Tomoe Kitao1, Kohei Arasaki2, Hiroki Nagai1,3, Tomoko Kubori1,3. 1. Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Gifu 501-1194, Japan. 2. School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan. 3. G-CHAIN, Gifu University, Gifu, Gifu 501-1194, Japan.
Abstract
The intracellular bacterial pathogen Legionella pneumophila exploits host cellular systems using approximately 300 effector proteins to establish a replicative niche known as the Legionella-containing vacuole (LCV). During infection, both host and bacterial proteins interactively function on the LCVs. Here, we describe a detailed step-by-step protocol to visualize proteins associated with LCVs in host cells. This protocol can aid in analyzing whether a protein of interest influences the subcellular localization of LCV-associated proteins during infection. For complete details on the use and execution of this protocol, please refer to Kitao et al. (2020).
The intracellular bacterial pathogen Legionella pneumophila exploits host cellular systems using approximately 300 effector proteins to establish a replicative niche known as the Legionella-containing vacuole (LCV). During infection, both host and bacterial proteins interactively function on the LCVs. Here, we describe a detailed step-by-step protocol to visualize proteins associated with LCVs in host cells. This protocol can aid in analyzing whether a protein of interest influences the subcellular localization of LCV-associated proteins during infection. For complete details on the use and execution of this protocol, please refer to Kitao et al. (2020).
In this manuscript, we describe a step-by-step protocol of immunofluorescence microscopy to analyze whether a target protein can localize to a bacteria-containing vacuole during infection using bacteria-infected cells. In addition, we describe an optional protocol to examine whether a protein of interest influences the subcellular localization of the bacterial vacuole-associated proteins during infection by using a semi-permeabilization technique (Kitao et al., 2020; Kubori et al., 2017).We use the intracellular bacterial pathogen Legionella pneumophila as a model organism for this protocol. According to biosafety guidelines, L. pneumophila is classified as a biosafety level 2 organism. Any experiments using L. pneumophila described here should be approved by the institutional biosafety committee and carried out according to the recombinant DNA committee guidance. Once approved, please make sure that the reagents and materials shown below are ready for use before starting the main protocol.
Preparation of bacterial strain(s)
Timing: 3 daysWhen analyzing whether a L. pneumophila effector protein of interest influences the subcellular localization of host protein that is already known to be recruited to the Legionella-containing vacuole (LCV) upon infection, use the L. pneumophila wild-type strain (positive control) and its isogenic mutant(s) lacking the gene(s) encoding the effector protein(s).Prepare the L. pneumophila deletion mutant strains as per the protocol described previously (Zuckman et al., 1999).Pick up a trace of the L. pneumophila wild-type strain Lp01 and its isogenic mutant(s) lacking the gene(s) of interest, from the stocks stored at −80°C, using autoclaved flat toothpicks, and streak them on charcoal yeast extract (CYE) agar plates.Incubate the plates at 37°C for 72 h (single colonies usually appear after 3 days).
