Research in the Robinson&#39;s lab is supported by funding from Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Germany (CECAD; funded by the DFG within the Excellence Initiative of the German federal and state governments) and grants from Deutsche Forschungsgemeinschaft (SFB 670), K&#246;ln Fortune, and Maria-Pesch Foundation of the University of Cologne, Germany.

All the steps involving the use of pathogenic S. typhimurium must be carried out in a BSL-2 or higher biological security level facility. The culture and coating of S. Typhimurium, as well as the infection of bone marrow-derived macrophages (BMDMs) must be performed under a laminar flow hood to prevent contamination. The isolation of S. typhimurium-containing phagosomes can be performed on any BSL-2 laboratory bench.
The extraction of bone marrow fr...

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella:+A+model+for+bacterial+pathogenesis.">Salmonella: A model for bacterial pathogenesis.</a> Annu Rev Med. 52, 259-274 (2001).</li><li>Mills, S. D., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+Salmonella+typhimurium+and+Yersinia+pseudotuberculosis-containing+phagosomes+from+infected+mouse+macrophages:+Y-pseudotuberculosis+traffics+to+terminal+lysosomes+where+they+are+degraded.">Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y-pseudotuberculosis traffics to terminal lysosomes where they are degraded.</a> Eur J Cell Biol. 77 (1), 35-47 (1998).</li><li>Buchmeier, N. A., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Macrophage+Phagosome-Lysosome+Fusion+by+Salmonella-Typhimurium.">Inhibition of Macrophage Phagosome-Lysosome Fusion by Salmonella-Typhimurium.</a> Infect Immun. 59 (7), 2232-2238 (1991).</li><li>Luhrmann, A., Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+purify+bacteria-containing+phagosomes+from+infected+macrophages.">A method to purify bacteria-containing phagosomes from infected macrophages.</a> Methods Cell Sci. 22 (4), 329-341 (2000).</li><li>Steinhauser, 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=Lipid-labeling+facilitates+a+novel+magnetic+isolation+procedure+to+characterize+pathogen-containing+phagosomes.">Lipid-labeling facilitates a novel magnetic isolation procedure to characterize pathogen-containing phagosomes.</a> Traffic. 14 (3), 321-336 (2013).</li><li>Weischenfeldt, J., Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Marrow-Derived+Macrophages+(BMM):+Isolation+and+Applications.">Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.</a> CSH Protoc. 2008, (2008).</li><li>Detilleux, P. G., Deyoe, B. L., Cheville, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+and+intracellular+localization+of+Brucella+spp.+in+Vero+cells:+fluorescence+and+electron+microscopy.">Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy.</a> Vet Pathol. 27 (5), 317-328 (1990).</li><li>Arenas, G. N., Staskevich, A. S., Aballay, A., Mayorga, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+trafficking+of+Brucella+abortus+in+J774+macrophages.">Intracellular trafficking of Brucella abortus in J774 macrophages.</a> Infect Immun. 68 (7), 4255-4263 (2000).</li><li>West, A. 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A new method for the isolation of S. typhimurium-containing phagosomes by coating the bacteria with biotin and streptavidin-conjugated magnetic beads is described here. After gentle disruption of the cell membrane, bacteria-containing phagosomes can be easily extracted using a magnetic rack. We show that labeling of the bacteria preserves the capacity of the pathogen to induce inflammation and does not alter the phagocytic property of the host cell. Importantly, phagosomes obtained by this method are en...

