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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+genomic+analysis+identifies+isoleucine+and+CodY+as+regulators+of+Listeria+monocytogenes+virulence.">Integrative genomic analysis identifies isoleucine and CodY as regulators of Listeria monocytogenes virulence.</a> PLoS Genet. 8 , e1002887 (2012).</li><li>Kaplan Zeevi, M., 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=Listeria+monocytogenes+multidrug+resistance+transporters+and+cyclic+di-AMP,+which+contribute+to+type+I+interferon+induction,+play+a+role+in+cell+wall+stress.">Listeria monocytogenes multidrug resistance transporters and cyclic di-AMP, which contribute to type I interferon induction, play a role in cell wall stress.</a> J Bacteriol. 195, 5250-5261 (2013).</li><li>Rabinovich, L., Sigal, N., Borovok, I., Nir-Paz, R., Herskovits, A. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+gamma-interferon+receptor+that+regulates+macrophage+tumoricidal+activity.">Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity.</a> J Exp Med. 160, 55-74 (1984).</li></ol>

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. ...

The overall goal of this procedure is to extract RNA from bacteria grown intracellularly in macrophage cells. This method can help answer key questions in the bacterial pathogenesis field, such as, what are the factors in genes that bacteria trigger in order to mediate infection? The main advantage of this technique is that it significantly enriches for bacterial RNA while minimizing contamination of host nucleic acids.Demonstrating the procedure will be Anna Pasechneck, a PhD student from my laboratory. To prepare cells for bacterial infection, on day one, use 145 millimeter dishes to seed two times ten to the seven bone marrow derived macrophage, or BMDM cells, in 30 milliliters of BMDM medium plus Pen/Strep. Incubate at 37 degrees Celsius and five percent carbon dioxide in a forced air incubator overnight.Inoculate a bacterial culture of wild type L.monocytogenes bacteria in 10 milliliters of brain heart infusion, or BHI, medium. Place tubes in an incubator in a slanted position and incubate at 30 degrees Celsius without shaking overnight. The following day, pre-warm BMDM medium without antibiotics and PBS to 37 degrees Celsius.Then use 25 milliliters of the warm PBS to wash the macrophage monolayer twice to remove the antibiotics before adding 30 milliliters of fresh BMDM medium to the cells. Next, wash the overnight L.monocytogenes culture by centrifuging 1.5 milliliters of the bacteria. Then use 1.5 milliliters of PBS to re-suspend the pellet.Then, using 0.5 milliliters of the washed bacterial suspension, infect each macrophage plate. If using multiple plates, space the infections 15 minutes apart to allow for harvesting each plate individually at the end of the infection. Following a 30 minutes incubation at 37 degrees Celsius, use PBS to wash the infected cells twice to remove unattached bacteria, and then add 30 milliliters of pre-warmed BMDM medium.Incubate for 30 minutes to allow bacteria to internalize. At one hour post infection, add gentamicin to a final concentration of 50 micrograms per milliliter to kill any remaining extracellular bacteria. Assemble the filter apparatus by placing a filter head on a collecting liquid flask with a vacuum outlet port.Then place a 0.45 micron filter on the filter head, followed by a cylinder funnel, and use a metal clamp to secure the different parts. At six hours post infection, examine the cell monolayer under the microscope before harvesting. Working with one plate at a time to harvest the bacteria, use PBS to wash the infected cells.Then, to lyse the macrophage cells, add 20 milliliters of ice cold RNAse free water and quickly but carefully use a cell scraper to scrape the cells off the plate. After collecting the cells into a 50 milliliter conical tube, vortex for 30 seconds, then centrifuge at 800 times g and four degrees Celsius for three minutes to remove macrophage cell nuclei. To collect bacteria, pass the supernatant through the filter using the vacuum system.Then, with tweezers, roll the filter and quickly transfer it to a 15 milliliter conical tube. Immediately use liquid nitrogen to snap freeze the tube. On the third day, prepare 400 microliters of a one to one mixture of acidified phenol and chloroform into separate 1.5 milliliter tubes for each sample.After mixing well, aspirate the top aqueous layer before adding 40 microliters of 10%SDS to the remaining solution. Thaw the