Maintenance of host cells
Timing: 3 daysWe use exponentially growing healthy HEK293T cells that constitutively express Fc gamma receptor II (HEK293T-FcγRII) or HeLa-FcγRII cells in this protocol for Legionella infection experiments. Both cell lines were maintained in 10-cm cell culture dishes containing 10 mL Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 200 μg/mL hygromycin.Thaw HEK293T-FcγRII or HeLa-FcγRII cells from the liquid nitrogen stocks and transfer them into a 15-mL conical tube containing 10 mL prewarmed DMEM supplemented with 10% FBS.Centrifuge the tube with swing buckets at 220 × g for 5 min at room temperature (15°C–25°C).Remove the supernatant and resuspend the precipitated cells in 10 mL of prewarmed DMEM supplemented with 10% FBS.Plate the cell suspension into 10-cm cell culture dish.Incubate at 37°C and 5% CO2 for 24 h.Detach the cells from the dish.HEK293T-FcγRII.Remove the cell culture medium from a dish with semi-confluent cells.Add 10 mL of prewarmed fresh DMEM supplemented with 10% FBS.Suspend the surface-attached cells by pipetting (detach by mechanical force).HeLa-FcγRII.Remove the cell culture medium from a dish with semi-confluent cells.Add 2 mL of trypsin/EDTA solution to the dish. (This treatment induces chemical detachment of the cells from the surface of the dish).Incubate the dish at 37°C for 5 min in a CO2 incubator.Gently tap the dish to completely detach the cells.Add 8 mL of prewarmed fresh DMEM supplemented with 10% FBS and suspend the cells by gentle pipetting.Cell passage: transfer the detached cells with an appropriate dilution to a new dish.HEK293T-FcγRII: Transfer 1 mL of the cell suspension to a new dish containing 9 mL of prewarmed DMEM supplemented with 10% FBS (1:10 split).HeLa-FcγRII: Transfer 2 mL of the cell suspension to a new dish containing 8 mL of prewarmed DMEM supplemented with 10% FBS (1:5 split).Add 40 μL of 50 mg/mL hygromycin.CRITICAL: Hygromycin is required for stable expression of FcγRII in our cell lines. We added hygromycin from the second passage.Incubate the dishes containing the diluted cells at 37°C and 5% CO2 in an incubator for 3 days to allow them to reach confluency.
Plasmid preparation for transfection
Timing: 24–36 hWe used endotoxin-free plasmids for transfection. Endotoxin-free plasmids can be prepared using EndoFree Plasmid MAXI prep kits (QIAGEN) according to the manufacturer’s instructions. This protocol describes how to detect GFP-tagged proteins of interest recruited to bacterial vacuoles during infection. A plasmid encoding a GFP-tagged protein must be constructed before starting this protocol. A plasmid encoding only GFP is required as a negative control.Pick up a trace of Escherichia coli DH5α carrying a plasmid encoding GFP or GFP-tagged protein from −80°C frozen stocks using a sterilized flat toothpick and streak them on LB agar plates containing 20 μg/mL kanamycin. (The vector used in this protocol encodes a β-lactamase gene that confers kanamycin resistance).Incubate the plate at 37°C overnight (18–20 h).Pick up a single colony and inoculate them in 100 mL LB medium containing 20 μg/mL kanamycin in a 200 mL flask.Shake at 37°C and 250 rpm, overnight (16–18 h).Transfer the bacterial culture into a 50-mL conical tube and centrifuge at 6,000 × g for 5 min at 4°C.Remove the supernatant.Pause point: The bacterial pellets can be stored at −80°C until purification.Purify the plasmid according to the manufacturer's instructions.
Purified protein
If you have a purified protein of interest that may influence the LCV-associated proteins, you can test the protein by using semi-permeabilized host cells infected with L. pneumophila. Prepare 100–200 μL of 1–2 mg/mL protein if necessary.
Key resources table
Materials and equipment
Preparation of bacterial strains
LB agar plates containing 20 μg/mL kanamycinSterilized swabsFlat toothpicksCYE platesDissolve ACES and yeast extract in approximately 800 mL of MilliQ water, then adjust the pH to 7.0 with KOH. After adjusting the volume to 1,000 mL, add it into a 2-L-flask containing charcoal and bacto agar. Autoclave it at 121°C for 20 min.After autoclaving, allow it to cool to around 60°C in a water bath. Prepare 40 mg/mL of L-cysteine solution (>10 mL) and 13.5 mg/mL of Iron(III) nitrate solution (>10 mL) separately in sterilized MilliQ water and filter them with a 0.22 μm disk filter. Add 10 mL of each to 1 L autoclaved CYE (The L-cysteine should be added first, followed by the iron solution to prevent precipitation.). If needed, add appropriate concentrations of antibiotics. Mix well using a magnetic stirrer and pour the media into sterilized petri dishes (approximately 30–40 mL/dish). Let the media cool down and dry the plates 2 overnights at room temperature (15°C–25°C). The CYE plates can be stored at 4°C. Remove moisture on the lids before the plates are stored.SpectrophotometerDisposable cuvette
Host cell maintenance
Cell culture-treated dishes (10-cm)Cell culture-treated plates (24-well)DMEM supplemented with 10% FBS filtered with 0.22 μm filterHygromycin (50 mg/mL)Trypsin/EDTA solutionCentrifuge for 24-well platesWater bath37°C CO2 incubator
Transfection
Polyethyleneimine solution (PEI)Dissolve PEI at a concentration of 2.0 mg/mL in MilliQ water, adjust the pH to 8.0 with 1N NaOH. Then, filter it with a 0.22 um disk filter. The solution can be dispensed into small aliquots (∼1 mL) and stored at −20°C.