Macrophages are circulating specialized phagocytic cells that detect, engulf, and degrade any foreign particle present in peripheral tissues, ranging from apoptotic cells to invading microorganisms such as bacteria. Upon surface receptor-mediated recognition of pathogen specific markers commonly present on the surface of microorganisms (known as pathogen-associated molecular patterns or PAMPs), macrophages initiate a complex reorganization of cellular membrane in order to surround and phagocytize the pathogen1.
The engulfed pathogen is then contained by the macrophage in an intracellular vesicle known as phagosome. Through a series of fusion and fission events with other vesicles such as endosomes and lysosomes, the pathogen-containing phagosome acquires a set of proteins required for the elimination of the phagosomal content. Therefore, the enzymatic composition of the phagosome is highly variable during the course of this process, known as phagosome maturation2.
Shortly after phagocytosis, the multimeric complex vacuolar ATPase (v-ATPase) is incorporated into the phagosome membrane by fusion with endosomes3. This complex utilizes ATP to pump protons from the cytosol to the lumen of the phagosome4. Acidification of the phagosome is essential for the fusion events with other vesicles5 and for the activation of a great number of pH-dependent degradative enzymes6. Another multimeric enzymatic complex that is quickly assembled on the phagosome membrane is the NADPH-oxidase (NOX) complex. NOX complex oxidizes NADPH in order to produce reactive oxygen species (ROS) that are secreted into the phagosome lumen and that significantly contribute to the killing of the engulfed microorganisms7.
During the initial steps of maturation, phagosomes present markers typically such as Rab5 and Rab7 of early and late endosomes respectively along with the V0 subunit of the v-ATPase8. Fusion of phagosomes with lysosomes and late endosomes results in the exposure of the phagocytized pathogen to a wide variety of hydrolytic enzymes such as cathepsin proteases, lipases, and &#946;-galactosidase9. Acidification of the lumen is also required for the activation of these enzymes. For example, the cleavage of cathepsin D to produce the active short form is pH-dependent10. These enzymes degrade the pathogen and mediate the production of pathogen-derived short peptides that are presented by the macrophage major histocompatibility complex (MHC) class II molecules to T cells to trigger an adaptive immune response11.
Hence, phagosome maturation is crucial for the innate immune response and links the innate and adaptive arms of the immune system. It is no surprise that the pathogens have evolved strategies to overcome elimination by macrophages through the above-described process of phagosome maturation. For example, the intracellular bacteria Mycobacterium tuberculosis and Legionella pneumophila prevent phagosome maturation by inhibiting v-ATPase assembly and consequent lumen acidification12,13. Other bacteria, such as Listeria monocytogenes or Shigella flexneri induce pore formation in the phagosome membrane to escape into the cytosol14,15. On the other hand, Salmonella enterica serovar typhimurium (S. typhimurium) is able to modify the properties of the phagosome within the vacuole to transform it into a suitable location for its replication16. This ability makes S. typhimurium a very interesting model to study pathogen-mediated interference of phagosome maturation.
S. typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. Typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained within phagosomes17. Some reports have previously described that S. typhimurium-containing phagosomes present makers for both endosomes and lysosomes18, and other studies have found phagosome-lysosome fusion prevented upon S. Typhimurium infection19.
Initially, phagosome maturation upon S. typhimurium infection has been investigated by immunofluorescence microscopy. The development of techniques for the isolation of bacteria-containing phagosomes enabled a more accurate study of the phagosome content in terms of endosome and lysosome markers. To date, the main method used for the isolation of bacteria-containing phagosomes is the subcellular fractionation on sucrose step gradients18,20. However, this method requires multiple centrifugation steps that can cause mechanical damage to phagosomes, can affect the stability of phagosomal components (proteins and lipids), and is time consuming. Moreover, it requires the use of an ultracentrifuge: a piece of specialized equipment that is not accessible for every laboratory.
Recently, a new approach has been applied to the isolation of bacterial-containing phagosomes, in which bacterial pathogens are labeled with biotinylated lipopetide (Lipobiotin) and later extracted using streptavidin-conjugated magnetic beads21. We propose an alternative complementary method by labeling bacterial surface amine-containing macromolecules with NHS-Biotin followed by streptavidin-conjugated magnetic beads. Phagosomes obtained by this method are highly enriched in endosome and lysosome markers and can be used for a broad range of assays, from protein analysis to omics analysis. Additionally, it does not require specialized equipment such as ultracentrifuges. Moreover, by eliminating the centrifugation steps, both the mechanical damage to phagosomes and the amount of time employed are considerably reduced. This method can be easily adapted for the isolation of phagosomes containing other bacteria, such as the Gram-positive Staphylococcus aureus, also included in this manuscript. In summary, this method for the isolation of S. typhimurium-containing phagosomes is a simple, cost-effective, and less time consuming than the classical isolation by sucrose gradient-ultracentrifugation, rendering highly enriched bacteria-containing phagosomes.

