Name: rna purification from intracellularly grown listeria monocytogenes in macrophage cells
Uploaded: 2016-06-04T14:00:00
Description: Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

The research in the Herskovits lab is supported by 335400 ERC and R01A/109048 NIH grants.

Note: During the entire experiment, macrophage cells are incubated at 37 &#176;C in a 5% CO2 forced-air incubator and taken out of the incubator only for experimental manipulations, which are performed in a Class II biological safety cabinet. Working with L. monocytogenes bacteria is according to biological safety level 2 regulations.
1. Cell Preparation and Bacterial Infection (Day 1 and 2)
<ol>
	<li>Day 1
	<ol>
		<li>Seed 2.0 x 107 bone marrow derived macrophage cells (BMDM) on a...

<ol>
	<li>Lobel, L., 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=The+metabolic+regulator+CodY+links+Listeria+monocytogenes+metabolism+to+virulence+by+directly+activating+the+virulence+regulatory+gene+prfA.">The metabolic regulator CodY links Listeria monocytogenes metabolism to virulence by directly activating the virulence regulatory gene prfA.</a> Mol Microbiol. , (2014).</li><li>Lobel, L., Sigal, N., Borovok, I., Ruppin, E., Herskovits, A. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prophage+excision+activates+Listeria+competence+genes+that+promote+phagosomal+escape+and+virulence.">Prophage excision activates Listeria competence genes that promote phagosomal escape and virulence.</a> Cell. 150, 792-802 (2012).</li><li>Swaminathan, B., Gerner-Smidt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+human+listeriosis.">The epidemiology of human listeriosis.</a> Microbes Infect. 9, 1236-1243 (2007).</li><li>Hamon, M., Bierne, H., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes:+a+multifaceted+model.">Listeria monocytogenes: a multifaceted model.</a> Nat Rev Microbiol. 4, 423-434 (2006).</li><li>Dussurget, O., Pizarro-Cerda, J., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+determinants+of+listeria+monocytogenes+virulence.">Molecular determinants of listeria monocytogenes virulence.</a> Ann Rev Microbiol. 58, 587-610 (2004).</li><li>Portnoy, D. A., Auerbuch, V., Glomski, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+Listeria+monocytogenes+infection:+the+intersection+of+bacterial+pathogenesis+and+cell-mediated+immunity.">The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity.</a> J Cell Biol. 158, 409-414 (2002).</li><li>Freitag, N. E., Port, G. C., Miner, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+-+from+saprophyte+to+intracellular+pathogen.">Listeria monocytogenes - from saprophyte to intracellular pathogen.</a> Nat Rev Microbiol. 7, 623-628 (2009).</li><li>Graham, J. E., Clark-Curtiss, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Mycobacterium+tuberculosis+RNAs+synthesized+in+response+to+phagocytosis+by+human+macrophages+by+selective+capture+of+transcribed+sequences+(SCOTS).">Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS).</a> Proc Natl Acad Sci U S A. 96, 11554-11559 (1999).</li><li>Toledo-Arana, A., 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=The+Listeria+transcriptional+landscape+from+saprophytism+to+virulence.">The Listeria transcriptional landscape from saprophytism to virulence.</a> Nature. 459, 950-956 (2009).</li><li>Schnappinger, D., 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=Transcriptional+Adaptation+of+Mycobacterium+tuberculosis+within+Macrophages:+Insights+into+the+Phagosomal+Environment.">Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment.</a> J Exp Med. 198, 693-704 (2003).</li><li>Albrecht, M., Sharma, C. M., Reinhardt, R., Vogel, J., Rudel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+sequencing-based+discovery+of+the+Chlamydia+trachomatis+transcriptome.">Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome.</a> Nucleic Acids Res. 38, 868-877 (2010).</li><li>La, M. V., Raoult, D., Renesto, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+whole+bacterial+pathogen+transcription+within+infected+hosts.">Regulation of whole bacterial pathogen transcription within infected hosts.</a> FEMS Microbiol Rev. 32, 440-460 (2008).</li><li>Westermann, A. J., Gorski, S. A., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+RNA-seq+of+pathogen+and+host.">Dual RNA-seq of pathogen and host.</a> Nat Rev Microbiol. 10, 618-630 (2012).</li><li>Rienksma, R. A., 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=Comprehensive+insights+into+transcriptional+adaptation+of+intracellular+mycobacteria+by+microbe-enriched+dual+RNA+sequencing.">Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing.</a> BMC Genomics. 16, 34 (2015).</li><li>Afonso-Grunz, F., 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=Dual+3'Seq+using+deepSuperSAGE+uncovers+transcriptomes+of+interacting+Salmonella+enterica+Typhimurium+and+human+host+cells.">Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells.</a> BMC Genomics. 16, 323 (2015).</li><li>Englen, M. D., Valdez, Y. E., Lehnert, N. M., Lehnert, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte/macrophage+colony-stimulating+factor+is+expressed+and+secreted+in+cultures+of+murine+L929+cells.">Granulocyte/macrophage colony-stimulating factor is expressed and secreted in cultures of murine L929 cells.</a> J Immunol Methods. 184, 281-283 (1995).</li><li>Becavin, C., 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=Comparison+of+widely+used+Listeria+monocytogenes+strains+EGD,+10403S,+and+EGD-e+highlights+genomic+variations+underlying+differences+in+pathogenicity.">Comparison of widely used Listeria monocytogenes strains EGD, 10403S, and EGD-e highlights genomic variations underlying differences in pathogenicity.</a> MBio. 5, e00969-e900914 (2014).</li><li>Celada, A., Gray, P. W., Rinderknecht, E., Schreiber, R. 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The protocol described here represents an optimized method for isolation of bacterial RNA from L. monocytogenes bacteria growing intracellularly in macrophage cells. This protocol is based on cell differential lysis and includes two major steps for enrichment of bacterial RNA: macrophage nuclei sedimentation using centrifugation and a rapid collection of bacteria by filtration. These steps are followed by a standard RNA extraction procedure. While this protocol describes purification of listerial RNA, it can be ...