Immunofluorescent staining
4% Paraformaldehyde (PFA)Phosphate buffer saline (PBS)2% Goat serum diluted with PBS (2% GS/PBS)0.2% Triton X-100Glass slidesSterilized round coverslipsProLong™ Diamond antifade mountantSemi-permeabilization buffer (Optional)Dissolve glucose in 450 mL water and add all the remaining reagents. After making-up the volume to 500 mL using MilliQ water, filter it with Stericup (0.22 μm).DTT (final concentration 1 mM) is added before use.
Step-by-step method details
Preparation of bacterial strains (day 1)
Timing: 30–45 minFor the infection experiment, on day 3, pick up a single colony of bacteria and re-streak on a new CYE agar plate to prepare “2-day-heavy patch” of the strain.Warm up a new CYE plate at 37°C to remove condensation.Pick up a single colony using a sterilized flat toothpick and re-streak it on the CYE plate.Incubate the plate at 37°C for 2 days (48 h).
Host cell preparation (day 1)
Timing: ∼2 hBefore starting this protocol, please make sure that you have semi-confluent cell culture plates. A single semi-confluent plate provides a sufficient number of cells to prepare one 24 well plate. For microscopy, we seed the cells in 24-well cell culture plates with round glass coverslips 24 h before transfection. For the HEK293T-FcγRII cells, the coverslips should be coated with poly-L-lysine (PLL) as described in steps 4–7 to increase the adherence of the cells. For the HeLa-FcγRII cells, steps 5–7 can be skipped.Place sterilized round glass coverslips in the 24-well cell culture plates. Set them up in triplicates for each sample. For example, if you analyze the subcellular localization of a GFP-tagged protein in host cells infected with two L. pneumophila strains, such as the wild-type and an isogenic mutant strain, you need to prepare the cells in six wells (three wells for the wild-type strain and three wells for the mutant strain).Add 250 μL of 0.001% (w/v) poly L-lysine solution to the coverslip in each well of the 24-well plates. Sink the coverslips using a sterilized pipet if they begin to float.Gently incubate the plates in an orbital shaker for 1 h at room temperature (15°C–25°C).Remove the poly-L-lysine solution and wash the coverslips in the wells with 500 μL/well of sterilized MilliQ water three times.Remove the cell culture medium from the semi-confluent 10 cm dish with cells.Add prewarmed 10 mL PBS.Detach the cells from the dish.For HEK293T-FcγRII cellsRemove PBS.Add 10 mL of prewarmed fresh DMEM supplemented with 10% FBS.Detach the cells by pipetting.Transfer the cells to a sterilized 50-mL conical tube.For HeLa-FcγRII cellsRemove PBS.Add 2 mL of the Trypsin/EDTA solution.Incubate the dish for 5–10 min in a 37°C CO2 incubator.Gently tap the dish to detach the cells.Add 8 mL of prewarmed fresh DMEM supplemented with 10% FBS and suspend the cells by gentle pipetting.Transfer the cells to a sterilized 50-mL conical tube.Count the cell number using a hemocytometer.Seed the cells into 24-well plates with PLL-coated coverslips (0.8 × 105 cells/well for HEK293T-FcγRII cells; 0.4 × 105 cells/well for HeLa-FcγRII cells).CRITICAL: Use hygromycin-free DMEM supplemented with 10% FBS.Incubate the plate at 37°C in a CO2 incubator for 24 h.