<table>
	<tbody>
		<tr>
			<td>EZ-Link NHS Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>20217</td>
			<td></td>
		</tr>
		<tr>
			<td>FluidMag Streptavidin</td>
			<td>Chemicell</td>
			<td>4205</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Carl Roth</td>
			<td>9156.2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>A537.4</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Carl Roth</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Carl Roth</td>
			<td>4175.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>43816</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase inhibitor cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>1861280</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin B</td>
			<td>Sigma</td>
			<td>C6762</td>
			<td></td>
		</tr>
		<tr>
			<td>DYNAL or DynaMag Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartSpec 3000 Spectrophotometer</td>
			<td>Bio-Rad</td>
			<td>170-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial loop (10&#181;l)</td>
			<td>Sarstedt</td>
			<td>86.1562.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella enterica serovar Typhimurium SL1344</td>
			<td>Leibniz Institute DSMZ-German collection of Microorganisms and Cell Cultures</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Biochrom</td>
			<td>FG1415</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biochrom</td>
			<td>L1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5-streptavidin</td>
			<td>Invitrogen</td>
			<td>SA1011</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-beta-actin antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-47778</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mCherry antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA5-34974</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rab5 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-46692</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rab7 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-10764</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-v-ATPase (V0) antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-28801</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-v-ATPase (V1) antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-20943</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-cathepsin D antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-6486</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Tomm20 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-17764</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-calnexin antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-46669</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GAPDH antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-20357</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of salmonella typhimurium-containing phagosomes from macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of bacteria-containing phagosomes by this protocol requires the biotinylation of the bacteria as a first step. We therefore assessed the effectiveness of S. typhimurium biotinylation by confocal microscopy analysis of BMDMs infected with biotinylated mCherry-S. typhimurium labeled with Cy5-Streptavidin. Briefly, BMDMs were infected as described in this protocol with mCherry-S. typhimurium previously biotinylated but not incub...

Watch this Scientific Journal Video about Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages at JoVE.com

