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 145 mm dish in 30 ml BMDM + Pen-Strep media (Table 2). Seed 3 plates for each bacterial strain to be analyzed. Incubate overnight at 37 &#176;C in a 5% CO2 forced-air incubator.</li>
		<li>Start an overnight bacterial culture of wild-type (WT) L. monocytogenes strain 10403S in 10 ml of brain heart infusion (BHI) medium, at 30 &#176;C in a standard incubator. Place culture tubes slanted without shaking. Note: These growth conditions up-regulate the expression of the flagella genes, which promotes efficient infection.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Pre-warm BMDM medium (without Pen-Strep antibiotics) (Table 2) and phosphate buffered saline (PBS) at 37 &#176;C.</li>
		<li>Wash macrophage monolayer twice with 25 ml of pre-warmed PBS to remove antibiotics. Add 30 ml fresh BMDM medium.</li>
		<li>Wash 1.5 ml of overnight bacterial culture with PBS by centrifuging bacteria at&#160; &#62; 14,000 x g for 1 min, discarding the supernatant, and resuspending bacteria gently in 1.5 ml of PBS by pipetting. Repeat twice.</li>
		<li>Infect each macrophage plate with 0.5 ml of a washed overnight culture of wild-type L. monocytogenes. If using multiple plates, infect each plate 15 min apart. This time interval will enable harvesting each plate individually at the end of the infection. 
		Note: The multiplicity of infection (MOI) is assayed by plating serial dilutions of bacterial culture used for infection on BHI agar plates and counting colony forming units (CFU) after 24 hr incubation of plates at 37 &#176;C. Using 0.5 ml of WT L. monocytogenes strain 10403S results in a MOI of ~ 100 (100 bacteria CFU per macrophage cell). When analyzing bacterial mutants defective in intracellular growth, a higher MOI should be considered.</li>
		<li>Following 0.5 hr incubation at 37 &#176;C, wash the infected cells twice with PBS, to remove unattached bacteria, and add 30 ml pre-warmed BMDM medium. Attached bacteria will internalize during next 0.5 hr.</li>
		<li>At 1 hr post infection, add 30 &#181;l of gentamicin (1:1,000 to reach a final concentration of 50 &#181;g/ml) to kill extra-cellular bacteria.</li>
		<li>Assemble the filter apparatus by placing a filter head on a collecting liquid flask with a vacuum outlet port. Then place a filter (0.45 &#181;m) on a filter head, followed by a cylinder funnel and secure the different parts with a metal clamp. Prepare ice-cold RNase-free water.</li>
		<li>At 6 hr post-infection, harvest bacteria from infected cells as follows. Treat each plate individually. 
		Note: Usually, L. monocytogenes completes 1-log of growth during 6 hr of infection in macrophage cells. Longer incubations could result in bacterial overgrowth and cell death of the macrophages, while shorter incubation periods could result in considerably lower bacterial loads. The MOI and the time of infection can be modified to reach optimal conditions.
		<ol>
			<li>Wash infected cells with PBS once. Add 20 ml of ice-cold RNase-free water to lyse macrophage cells. Using a cell scraper, scrape the cells off the plate quickly but carefully.</li>
			<li>Collect lysed cells to a 50 ml conical tube. Vortex for 30 sec. Centrifuge at 800 x g for 3 min at 4 &#176;C.</li>
			<li>Pass the supernatant through the filter apparatus using a vacuum system. Using tweezers, roll the filter and quickly transfer it to a 15 ml conical tube. Snap-freeze the tubes with the filters in liquid nitrogen.</li>
			<li>Assemble the filtration apparatus with a new filter for the next sample, as described in 1.2.7.</li>
			<li>Store the frozen filters at -80 &#176;C for the next day. Alternatively, proceed directly to nucleic acid extraction.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Nucleic Acids Extraction (Day 3)
Note: Perform all manipulations with phenol and chloroform solutions in a fume hood.
<ol>
	<li>Prepare a 1:1 mix of acidified phenol: chloroform, 400 &#181;l for each sample. Mix in separate tubes, and then withdraw the resulting aqueous layer by aspiration. Add 40 &#181;l of 10% SDS.</li>
	<li>Thaw the filter-containing tubes on ice to keep them cold. To each filter-containing tube add 650 &#181;l of acetate-EDTA (AE) buffer (Table 2).</li>
	<li>Perform the following steps as quickly as possible.
	<ol>
		<li>Vigorously vortex the tube containing the filter so that the filter whisks to the periphery of the tube and the buffer fully washes the filter. Always keep the tube cold by placing it back on ice. Note: It may be necessary to additionally vortex the tube while inverted to fully wash the bacteria off the filter.</li>