Plasmid transfection (day 2)
Timing: 30–50 minReplace the cell culture medium with fresh and prewarmed DMEM supplemented with 10% FBS.Twenty-four hours prior to infection, transfect the host cells with plasmid DNA encoding a GFP-tagged desired protein using PEI. In the following protocol, the indicated amount of reagents is required for cells in a single well.Dilute 1 μL of PEI with 50 μL of Opti-MEM (Gibco) in a 1.5-mL microfuge tube.Incubate the tube for 15 min at room temperature (15°C–25°C).Add 1.25 μL of a 100 ng/μL endotoxin-free plasmid.Incubate the tube at room temperature (15°C–25°C) for 15 min.Drop PEI/plasmid/Opti-MEM mixture into cells seeded in the 24-well plates.We used PEI for transfection to prevent saturated signals of GFP-tagged protein. Other transfection reagents, such as lipofectamine, can also be used to transfect host cells. Follow the manufacturer’s instructions for each product.Incubate the plates at 37°C in a CO2 incubator for 24 h.
Legionella infection (day 3)
Timing: 2 h-1 day ∗Varies with time of infectionThe following protocol is an example of a case in which host cells are infected with L. pneumophila at a multiplicity of infection (MOI) of 5.Prepare L. pneumophila suspension in sterilized MilliQ water.Scrape the appropriate amount of a 2-day heavy patch of L. pneumophila grown on a CYE plate using a sterilized pipette tip (1,000 μL size) or flat toothpick and resuspend in 500 μL of sterilized MilliQ water.Mix the bacterial solution well using a vortex and measure the absorbance of the suspension using a spectrophotometer at 600 nm (OD600).Prepare the bacterial suspension with an OD600 value of 0.1, which approximately corresponds to 1 × 105 bacteria/μL, in a new 1.5-mL microfuge tube.Opsonize L. pneumophila with anti-Legionella antibody to enhance FcγRII-mediated bacterial internalization. The preparation shown below is sufficient for cells in three wells.Prepare 2 mL of prewarmed DMEM supplemented with 10% FBS in a sterilized 10-mL conical tube.Add 0.7 μL (1/3,000 volume) of anti-Legionella rabbit serum.Add L. pneumophila (16 μL of 1 × 105 cells/μL L. pneumophila for HEK293TFcγRII; 8 μL of 1 × 105 cells/μL L. pneumophila for HeLa-FcγRII).Shake the tubes at 37°C at 250 rpm for 30 min.Infect cells with the opsonized L. pneumophila.Remove the cell culture medium from the wells of the 24-well cell culture plates and dispense DMEM containing the opsonized L. pneumophila prepared in step 18 to the wells (500 μL/well).Centrifuge the cell culture plate at 220 × g for 5 min at room temperature (15°C–25°C) with a swing bucket rotor. This procedure involves the attachment of bacteria to the surface of the adherent cells because the L. pneumophila strains used here are non-motile.To initiate infection, float the plate in a 37°C water bath for 5 min immediately after the centrifuge is stopped (Time zero of infection).Incubate the plate at 37°C in a CO2 incubator for 55 min.At 1-h post-infection, gently wash the cells with prewarmed PBS (1 mL/well) three times to remove extracellular bacteria, and then add prewarmed DMEM supplemented with 10% FBS. Resume the incubation of the plate at 37°C in a CO2 incubator (time can vary as per the requirement).You can skip step 20 if the sample is taken at 1 h or less post infection.Gently wash the cells with prewarmed PBS (1 mL/well) three times at the time of sampling.Remove PBS and add 500 μL/well of 4% PFA/PBS solution to fix the cells and incubate the plates at room temperature (15°C–25°C) for 15 min.Wash the cells with PBS (1 mL/well) three times.The following protocol is to analyze whether a protein of interest may influence another protein, which is already known to be recruited to LCVs, by using the combination of a purified protein and semi-permeabilized host cells. Digitonin is used for semi-permeabilization in this protocol. At a given concentration of digitonin permeabilizes only plasma membrane (Arasaki et al., 2012). In some cases, the concentration of digitonin may be required to be optimized.After L. pneumophila infection, wash the cells on the coverslips in the 24-well cell culture plates with prewarmed 500 μL/well of the semi-permeabilization buffer twice.Remove the semi-permeabilization buffer and add 500 μL/well of fresh semipermeabilization buffer containing 3 μg/mL digitonin.Incubate at room temperature (15°C–25°C) for 4 min.Gently wash the cells with 500 μL/well of semi-permeabilization buffer three times.Add 250 μL/well of semi-permeabilization buffer containing 1–10 μg/mL protein to be analyzed. Pilot tests should be done to determine the optimal concentration of protein in advance.Incubate the plates at 37°C in a CO2 incubator for 1 h to allow the protein to access to the cell cytosol where LCVs are located.Wash the cells with prewarmed 500 μL/well of the semi-permeabilization buffer twice.Remove the buffer and add 500 μL of 4% PFA/PBS solution and incubate the plates at room temperature (15°C–25°C) for 15 min to fix the cells.Wash the cells with PBS (1 mL/well) three times.Pause point: You can pause the experiment here if needed. In this case, cover the plates with aluminum foil to protect them from light and evaporation and store them at 4°C.
