Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Summary

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.

Abstract

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.

Introduction

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 β-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 acidification^12,13. Other bacteria, such as Listeria monocytogenes or Shigella flexneri induce pore formation in the phagosome membrane to escape into the cytosol^14,15. 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 gradients^18,20. 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.

Protocol

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 from mice for its differentiation into macrophages was performed in accordance with institutional guidelines on animal welfare and approved by the North Rhine-Westphalian State Agency for Nature, Environment, and Consumer Protection [Landesamt für Natur, Umwelt and Verbraucherschutz (LANUV) Nordrhein-Westfalen; File no: 84-02.05.40.14.082 and 84-02.04.2015.A443] and the University of Cologne.

1. Culturing S. typhimurium

1. Inoculate S. typhimurium (SL1344 strain) from a single bacterial colony into 5 mL of brain heart infusion (BHI) broth using a bacterial loop.
2. Incubate the bacterial suspension in an incubator at 37 °C overnight with shaking.
3. On the following day, transfer 1 mL of the bacterial suspension into 19 mL of BHI broth in a conical flask and incubate at 37 °C in an incubator with shaking.
4. Monitor the S. typhimurium growth by measuring the optical density (OD) at 600 nm (OD₆₀₀) with a spectrophotometer. Measurements should be taken approximately every 30 min.
5. When OD₆₀₀ reaches 1.0, remove bacterial suspension from the incubator and transfer into 50 mL tube. Centrifuge bacteria at 5,400 x g for 15 min at 4 °C.
6. Remove supernatant and resuspend in 10 mL of sterile phosphate-buffered saline (PBS).
7. Centrifuge at 5,400 x g for 15 min at 4 °C.
8. Remove the supernatant and resuspend the bacteria in 4.9 mL of sterile PBS.

2. Coating of S. typhimurium with NHS-Biotin/Streptavidin-conjugated Magnetic Beads

1. Add 100 µL of 10 mg/mL biotin linking solution (NHS-Biotin dissolved in dimethyl sulfoxide, DMSO) freshly prepared, to the bacterial suspension prepared in the previous step. For example, dilute 5 mg of NHS-Biotin into 0.5 mL of sterile DMSO. Mix properly by pipetting up and down several times.
2. Split the mix into five 1.5 mL tubes.
3. Incubate for 2 h at room temperature (RT) on a thermoblock with constant shaking at 350 rpm.
4. Centrifuge tubes at 15,000 x g for 10 min at RT and discard the supernatant.
5. Resuspend in 1 mL of sterile PBS, centrifuge at 15,000 x g for 10 min at RT and discard the supernatant.
6. Repeat step 2.5 two more times to completely remove the excess biotin linking solution.
7. After the last wash, resuspend the pellet in 1 mL of sterile PBS.
8. Transfer 100 µL of 10 mg/mL streptavidin-conjugated magnetic beads solution into a 1.5 mL tube and leave it on the magnetic rack for 5 min.
9. Remove the solvent with a pipette, take the streptavidin- conjugated magnetic beads solution from the magnetic rack, and resuspend it with 1 mL of biotin coated-bacterial suspension prepared in step 2.7.
10. Incubate for 1 h at RT on a thermoblock with constant shaking at 350 rpm.
11. Place the solution on the magnetic rack; wait for 5 min and then remove with a pipette the bacteria that did not adhere onto the wall (keep it in a tube labeled as "non-coated bacteria"). The fraction adhering to the wall of the tube in contact with the magnet is the biotin/streptavidin-coated bacteria.
12. Remove the tube containing the labeled bacteria from the magnetic rack and resuspend in 1 mL of sterile PBS.
13. Place it again on the magnetic rack, wait for 5 min, and use a pipette to remove the PBS.
14. Repeat steps 2.13 and 2.14 two more times to remove all the bacteria that are not coated with the streptavidin-conjugated magnetic beads.
15. After the last wash, resuspend the coated-bacteria in 500 µL of sterile PBS and label the tube as "coated bacteria".
16. Count the colony forming units (cfu) of both "non-coated bacteria" and "coated bacteria" solutions by plating serial dilutions on BHI agar plates. Approximately 2 x 10⁸ cfu/mL of coated-bacteria are obtained from 20 mL of initial bacterial suspension with OD₆₀₀ of 1.0. Keep the coated-bacterial suspension at 4 °C to be used on the following day to infect macrophages.

