spheroplasts and Gram-positive B.megaterium protoplast should be prepared in a biological safety cabinet or laminar flow hood using appropriate sterile technique. If desired, bacterial strains can contain an antibiotic resistant plasmid, and spheroplasts and protoplasts can be prepared at an open lab bench. In this video spheroplasts and protoplasts are prepared at an open lab bench using antibiotic resistant bacterial strains.To set up an overnight culture, use a sterile micropipette tip to transfer a single bacterial colony from a solid agar culture plate to a 14 milliliter culture tube, containing two to three milliliters of three percent trypticase soy broth, TSB, for a 16 to 21 hour incubation at 37 degrees celsius with shaking. To prepare Gram-negative E.coli spheroplasts, dilute the overnight culture at one to 100 ratio, and 25 milliliter of TSB in a 250 milliliter flask, and return the bacteria to the shaking incubator for another 2.5 hours. When the solution has reached an optical density of 0.5 to 0.8 at 600 nanometers, dilute the culture at a one to ten ratio in fresh TSB in a new 250 milliliter flask for a second 2.5 hour incubation with a final effective concentration of 60 micrograms per milliliter of cephalexin.To produce single cell filaments of about 50 to 150 micrometers in length, as visualized under a light microscope at a 1000x magnification. Centrifuge the bacterial solution to harvest the filaments and decant the supernatant. Wash the filaments with the gentle addition of one milliliter of 0.8 molar sucrose, taking care not to disturb the pellet.After one minute discard the supernatant without disturbing the pellet, and add the indicated reagents in their respective order to the filaments. After ten minutes at room temperature, gradually add one milliliter of solution A over a period of one minute, while gently swirling the solution by hand. Incubate the solution for four minutes at room temperature, then split the filament suspension into to two equal aliquots between two 15 milliliter conical tubes containing seven milliliters of four degree Celsius solution B.Next, pellet the filaments by centrifugalization, and use assyriological pipet to carefully remove all but one to two milliliters of the supernatant without disturbing the pellets.Use a p1000 micropipette to gently re suspend the pellets. Check a small aliquots of the sample for spheroplast formation by light microscopy. The spheroplast can then be stored at minus 20 degrees Celsius for up to a week or until they have gone through three freeze thaw cycles.To prepare Gram-positive B.megaterium protoplasts, dilute the overnight bacterial culture at a one to 1000 ratio in 100 milliliters of three percent TSB in a 250 milliliter flask for a 4.5 hour incubation at 37 degrees Celsius with shaking. When the liquid culture reaches an optical density of 0.9 to 1.0 at 600 nanometers, split the bacteria between two 50 milliliter conical tubes for their centrifugation, and use assyriological pipet to asperate the supernatants. Re suspend the pellets in 2.5 milliliters of protoplast medium per tube, and pull the suspension into a single conical tube for culture in a 125 milliliter flask.Add one milliliter of five milligrams per milliliter lysozyme to the culture for a one hour incubation at 37 degrees Celsius with shaking. Monitoring the growth of the protoplast under the light microscope and noting any irregularities, such as bacteria that appear as swollen rods instead of spheres. At the end of the incubation, transfer the suspension to a 15 milliliter conical tube for centrifugation, and re suspend the pellet in five milliliters of protoplast medium.The protoplast can then be stored at minus 20 degrees Celsius for up to a week or until they have gone through three freeze thaw cycles. For visualization of the spheroplast or protoplast transfer five microliters of the suspension of interest onto a Poly L Lysine coated glass slide, followed by two microliters of fitc labeled 100 to 200 micromolar antimicrobial peptide or AMP. After a three minute incubation protected from light add one microliter of an appropriate membrane die to the sample for another three minute incubation in the dark.At the end of the incubation cover the labeled spheroplast or protoplast with a glass cover slip, and seal the cover slip with the nail polish. To image the cells, turn on the confocal microscope and appropriate laser and select the 63x objective, then use the appropriate imaging software to obtain eight bit 512 by 512 composite Z stack images composed of 0.5 micrometer slices of the entirety of each bacterial cell of interest. To visualize localization of the fluorescently labeled AMP open the composite Z stack images in an appropriate imaging analysis program and locate the center most slice of the first spheroplast or protoplast of interest.Place one circular 0.3 micrometer diameter region of interest on the membrane, one at the center of the cell, and one away from the cell to measure the background fluorescence, taking care to avoid saturated pixels. The mean fluorescence intensity in each region of interest can then be calculated by the software. The resolution limits of conventional light microscopes make it challenging to distinguish whether fluorescently labeled peptide signals arise from the membrane or the intracellular space in normal bacteria.As the signals localize to the membrane will appear to overlap with the intracellular space. In contrast the enlarged size of the spheroplast and protoplast compared to normal bacteria, results in a clear resolution between the membrane marker and the intracellular space. Making it easier to distinguish peptide localization.For example, the localization to the membrane and translocation across the cell membrane of fluorescently labeled peptide can be visualized in both E.coli spheroplast and B.megaterium protoplast, by confocal fluorescence microscopy as just demonstrated. The placement of regions of interest on the membrane and intracellular space of the spheroplast or protoplast and in the background, allows calculation of the ratio of intracellular peptide fluorescence intensity to peptide fluorescence intensity on the membrane of each spheroplast or protoplast interacting with AMP. For example, this method can be used to visualize the interactions of a histone derived microbial peptide mutant with E.coli spheroplast and B.megaterium protoplasts.In this experiment the AMP demonstrated a similar localization pattern in both strains of bacteria. Highlighting it's reduced translocation relative to the wild type peptide. Ultimately this technique enables researchers to explore peptide localization patterns on a single cell level.Offering insights into the mechanism of action of a peptide of interest. In addition to allowing researchers to collect larger numbers of images for the systematic characterization of peptide mechanisms, images of larger spheroplasts and protoplasts are more manageable for quantitative analysis than those of smaller bacterial cells.

Research

JoVE Journal

Biology

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

عزلة Protoplasts من أنسجة لمدة 14 يوما من عمره الشتلات thaliana Arabidopsis</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

مضان الفرز الخلية النباتية المنشط من Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

نانو FEM: تعريب البروتين عن طريق الصور التي تنشط المجهر التعريب والمجهر الإلكتروني

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

استهدفت RNA تسلسل الفحص لتحديد خصائص التعبير الجيني والجينوم التعديلات

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

التصور-المربوطة السطحية الكبيرة جزيئات الحمض النووي مع الحمض النووي البروتين الفلوري ملزم الببتيد

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

باستخدام مستقبلات الموسومة pHluorin لمراقبة التحت خلوية التعريب والاتجار

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

قياس اندوريدوبليكيشن بقياس التدفق الدرنات المعزولة بروتوبلاستس

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

طريقة بسيطة لعزل بروتوبلاستس فول الصويا وتطبيق لتحليل التعبير الجيني عابر

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

تقييم التعريب الخاص بنواه عدسه Zebrafish والنزاهة التكوينية

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...