Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Summary

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Abstract

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Introduction

Intracellular bacterial pathogens replicate either directly in the host cell cytosol, or in specialized vacuolar compartments¹. During the initial stage of the infection most pathogens get internalized either by phagocytosis by specialized cells (such as macrophages) or by actively promoting their own uptake into non-phagocytic cells. In phagocytic cells, the phagosome normally fuses with lysosomes to form a degradative compartment, where the phagocytosed particles are digested. Specialized cytosolic bacteria, such as Francisella novicida or Shigella flexneri, escape phagosomal degradation by inducing the rupture of the phagosome and subsequently escape into the cytosol^2,3. This requires virulence-associated mechanism such as the Francisella Pathogenicity Island (FPI) or the Shigella flexneri T3SS, which injects effector proteins that promote vacuolar rupture^2-4. The host cell cytosol features a number of conserved pattern recognition receptors that normally recognize the presence of pathogens and induce innate immune signaling⁵. In addition, xenophagy, an anti-microbial form of autophagy can degrade bacteria that enter the cytosol. Cytosolic bacteria are normally well adapted to blunt or circumvent these responses using different strategies. For example, Francisella modifies its LPS to avoid host recognition and Shigella prevents autophagy recruitment using secreted effector proteins^6,7.

Another strategy to escape lysosomal degradation is employed by the model vacuolar pathogen Salmonella enterica serovar Typhimurium, thereafter referred to as Salmonella typhimurium. Salmonella uses a T3SS to inject effectors that remodel the initial phagosome into its intracellular niche, the Salmonella-containing vacuole (SCV) ^8,9. Continuous manipulation of host pathways is necessary to maintain this vacuole by recruiting lipids and other nutrients to the vacuole. Indeed, a sifA mutant of Salmonella fails to maintain vacuolar stability, and enters the cytosol of host cells within hours after infection, which results in activation of innate immune pathways and autophagy recruitment⁸. Vacuolar escape of Salmonella varies depending on the cell type that is studies, and several reports have shown that in epithelial cells even WT Salmonella can escape the SCV and hyperreplicate in the cytosol¹⁰. Recent reports show that innate immune detection of vacuolar pathogens also depends on the ability of the host to recognize and destabilize pathogen-containing vacuoles (PCVs) ^11-14.

Given that both the host or bacteria genotype can affect the distribution of intracellular bacteria between the vacuolar and cytosolic compartment, it is necessary to quantify the number of vacuolar vs. cytosolic bacteria. Since, in most experimental setups host cells are infected with more than one bacterium, and subsequently can harbor several bacteria in different subcellular compartments, a technique is needed that allows quantification on the level of single bacteria. Differential permeabilization (also known as phagosomal protection assay) provides this resolution ^2,4,10,12. The assay is based on selective permeabilization of the host cell plasma membrane with the detergent digitonin, which leaves vacuolar membranes intact and thus allows selective staining of cytosolic bacteria with antibodies. Here we provide two protocols for the quantification of cytosolic and vacuolar Salmonella typhimurium using differential permeabilization. The principle of this method is described in Figure 1: Bone-marrow derived macrophages (BMDMs) are infected with stationary phase mCherry-expressing Salmonella. Stationary phase Salmonella need to be used because they downregulate the expression of the SPI-1 T3SS, whose activity would otherwise be recognized by the NLRC4 inflammasome and induce rapid cell death (pyroptosis) of the BMDMs¹⁵. Following the infection cells are washed and treated with 50 μg/ml of digitonin for 1 minute. Cells are washed again immediately and incubated with anti-Salmonella antibodies coupled to FITC to mark bacteria with access to the cytosol. After an additional washing step cells are lysed and the percentage of mCherry+ (vacuolar) and FITC+ / mCherry+ (cytosolic) bacteria is determined by FACS. We also report an adaptation of this method that can be used if no fluorescent protein-expressing strains are available (Figure 2). Additional steps are introduced after the FITC labeling in which cells are fixed and completely permeabilized. Thereafter all bacteria are stained with anti-Salmonella antibodies and corresponding secondary antibodies. Detection is then done by microscopy instead of FACS analysis.