3. Infection of BDMDs

1. On the same day when the bacteria are coated with biotin and streptavidin-conjugated magnetic beads, plate fully the differentiated BMDMs²² in 6 cm dishes at a density of 5 x 10⁶ cells per dish.
2. On the next day, centrifuge the coated-bacterial suspension at 15,000 x g for 5 min at 4 °C.
3. Remove the supernatant and resuspend in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS) at a concentration of 10 x 10⁶ cfu/mL.
4. To infect macrophages at a multiplicity of infection (MOI) of 10, remove the medium from the BMDMs and add 5 mL of coated-bacterial suspension prepared. Optimal MOI may be assessed for every cell type or experimental purpose of the isolated phagosomes.
5. Incubate at RT for 10 min to synchronize phagocytosis.
6. Incubate at 37 °C in a 5% CO₂ incubator for 30 min to initiate phagocytosis. Other cell types may require different incubation times to ensure internalization of the bacteria.
7. Remove the bacteria-containing medium from the dishes and wash the cells with RPMI three times to remove non-internalized bacteria.
8. Finally add 10 mL of RPMI containing 10% FBS and 50 µg/mL gentamycin to kill any non-phagocytized bacteria.
9. Incubate the infected BMDMs at 37 °C with 5% CO₂ until the desired time point. The desired time point must be experimentally determined according to the bacteria and cell type used and the purpose of analysis. For the study of early phagosome-endosome fusion events in S. Typhimurium-infected BMDMs, a 30 min incubation is recommended. For the analysis of later events, phagosomes can be extracted after 2 h or 4 h of incubation. Prolonged incubation of macrophages with S. Typhimurium beyond 24 h results in death of the macrophages. Extended intracellular infection before the phagosome extraction could probably lead to degradation of biotin molecules. However, we did not observe loss of biotin on coated-bacteria until 24 h.

4. Isolation of S. typhimurium-containing Phagosomes

1. Prepare the volume of required phagosome isolation buffer A (50 mM PIPES, pH.7, 50 mM MgCl₂, 5 mM EGTA) by adding dithiothreitol (DTT) to a final concentration of 1 mM, Cytochalasin B to a final concentration of 10 µM, and protease and phosphatase inhibitors as recommended by the manufacturer.
   NOTE: DTT and Cytochalasin B must be added to the phagosome isolation buffer A always immediately before use. Cytochalasin B disrupts the cytoskeleton, facilitating the rupture of the cell membrane.
2. At the desired time point, remove the medium from the infected cells and wash with sterile PBS warmed to RT.
3. Add 750 µL of phagosome isolation buffer A per dish and incubate 20 min on ice. When using different cell numbers, the volume of isolation buffer A should be adjusted proportionally.
4. Add 250 µL of phagosome isolation buffer B (50 mM PIPES, pH.7, 50 mM MgCl₂, 5 mM EGTA, 220 mM Mannitol, and 68 mM sucrose).Rock the plate to ensure that the buffer reaches the complete surface of the dish. When using different cell numbers, the volume of isolation buffer B should be adjusted proportionally.
5. Remove the cells from the dish by gently scraping using a rubber policeman and transfer them to a pre-chilled 1.5 mL tube.
6. Pass the cell suspension through a 26G needle using a 1 mL syringe at least 15 times (one aspiration plus one ejection of the cell suspension counts as one time). This is enough to release the cytosolic content of BMDMs. The number of passes through the needle must be optimized for every cell type.
7. Place the cell suspension on the magnetic rack and wait 5 min. The particles attached to the magnet are the phagosomes containing coated-S. Typhimurium. The suspension contains the rest of the cellular components.
8. Transfer the suspension into a 1.5 mL tube labeled as "cytosol".
9. Remove the tube with the isolated phagosomes from the magnetic rack and resuspend them in 1 mL of sterile PBS.
10. Place the phagosome suspension on the magnetic rack, wait for 5 min, and remove the PBS.
11. Repeat steps 4.9 and 4.10 to wash the isolated S. typhimurium-containing phagosomes.
12. Finally remove the PBS and resuspend the phagosomes in the required buffer; for example, in a radioimmunoprecipitation assay (RIPA) buffer for protein analysis. Approximately 50-200 µg of protein is obtained per sample following this protocol, depending on the initial amount of cells and the MOI of infection.

Results

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...

Discussion

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...

Materials

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