		<li>Repeat for all tubes. It may be useful to spin-down the suspension shortly (1 min at 120 x g) at the end of the process.</li>
	</ol>
	</li>
	<li>Transfer the bacteria-containing buffer to a 1.5 ml microcentrifuge tube containing the SDS and phenol/chloroform mix (as prepared in 2.1). Repeat for all tubes. It may be necessary to spin the filter containing tubes (1 min at 120 x g) again to get residual buffer from the filter.</li>
	<li>Place all the 1.5 ml tubes in a multi-tube vortex device, and vortex at full speed for 10 min.</li>
	<li>Incubate the tubes in a 65 &#176;C heat block for 10 min. Centrifuge at maximum speed (&#62; 14,000 x g) for 5 min.</li>
	<li>Transfer the aqueous layer (about 400 &#181;l) from each tube to a new 1.5 ml tube containing 40 &#181;l of 3 M sodium acetate (pH 5.2) and 1.0 ml of 100% ethanol. Vortex each tube thoroughly. 
	Note: Glycogen can be added at this step to improve visualization of the precipitated pellet.</li>
	<li>Incubate the samples at -80 &#176;C for 1 hr. Alternatively, incubate samples at -20 &#176;C overnight. Then, centrifuge the samples at 4 &#176;C for 20 min at maximum speed (&#62; 14,000 x g).</li>
	<li>Carefully aspirate off the ethanol (upper layer) from each tube. Add 500 &#181;l cold 70% ethanol to each sample and vortex thoroughly. Centrifuge at 4 &#176;C for 20 min at maximum speed (&#62; 1 4,000 x g).</li>
	<li>Carefully aspirate off the ethanol from each tube. Dry the samples for approximately 2 min using a vacuum evaporator with no heat. Do not over-dry the samples.</li>
	<li>Add 25 &#181;l RNase-free water to each sample. Incubate at room temperature for 20 min. Carefully vortex and spin-down.</li>
	<li>Measure RNA concentration in the samples using a microvolume UV-Vis spectrophotometer. Expect to extract about 0.5 - 1 &#181;g of nucleic acids per plate. 
	Note: Technical replicates can be combined at this stage.</li>
</ol>
3. DNase Treatment
<ol>
	<li>Set up the reaction according to Table 1 in a microfuge tube. Incubate at 37 &#176;C for 45 min. Add 450 &#181;l RNase-free water and 500 &#181;l of Phenol-Chloroform-IAA mix (Table 2), separate phases by centrifuging for 2 min at maximal speed (&#62; 14,000 x g).</li>
	<li>Transfer the aqueous layer to a new tube and add 500 &#181;l chloroform-IAA mix (Table 2), vortex and separate phases by 2 min centrifugation at maximal speed (&#62; 14,000 x g).</li>
	<li>Transfer the aqueous layer to a new tube and add 1 ml of ethanol and 50 &#181;l of 3 M sodium acetate (pH 5.2). Vortex. 
	Note: Glycogen can be added at this step to improve visualization of the precipitated pellet.</li>
	<li>Incubate for 1 hr at -80 &#176;C. Alternatively, incubate samples at -20 &#176;C overnight. Centrifuge at 4 &#176;C for 20 min at maximal speed.</li>
	<li>Carefully aspirate off the ethanol from each tube. Add 500 &#181;l cold 70% ethanol to each sample and vortex thoroughly. Centrifuge at 4 &#176;C for 20 min at maximum speed. Carefully aspirate off the ethanol from each tube.</li>
	<li>Dry the samples for approximately 2 min using a vacuum evaporator with no heat. Do not over-dry the samples.</li>
	<li>Add 12 &#181;l of RNase-free water, incubate for 2 min at room temperature, vortex, and spin-down. The samples contain purified RNA, keep them on ice. Alternatively, dissolve RNA in RNase-free H2O with 0.1 mM EDTA or TE buffer (10 mM Tris, 1 mM EDTA).</li>
	<li>Measure RNA concentrations using a microvolume UV-Vis spectrophotometer. Expect ~ 100 ng of RNA per sample (if technical replicates are combined). 
	Note: RNA extracted according to this protocol is suitable for gene transcription analysis by multiple techniques. We usually employ RT-qPCR analysis to study L. monocytogenes gene transcription during infection of macrophage cells 1-4.</li>
</ol>

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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 authors have nothing to disclose.

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. ...

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

Research

JoVE Journal

Immunology and Infection

9.1K Views.  Tel Aviv University. 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.

Video: RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

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 ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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.

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 ...