Immunofluorescent staining (day 4)
Timing: 1 dayWhen the infected cells are semi-permeabilized and treated with the protein as described in the previous section, begin the procedure from step 29. If not, begin from step 24. If the semi-permeabilization technique is not used, the extracellular bacteria should be immunologically stained with a blue signal to distinguish them from the intracellular bacteria (Figure 1). This procedure can help exclude the extracellular uninfected bacteria in the imaging analysis step and the LCV counting process.
Figure 1
Example of extracellular uninfected bacteria staining
HeLa-FcγRII cells transfected with plasmids encoding GFP-LotB and the plasmid encoding FLAG-β’-COP were infected with Lp01 at an MOI of 5. After 4 h, the cells were fixed, immunostained and visualized using a fluorescence microscope. White and yellow arrows indicate extracellular and intracellular L. pneumophila cells, respectively. By using this protocol, all of L. pneumophila cells used to infect host cells can be stained with red fluorescence signal, whereas extracellular uninfected cells can be stained with both red and blue fluorescence signals. Scale bars, 10 μm.
Blocking: Add 500 μL of 2% GS/PBS (2% goat serum prepared in PBS) per well to the fixed cells.Incubate the plates at room temperature (15°C–25°C) for 30 min.Remove the coverslips using sharp-tip tweezers from the wells of the plate and place them upside down on the drops (40 μL) of diluted anti-Legionella rat serum (1:5,000 dilution) prepared in 2% GS/PBS to stain uninfected extracellular bacteria. (Set up reactions on the parafilm placed on the flat surface.)Incubate the coverslips at room temperature (15°C–25°C) for 1 h in a closed container.Replace the coverslips in the wells with 1 mL of PBS (cell-side up) in the 24-well plates. Wash the coverslip with PBS (1 mL/well) three times.Remove PBS and add 500 μL of 0.2% Triton X-100 in PBS to permeabilize the cell organelles so as to stain bacteria residing inside the LCV.Gently rock the plates using an orbital shaker at room temperature (15°C–25°C) for 20 min.Wash the cells with PBS (1 mL/well) three times.Add 500 μL of blocking solution (2% GS/PBS) per well.Incubate the plates at room temperature (15°C–25°C) for 30 min.Remove the coverslips from the wells of the plates and place them on drops (40 μL) of diluted anti-Legionella rabbit serum (1:5,000 dilution) prepared in 2% GS/PBS to stain intracellular bacteria.Incubate the coverslips at room temperature (15°C–25°C) for 1 h in a closed container.Replace the coverslips in the wells with 1 mL of PBS (cell-side up) in the 24-well plates. Wash the coverslips with PBS (1 mL/well) three times.Secondary antibody reaction.Cells without semi-permeabilization.Remove the coverslips from the wells of the plates and place them on drops (40 μL) of the secondary antibody solution containing Alexa Fluor 350-conjugated anti-rat IgG (1: 500 dilution) and Rhodamine Red-X-conjugated anti-rabbit IgG (1: 1000 dilution) prepared in 2% GS/PBS.Incubate the coverslips at room temperature (15°C–25°C) for 30 min in a closed container.Semi-permeabilized cells.Remove the coverslips from the wells of the plates and place them on drops (40 μL) of the secondary antibody solution containing DAPI (10,000 times dilution) and Rhodamine Red-X-conjugated anti-rabbit IgG (1000 times dilution) prepared in 2% GS/PBS.Incubate the coverslips at room temperature (15°C–25°C) for 30 min in a closed container.Replace the coverslips in the wells with 1 mL of PBS (cell-side up) in the 24-well plates.Wash the coverslips with PBS (1 mL/well) three times.Briefly wash the