Intracellular bacterial pathogens &#9472; bacteria causing infectious diseases and capable of growing and reproducing inside cells of human hosts &#9472; are a major health concern worldwide5. To invade and replicate within a mammalian cell, intracellular pathogens have acquired sophisticated virulence mechanisms and factors. While these mechanisms are fundamental to the ability to cause a disease, we know little about their regulation and dynamics. Since gene expression profiles of bacteria grown in liquid media do not reflect the actual environment within host cells, there is a growing need for transcriptome analyses of bacteria grown in their intracellular niches. Such analyses will enable the deciphering of specific bacterial adaptations triggered by the host and will help to identify new targets for therapeutic design. Transcriptome analysis of intracellularly grown bacteria is highly challenging since mammalian RNA outnumber bacterial RNA by at least ten fold. In this manuscript, we describe an experimental method to isolate bacterial RNA from Listeria monocytogenes bacteria growing inside murine macrophage cells. The extracted RNA can be used to study intracellular adaptations and virulence mechanisms of pathogenic bacteria by various techniques of transcription analysis, such as RT-PCR, RNA-Seq, microarray and other hybridization based technologies.
L. monocytogenes is the causative agent of listeriosis in humans, a disease with clinical manifestations targeting primarily immunocompromised individuals, elderly people and pregnant women6. It is a Gram-positive facultative intracellular pathogen that invades a wide array of mammalian cells that have been used for decades as a model in host-pathogen interactions studies7. Upon invasion, it resides initially in a vacuole or a phagosome (in the case of phagocytic cells), from which it must escape into the host cell cytosol in order to replicate. Several virulence factors have been shown to mediate the escape process, primarily the pore-forming hemolysin, Listeriolysin O (LLO) and two additional phospholipases8. In the cytosol the bacteria use the host actin polymerization machinery to propel themselves on actin filaments within the cell and to spread from cell to cell (Figure 1). All major virulence factors of L. monocytogenes involved in invasion, intracellular survival and replication, are activated by the master virulence transcription regulator, PrfA. 8-10.
In the last decade, several studies conducted by us and others have successfully applied methods for transcriptome analysis of intracellularly grown bacteria inside host cells2,11-15. Two main approaches are used to separate bacterial RNA from host-RNA which are based on: 1) Selective enrichment of bacterial RNA and 2) RNA isolation by differential cell lysis. The first approach relies on subtractive hybridization of total RNA extracts to mammalian RNA molecules (for example using commercially available kits) or selective capture of bacterial transcribed sequences (SCOTS)11. The second approach relies on differential lysis of bacteria and host cells, in which the host cells are lysed while bacterial cells remain intact. Bacterial cells are then separated from the host cell lysate, usually by centrifugation, and the RNA is extracted using standard techniques. The main problem using this approach is that together with the intact bacteria, host cells nuclei are also isolated, thus the RNA preparations still contain mammalian RNA. One way to overcome this problem is to separate intact bacteria from host cells nuclei using differential centrifugation, though this procedure usually takes time raising the concern of changes in gene expression profile during extraction. In this paper we present an improved and rapid bacteria RNA extraction protocol, which is based on cell differential lysis approach. First, L. monocytogenes infected macrophage cells are lysed with cold water. Next, macrophage nuclei are removed by a brief centrifugation and intact bacteria are rapidly collected on filters, from which RNA is isolated using hot phenol-SDS extraction of bacterial nucleic acids.