tubes containing the filters on ice to keep them cold. Then to each tube, add 650 microliters of cold acetate EDTA or AE buffer.Working as quickly as possible, vigorously vortex the filter tube so that the filter whisks to the periphery of the tube and the buffer fully washes the filter. It may be necessary to invert the tube and vortex to fully wash off the bacteria from the filter. The most critical step of the procedure is to ensure the bacteria are efficiently removed from the filter.To do so, we repeat vortexing the tubes multiple times in different positions. Once the rest of the samples have been vortexed, briefly spin down the tubes. Next, after transferring the bacteria containing buffer from each tube to the tubes of SDS and phenol chloroform, place the tubes in a multi-tube vortex device and vortex at full speed for 10 minutes.Then, incubate the tubes in a heat block at 65 degrees Celsius for 10 minutes before spinning at maximum speed for five minutes. Transfer the aqueous layer to a fresh tube containing 40 microliters of three molar sodium acetate and one milliliter of 100%ethanol. After thoroughly vortexing the samples, incubate the tubes at minus 80 degrees Celsius for one hour or at minus 20 degrees Celsius overnight.Then, after centrifuging the samples, carefully aspirate ethanol from each tube before adding 500 microliters of cold 70%ethanol to each sample. After vortexing and spinning the samples again, carefully aspirate the ethanol from each tube and use a vacuum evaporator to dry the pellets for two minutes. Next, add 25 microliters of RNAse free water to each tube, and incubate at room temperature for 20 minutes.After vortexing and spinning down the samples, with a micro-volume UV/VIS spectrophotometer, measure the RNA concentration. Expect to extract about 0.5 to one microgram of nucleic acids per plate. To carry out Dnase treatment, set up the reactions in microcentrifuge tubes according to the text protocol.Incubate the samples at 37 degrees Celsius for 45 minutes. Then add 450 microliters of RNAse free water and 500 microliters of phenol chloroform IAA mix. Once the samples have been vortexed and spun at maximum speed for two minutes, transfer the aqueous layer to a new tube and add 500 microliters of chloroform IAA mix.After vortexing and spinning the samples again, transfer the aqueous layer to a new tube, before adding one milliliter of ethanol and 15 microliters of three molar sodium acetate. Following another vortex, incubate the tubes at minus 80 degrees Celsius for one hour or minus 20 degrees Celsius overnight. After the incubation, centrifuge at maximum speed and four degrees Celsius for 20 minutes.Carefully aspirate off the ethanol from each tube. Then add 500 microliters of cold 70%ethanol to each sample. After vortexing and spinning as before, carefully aspirate the ethanol from the tubes and use an unheated vacuum evaporator to dry the samples for two minutes without over drying.Then, add 12 microliters of RNAse free water, incubate at room temperature for two minutes, and vortex the samples before spinning them down. Immediately place the purified RNA samples on ice. Finally, use a micro-volume UV/VIS spectrophotometer to measure the RNA concentrations.Expect approximately 100 nanograms of RNA per combined samples. The Listeria RNA purified from macrophage cells is suitable for downstream transcription analysis by multiple available techniques. Shown here are the results of an RT qPCR experiment used to validate the transcription of known virulence genes during wild type L.monocytogenes growth in macrophages in comparison to exponential growth in the rich laboratory medium, BHI.In particular, the results of the hly and actA genes at six hours post infection are illustrated. Transcription levels were normalized to the levels of 16S ribosomal RNA as a reference gene. The data shown are representative of three independent biological repeats.Once mastered, this technique yields about 100 nanogram of total RNA, which is suitable for transcriptomic analysis by different methods, such as RT qPCR, microarray, and RNA-seq. While attempting this procedure, it is important to remember to work quickly as possible to avoid changes in gene transcription. While this protocol describes the purification of Listeria RNA, it can be easily modified to other bacterial pathogens.

rna-purification-from-intracellularly-grown-listeria-monocytogenes

Research

JoVE Journal

Immunology and Infection

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...