coverslips with sterilized MilliQ water (1 mL/well) in 24 well platesMount the coverslips on glass slides with ProLong™ Diamond Antifade Mountant without DAPI.Let the samples dry in a dark place at room temperature (15°C–25°C) overnight (18–20 h).Pause point: The slide glass sample can be stored at −20°C until imaging process. Please make sure to defrost before starting observation.Example of extracellular uninfected bacteria stainingHeLa-FcγRII cells transfected with plasmids encoding GFP-LotB and the plasmid encoding FLAG-β’-COP were infected with Lp01 at an MOI of 5. After 4 h, the cells were fixed, immunostained and visualized using a fluorescence microscope. White and yellow arrows indicate extracellular and intracellular L. pneumophila cells, respectively. By using this protocol, all of L. pneumophila cells used to infect host cells can be stained with red fluorescence signal, whereas extracellular uninfected cells can be stained with both red and blue fluorescence signals. Scale bars, 10 μm.
Imaging and counting the number of GFP-positive LCVs (day 5)
Timing: 2–3 hWe used an inverted microscope (TE2000-U; Nikon) equipped with a digital ORCA-ERA camera (Hamamatsu) to observe the LCVs as described previously (Kitao et al., 2020). Data can be collected using any fluorescent microscope with RGB channels and filters to visualize red, green, and blue signals. A confocal microscope also can be used to observe the LCVs alternatively.Clean coverslips using a paper moistened with water.Set up a glass slide on a 100× oil-immersion objective lens to observe the sample.Observation.Cells without semi-permeabilization.Find an intracellular L. pneumophila, i.e., LCV (positive for red signal, negative for blue signal).Check whether the LCV is associated with green signals (GFP-positive) or not.Semi-permeabilized cells.Find an LCV (positive for red signal) and DAPI staining (blue signal).Check whether the LCV is associated with green signals (GFP-positive) or not.Count at least 100 LCVs for each sample. Perform statistical analysis using Student’ s t tests for the data obtained from three independent experiments.
Expected outcomes
Here, we present results of three experiments performed using this protocol, which include the host t-SNARE GFP-Stx3 and the L. pneumophila effector protein LotB. These data are from the original Figure 7 in Kitao et al., 2020.Example outcome #1: Result of an analysis to determine whether LotB influences the association of GFP-Stx3 with the LCVs.Syntaxin 3 (Stx3) is a host t-SNARE protein that is known to form non-canonical SNARE paring with the v-SNARE Sec22b upon L. pneumophila infection (Arasaki and Roy, 2010). Given that the deubiquitinase (DUB) activity of LotB toward Sec22b promotes dissociation of Stx3 from Sec22b during the infection, we examined whether the LotB-mediated dissociation of Stx3 from Sec22b affects the level of Stx3 on the LCVs. As shown in Figure 2, the number of Stx3-positive LCVs in the cells infected with the ΔlotB mutant strain was higher than that of the Stx3-positive LCVs in the cells infected with the wild-type strain 4 h after infection.
Figure 2
Example outcome #1: LotB influences the association of GFP-Stx3 with LCVs during L. pneumophila infection
(A) HEK293T-FcγRII cells transiently expressing GFP-Stx3 were infected with the wild-type Lp01 or ΔlotB mutant strains at an MOI of 5. After 4 h, the cells were fixed and visualized using a fluorescence microscope. The number of GFP-Stx3-positive vacuoles containing L. pneumophila was assessed. Values represent the means from three independent experiments in which 100 LCVs were assessed. ∗∗p < 0.001.