<table>
	<tbody>
		<tr>
			<td rowspan="2">Listeria monocytogenes 10403S</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">20</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Bone marrow derived macrophages prepared from C57B/6 female mice</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">21</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>H2O, RNAse free</td>
			<td>Thermo Scientific</td>
			<td>10977-015</td>
			<td>DEPC-treated water can be used</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965039</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-088</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol&#160;&#160;</td>
			<td>Gibco</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline-PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion (BHI)</td>
			<td>Merckmillipore</td>
			<td>1104930500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol saturated pH 4.3</td>
			<td>Fisher</td>
			<td>BP1751I-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iso-amyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>W205702</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>W302406</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Fermentas,</td>
			<td>EN0521</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS 10%</td>
			<td>Sigma-Aldrich</td>
			<td>L4522</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck Millipore</td>
			<td>1070174000</td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C, 5% CO2 forced-air incubator</td>
			<td>Thermo Scientific</td>
			<td>Model 3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Nunc</td>
			<td>179693</td>
			<td></td>
		</tr>
		<tr>
			<td>Kontes glass holder for 45 mm filters</td>
			<td>Fisher</td>
			<td>K953755-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>MF-Millipore filters 45 mm, 0.45 &#181;m</td>
			<td>Merck Millipore</td>
			<td>HAWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac system</td>
			<td>Thermo Scientific</td>
			<td>SPD131DDA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>Model G560E</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>145 mm cell culture dishes</td>
			<td>Greiner</td>
			<td>639 160</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml tubes, RNase-free</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr>
			<td>30&#176;C incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>65 &#176;C heat block</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 &#176;C table centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipettes, 25 ml</td>
			<td>Greiner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 50 ml</td>
			<td>Greiner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

rna purification from intracellularly grown listeria monocytogenes in macrophage cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

The model system is shown in Figure 1 and includes macrophage cells infected with L. monocytogenes bacteria, which replicate in the macrophage cytosol. Figure 2 represents the experimental scheme. Figure 3 represents typical results of such RT-qPCR analysis of virulence genes during WT L. monocytogenes growth in macrophages in comparison to growth in rich laboratory medium BHI. The results show the transcription levels o...

Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

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