Protocol

1. Digitonin Assay with mCherry-expressing Salmonella (FACS-based Analysis)

1. Preparing bacteria
   Note: Salmonella wild-type SL1344 (Streptomycin resistant) expressing mCherry from a plasmid (pFPV encoding mCherry under the rpsM promoter) is used in this assay. Bacterial phagocytosis and proliferation where not altered by the constitutive expression of mCherry (data not shown) ¹⁶.
   1. Inoculate the strain 1 day before the infection in 3 ml of LB medium supplemented with Streptomycin (90 µg/ml) and Ampicillin (100 µg/ml, mCherry expressing vector) in a shaker at 37 °C O/N.
2. Plating cells for infection.
   1. Seed wild-type BMDMs in a 24 well plate at a density of 1.25 x 10⁵ cells/well in 1 ml of primary macrophage medium (DMEM supplemented with 10% Fetal Calf Serum (FCS, heat-inactivated, 56 °C, 30 min), 1% HEPES, 1% Non-essential amino acids (NEAA), 1% L-Glutamine and 10% L929 supernatant (conditioned medium from macrophage colony-stimulating factor (M-CSF) producing L929 cells). Seed wells according to Table 1 to account for the individual conditions. Add coverslips to the wells used as controls (Table 1).
   2. Incubate the cells overnight in an incubator at 37 °C and 5% CO₂.

			Permeabilization		Staining
	Glass coverslip	Salmonella	Digitonin	Saponin	anti-Salmonella	anti-Calnexin	anti-PDI
A1 (sample)	-/+*	+	+		+
A2 (sample)	-/+*	+	+		+
A3 (sample)	-/+*	+	+		+
A4 (no permeabilization control)	-/+*	+			+
A5 (complete permeabilization control)	-/+*	+		+	+
B1 (stained for calnexin)	+					+
B2 (stained for calnexin)	+		+			+
B3 (stained for calnexin)	+			+		+
C1 (stained for PDI)	+						+
C2 (stained for PDI)	+		+				+
C3 (stained for PDI)	+			+			+

Table 1: Plating scheme for bone-marrow derived macrophages (BMDMs) in a 24-well format for subsequent infection with Salmonella, permeabilization, and staining. *Depending on whether protocol 1 or protocol 2 is done.

3. Preparing the bacteria for infection.
   1. The next day, determine the concentration of Salmonella in the overnight culture by diluting the culture 1:10 in PBS and measuring the OD₆₀₀ against a PBS only control. This ensures that the OD₆₀₀ is measured in the linear range of the spectrophotometer.
   2. Calculate the concentration of Salmonella per ml. An OD600 of 1 corresponds to 1 x 10⁹ bacteria.
   3. Dilute the bacteria in macrophage medium to reach a Salmonella concentration of 1.25 x 10⁶ cells/ml.
4. Infection of cells with Salmonella.
   1. Remove medium from BMDMs. Add 1 ml of macrophage medium to uninfected control wells (B1-B3, C1-C3). Add 1 ml of Salmonella suspension to wells A1 - A5 to reach a multiplicity of infection (m.o.i.) of 10 bacteria per cell.
   2. Centrifuge the plate for 15 min at 300 x g at 37 °C to synchronize the infection. Transfer the plate in an incubator at 37 °C and 5% CO₂.
5. Killing extracellular Salmonella with Gentamicin.
   1. At 1 hr post-infection, remove plate from incubator and add 0.1 ml of macrophage medium containing 0.1 mg/ml Gentamicin to kill extracellular bacteria. Add Gentamicin to all wells, even to uninfected controls, to ensure that all cells are treated the same way. Transfer the plate in an incubator at 37 °C and 5% CO₂.
6. Washing the cells.
   1. At 2 hr post infection, remove plate from incubator and wash all wells 2x with 1 ml of plain DMEM (pre-warmed to 37 °C) and replace it with macrophage medium (pre-warmed to 37 °C) containing 10 µg/ml Gentamycin to prevent growth of any remaining extracellular bacteria. Transfer the plate in an incubator at 37 °C and 5% CO₂.
7. Preparing fresh buffers with detergents and antibodies.
   1. Just before the desired time point of analysis, prepare a fresh stock solution of digitonin at 1 mg/ml in KHM Buffer (110 mM potassium acetate, 20 mM HEPES, 2 mM MgCl₂, pH 7.3). Filter the stock and dilute in KHM buffer to a working concentration of 50 µg/ml of digitonin. In addition, prepare a solution of 0.1% Saponin in KHM Buffer, anti-Salmonella antibody coupled to FITC (CSA-1-FITC) at 1/500, anti-calnexin at 1/100 or anti-PDI at 1/100 in KHM Buffer supplemented with 3% BSA.
8. Cell permeabilization.
   1. Remove plate from incubator and wash wells 3x with 0.5 ml of KHM Buffer (pre-warmed to 37 °C). Remove KHM Buffer and add 0.25 ml plain KHM Buffer to wells A4 and B1/C1, KHM Buffer with digitonin to wells A1 - A3 and B2/C2 or KHM Buffer with saponin to wells A5 and B3/C3 (according to Table 1). Incubate for exactly 1 min at RT and immediately wash all wells 3x with 0.5 ml of KHM Buffer (pre-warmed to 37 °C).
9. Primary antibody staining.
   1. Remove the KHM Buffer and add the primary antibody solutions (Goat anti Salmonella-FITC, 250 µl/well, 0.1 µg/ml) to the appropriate wells (Figure 1, Table 1). Incubate the plate for 15 min in an incubator at 37 °C and 5% CO₂.
   2. Wash the cells 1x with 1 mL PBS.
10. Infected Cells (wells A1 - A5): FACS analysis
    1. Wash cells 3x with PBS and add 0.5 mL of ice-cold PBS containing 0.1% Triton. Transfer to a 5 mL FACS tube with cell strainer cap to break up cell aggregates. Keep samples on ice.
    2. Analyze the samples of a FACS. Using the fully permeabilized controls, set the gate for total bacteria based on forward and side scatter (FSC/SSC) first, and the mCherry signal (610 nm ± 20 nm filter).
    3. Next, analyze the FITC signal in the total bacterial population using the fully permeabilized and not permeabilized controls to set the gates for cytosolic bacteria (FITC+, mCherry+) and vacuolar bacteria (FITC-, mCherry+) (Figure 3).
    4. With the gates set, analyze the samples and determine the percentage of cytosolic vs. vacuolar bacteria using FlowJo software according to manufacturer’s protocol.
11. Uninfected permeabilization controls (wells B1 - B3, C1 - C3): microscopy
    1. Fixation
       1. Remove PBS and add 250 µl of PFA 4% in PBS. Incubate for 10 min at 37 °C.
       2. Wash wells 2x with 1 ml of PBS and add 250 µl of 0.1 M glycine in PBS for 10 min to quench the fixative. Wash wells 2x with 1 ml of PBS.
    2. Secondary antibody staining.
       1. Stain permeabilization controls for 1 hr at RT with appropriate secondary antibodies coupled to fluorophores in PBS supplemented with 0.1% Saponin and 3% BSA to fully permeabilize cells.
       2. Wash coverslips 3x with 1 ml of PBS and mount the coverslips for analysis in mounting medium with DAPI (1.5 μg/ml).
    3. Microscopy analysis.
       1. Analyze the coverslips with the controls at a 40X or 63X magnification using a confocal fluorescence microscope. If the permeabilization has worked, there should be no staining for non-permeabilized cells, Calnexin staining for both digitonin- and Saponin-permeabilized cells and PDI staining only for saponin-permeabilized cells (Figure 4).