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

The overall goal of this procedure is to isolate intact and enriched bacteria containing Phagosomes. This method can help us answer key questions in the field of Immunology such as changes in Phagosome maturation and antigen processing when pathogenic microorganisms invade host cells. The main advantage of the technique is that it produces high quality bacteria containing Phagosomes using simple steps and without a requirement of specialized equipment.Individuals new to the method would find it technically approachable in comparison to the non methodologies. We first had the idea to develop this method when we were studying the mechanisms by which intracellular pathogens hijack the Phagosomes to their advantage. Demonstrating this procedure will be Saray Gutierrez, a post-doc from my laboratory.To start Salmonella Typhimurium culture, use a bacteria loop to inoculate a single bacterial colony into 5 milliliters of brain heart infusion broth. Then incubate the suspension in a 37 degrees Celsius incubator overnight while shaking. Transfer one milliliter of this bacterial suspension into 19 milliliters BHI broth in a conical flask on the following day.Incubate again in 37 degrees Celsius incubator while shaking. When the OD600 reaches one, remove the flask from the incubator and transfer the culture into a 50 milliliter tube. Then centrifuge the tube at 5400 x G at 4 degrees Celsius for 15 minutes.After removing the supernatant, re-suspend the bacterial pellet in 10 milliliters sterile PBS. After repeating the centrifugation once, re-suspend the pellet in 4.9 milliliters of PBS. Next, add 100 microliters of freshly prepared Biotin linking solution to the bacterial suspension.After splitting this suspension into five 1.5 milliliter tubes, incubate in a thermal block at room temperature with constant shaking at 350 rpm for two hours. After centrifuging the tubes at 15, 000 x G at room temperature for ten minutes, discard the supernatant. After two more centrifugation steps and complete removal of the Biotin linking solution, re-suspend this Biotin-coded bacteria in one milliliter sterile PBS in a single 1.5 milliliter tube.Transfer 100 microliters of streptavidin-conjugated magnetic beads solution into a fresh 1.5 milliliter tube and place it in a magnetic rack for five minutes. After removing the solvent from the tube, remove the tube from the magnetic rack. Then re-suspend the beads that remained in the tube with one milliliter of Biotin coded bacterial suspension.Incubate the tube on a thermo block at room temperature for one hour while shaking at 350 rpm. Next, place the tube on the magnetic rack for five minutes. Use a pipette to remove the bacteria that did not adhere to the wall and transfer to a new tube labeled non-coded bacteria.The tube with magnetic beads adhering to the wall contains Biotin Streptavidin coded bacteria. Remove the tube with Biotin Streptavidin coded bacteria from the magnetic rack and re-suspend in one milliliter sterile PBS. After the tube has been on the magnetic rack for five minutes, remove the PBS.Finally, re-suspend the washed Biotin Streptavidin coded bacteria in 500 microliters of sterile PBS and label the tube as coded bacteria. On the day prior to infection, plate fully differentiated BMDMs in a six centimeter dish in RPMIs supplemented with ten percent FBS. On the following day, centrifuge the coded bacterial suspension at 15, 000 x G at four degrees Celsius for five minutes.After removing the supernatant, re-suspend the bacterial pellet in RPMI medium supplemented with ten percent FBS. Remove the medium from the BMDMs and add five milliliters of coded bacterial suspension in RPMI medium in order to infect the cells. To synchronize Phagocytosis, incubate at room temperature for ten minutes.Then to initiate Phagocytosis, incubate at 37 degrees Celsius in a five percent Carbon Dioxide incubator for 30 minutes. After incubation, remove the medium from the dishes and wash the cells three times at RPMI to remove non internalized bacteria. To kill any non-Phagocytized bacteria at ten milliliters of RPMI containing ten percent FBS in 50 micrograms per milliliter Gentamicin.Finally, incubate the infected BMDMs at 37 degrees Celsius in a five percent Carbon Dioxide incubator until the desired time point. First, prepare the volume of required Phagosome Isolation Buffer A by adding DTT and Cytochalasin B just before use and Protease and Phosphatase Inhibitors as recommended by the manufacturer. Remove the medium from the infected cells at the desired time point and wash the cells with sterile PBS pre-warmed to room temperature.Then add 750 microliters of Phagosome Isolation Buffer A to the dish and incubate on ice for 20 minutes. Remember to rock the plate occasionally. Next, add 250 microliters of Phagosome Isolation Buffer B and then rock the plates to ensure that the buffer completely covers the surface.Gently scrape the cells using a rubber policeman to remove the cells from the dish and transfer them to a pre-chilled 1.5 milliliter tube. Then use a one milliliter syringe to pass the cell suspension thru a 26-gauge needle at least 15 times. Next, place the cell suspension on the magnetic rack for five minutes.Particles attached to the magnetic beads are Phagosomes containing coded S.Typhimurium. Transfer then the suspension which contains the rest of the cellular components into a new 1.5 milliliter tube labeled as Cytosol. Remove the tube containing isolated Phagosomes from the rack and re-suspend in one milliliter sterile PBS.Place the tube back on the magnetic rack for five minutes and then remove the PBS. After repeating this washing with PBS once, remove the PBS and finally, re-suspend S.Typhimurium containing Phagosomes in the required buffer. To assess effectiveness of S.Typhimurium Biotinylation by confocal microscopy, BMDMs were infected with mCherry S.Typhimurium labeled with Biotin.After fixation and incubation with Cy5-Streptavidin, successful Biotinylation was confirmed by Cy5-Streptavidin fluorescent signal. This signal was observed exclusively in Biotinylated mCherry S.Typhimurium whereas there was no signal in not Biotinylated mCherry S.Typhimurium. Immunoblock analysis of isolated Phagosomes showed significant enrichment of mCherry expressing S.Typhimurium in Phagosomes in comparison to the cytosolic fraction.Immunoblock analysis using endosome and lysosome markers in isolated Phagosomes showed enrichment of the tested markers in Phagosomes isolated four hours after infection compared to 30 minutes after infection. When this protocol was applied to Staphylococcus aureus, successful Biotinylation of GFP expressing S.aureus was shown. Once mastered, this technique can be done in approximately one hour and a half depending on the incubation time chosen for infection by the investigator.To ensure the success of the isolation of bacteria containing Phagosomes, please make sure that the buffers contain the indicated concentrations and that the plates are rocked to ensure the homogeneous coverage of the cells. Also, be gentle when scraping the cells and when passing the cell suspension through the needle. Following this procedure, other techniques such as proteomics or metabolomics can be performed with isolated Phagosomes to study alteration caused by pathogens under pathogenic factors involved.

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Research

JoVE Journal

Immunology and Infection

10.5K 조회수.  University of Cologne. The overall goal of this procedure is to isolate intact and enriched bacteria containing Phagosomes. This method can help us answer key questions in the field of Immunology such as changes in Phagosome maturation and antigen processing when pathogenic microorganisms invade host cells. The main advantage of the technique is that it produces high quality bacteria containing Phagosomes using simple steps and without a requirement of specialized equipment.Individuals new to the method woul...