(B) Images of LCVs containing the Lp01 or ΔlotB mutant strains 4 h post infection. Magnified images of the insets are shown at the bottom right of each panel. White arrows indicate LCVs. Scale bars, 10 μm.
Example outcome #2: Result of an analysis using the semi-permeabilization technique to determine whether LotB can induce the release of GFP-Stx3 from the LCVs.Example outcome #1: LotB influences the association of GFP-Stx3 with LCVs during L. pneumophila infection(A) HEK293T-FcγRII cells transiently expressing GFP-Stx3 were infected with the wild-type Lp01 or ΔlotB mutant strains at an MOI of 5. After 4 h, the cells were fixed and visualized using a fluorescence microscope. The number of GFP-Stx3-positive vacuoles containing L. pneumophila was assessed. Values represent the means from three independent experiments in which 100 LCVs were assessed. ∗∗p < 0.001.(B) Images of LCVs containing the Lp01 or ΔlotB mutant strains 4 h post infection. Magnified images of the insets are shown at the bottom right of each panel. White arrows indicate LCVs. Scale bars, 10 μm.We also examined whether Stx3 can physically dissociate from the LCVs due to LotB. We used the purified protein and semi-permeabilized cells after infection (Figure 3). The number of GFP-Stx3-positive LCVs significantly decreased in the cells treated with fulllength LotB compared to the untreated cells, suggesting that LotB induces the release of Stx3 from the LCVs.
Figure 3
Example outcome #2 using the semi-permeabilization technique: Influence of LotB on the association of GFP-Stx3 with LCVs during L. pneumophila infection
(A) GFP-Stx3-expressing HEK293T-FcγRII cells were semi-permeabilized with buffer containing digitonin 4 h after infection with the ΔlotB mutant strain at an MOI of 5. After treatment with or without full-length LotB, the cells were fixed and visualized with an immunofluorescence microscope.
(B) Percentages of LCVs positive for the GFP-Stx3 proteins are shown. Values represent means from three independent experiments in which 100 LCVs were assessed. ∗∗p < 0.001.
Example outcome #3: Result of an analysis to determine whether LotB can localize to the LCVs.Example outcome #2 using the semi-permeabilization technique: Influence of LotB on the association of GFP-Stx3 with LCVs during L. pneumophila infection(A) GFP-Stx3-expressing HEK293T-FcγRII cells were semi-permeabilized with buffer containing digitonin 4 h after infection with the ΔlotB mutant strain at an MOI of 5. After treatment with or without full-length LotB, the cells were fixed and visualized with an immunofluorescence microscope.(B) Percentages of LCVs positive for the GFP-Stx3 proteins are shown. Values represent means from three independent experiments in which 100 LCVs were assessed. ∗∗p < 0.001.In this experiment, we assessed whether LotB could localize to the LCV (Figure 4).
Figure 4
Example outcome #3: L. pneumophila LotB can localize to the LCVs during infection
(A) HeLa-FcγRII cells transfected with plasmids expressing GFP-LotB, GFP-LotBC29S, or GFP-LotBΔTM together with the plasmid expressing FLAG-β′-COP were infected with Lp01 at an MOI of 5. After 4 h, the cells were fixed and visualized using a fluorescence microscope. The areas indicated with white squares are magnified and shown in the bottom panels. White arrows indicate the location of LCVs containing L. pneumophila. Scale bars, 10 μm.
(B) Percentage of GFP-LotB-positive LCVs 4 h after infection. Values represent means from three independent experiments in which 100 LCVs were assessed for each sample. ∗∗∗p < 0.0001, ∗∗∗∗p < 0.00001.
Example outcome #3: L. pneumophila LotB can localize to the LCVs during infection(A) HeLa-FcγRII cells transfected with plasmids expressing GFP-LotB, GFP-LotBC29S, or GFP-LotBΔTM together with the plasmid expressing FLAG-β′-COP were infected with Lp01 at an MOI of 5. After 4 h, the cells were fixed and visualized using a fluorescence microscope. The areas indicated with white squares are magnified and shown in the bottom panels. White arrows indicate the location of LCVs containing L. pneumophila. Scale bars, 10 μm.(B) Percentage of GFP-LotB-positive LCVs 4 h after infection. Values represent means from three independent experiments in which 100 LCVs were assessed for each sample. ∗∗∗p < 0.0001, ∗∗∗∗p < 0.00001.Four hours after infection, ectopically expressed GFP-LotB was readily detected on the LCVs in the presence of nocodazole, which prevents microtubule-dependent vesicular trafficking between the ER-Golgi intermediate compartment (ERGIC) and Golgi, thereby disrupting the later stage of the early secretory pathway. The number of LotB-positive LCVs was not affected by the mutation of catalytic cysteine. However, LotB lacking the transmembrane (TM) domain (GFP-LotBΔTM) was not detected on LCVs, which indicates that the ability of LotB to associate with the LCVs requires the TM domain.
Limitations
In this protocol, we have described a method to analyze whether a protein of interest can localize to the LCVs, a vacuole of L. pneumophila established during infection in host cells. This protocol can be used to analyze both host proteins and L. pneumophila effector proteins. In addition, as an optional experiment, our protocol includes a semi-permeabilization technique to analyze the effect of a target protein on the localization of other proteins of interest. However, these procedures are not specific to L. pneumophila and can be used for other intracellular pathogens with proper modifications. For example, selection of a detergent and optimization of the buffer condition to semi-permeabilize the host plasma membrane without disrupting the vacuoles would be a critical factor. For detection of a specific pathogen, antibodies against the target pathogen or alternative detection tools are required.
Troubleshooting
Problem 1
A target protein is not detected on the LCVs (This problem may arise in step 45).
Potential solution
When analyzing for the first time, whether the target host protein is associated with the LCV, a positive control should be included. GFP-Stx3 can be used as a positive control, but the association of this protein during infection does not depend on the Dot/Icm type IV secretion system (T4SS), which is a bacterial apparatus essential for L. pneumophila infection (Arasaki and Roy, 2010). To determine whether your target host protein is recruited to the LCV in a T4SS dependent manner, host v-SNARE Sec22b could act as an alternative positive control protein (Kagan et al., 2004). In addition, you may also consider planning a time-course experiment to see how your target protein behaves at different time points, such as 0.5, 1, 2, 4, and 7 h post infection (step 20).In the protocol, we used GFP-tagged proteins to monitor cellular localization during infection. Alternatively, other fluorescent protein tags, such as mRFP, can also be used. Protein tagging can alter the function of the protein. In that case, smaller epitope tags, such as FLAG, Myc, or HA, can be used. The combination of an antibody with a target protein and a fluorescence-labeled secondary antibody, such as Alexa Fluor 488, can also be substituted for GFP-tagging of the target protein. Modify the protocol as per the detection system (step 37).
Problem 2
The fluorescence intensity of GFP-tagged protein is too low or too high (This problem may arise in step 45).If the fluorescence intensity of GFP-tagged protein is too low, the amount of plasmid used for transfection needs to be increased (step 15). Also, the lipofection method can be used alternative to PEI. If the fluorescence intensity of GFP-tagged protein is too high, reduction of the amount of plasmid used for transfection may help to improve the signal intensity (step 15).
Problem 3
An experiment using the L. pneumophila isogenic mutant lacking a gene encoding the effector protein of interest revealed that the effector protein influences the subcellular localization of host protein recruited to the LCVs upon infection. However, the semi-permeabilization experiment using the purified effector protein didn’t generate the supportive result (This problem may arise in the optional protocols after step 23).To influence the subcellular localization of host protein, biochemical events such as the physical interaction between the Legionella effector and host protein, the enzymatic activity and the appropriate subcellular localization of the effector protein are considered to be required. If you used partially truncated protein in semi-permeabilization experiment for some reasons, such as to increase the yield of soluble protein, try to use the full-length protein.
Resource availability
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Tomoe Kitao (kitao@gifu-u.ac.jp).
Materials availability
This study did not generate new unique reagents.
Data and code availability
This study did not generate and/or analyze any datasets.
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