2. Digitonin Assay with Unlabeled Salmonella (Microscopy-based Analysis)

1. Preparing bacteria for O/N cultures.
   1. Inoculate unlabeled Salmonella wild-type SL1344 (Streptomycin resistant) 1 day before the infection in 3 ml of LB medium supplemented with Streptomycin (90 µg/ml) in a shaker at 37 °C O/N.
   2. Plating cells for infection.
      1. Seed BMDMs in a 24 well plate containing sterile glass coverslips at a density of 1.25 x 10⁵ cells/well in 1 ml of macrophage medium (DMEM supplemented with 10% Fetal Calf Serum (FCS, de-complemented, 56 °C, 30 min), 1% HEPES, 1% L-Non-essential amino acids (NEAA), 1% L-Glutamine and 10% L929 supernatant). Seed wells according to Table 1 to account for the individual conditions.
      2. Incubate the cells O/N in an incubator at 37 °C and 5% CO₂.
   3. Preparing the bacteria for infection.
      1. The next day, determine the concentration of Salmonella in the O/N culture by diluting the culture 1:10 in PBS and measuring the OD₆₀₀ against a PBS only control. This ensures that the OD₆₀₀ is measured in the linear range of the spectrophotometer.
      2. Determine the concentration of Salmonella per milliliter. An OD₆₀₀ of 1 corresponds to 1 x 10⁹ bacteria.
      3. Dilute the bacteria in macrophage medium to reach a Salmonella concentration of 1.25 x 10⁶ cells/ml.
   4. Infection of cells with Salmonella.
      1. Remove medium from all wells. Add 1 ml of plain macrophage medium to uninfected control wells (B1 - B3, C1 - C3). Add 1 ml of Salmonella suspension to wells A1 - A5 to reach a multiplicity of infection (m.o.i.) of 10 bacteria per cell.
      2. Centrifuge the plate for 15 min at 300 x g at 37 °C to synchronize the infection. Transfer the plate in an incubator at 37 °C and 5% CO₂.
   5. Killing extracellular Salmonella with Gentamycin.
      1. At 1 hr post-infection, remove plate from incubator and add 0.1 ml of macrophage medium containing 1 mg/ml Gentamycin to kill extracellular bacteria to all wells. Transfer the plate in an incubator at 37 °C and 5% CO₂.
   6. Washing the cells. 
      1. At 2 hr post-infection, remove plate from incubator and wash each well 3x with 1 ml of fresh macrophage medium containing 10 µg/ml Gentamycin to prevent growth of any remaining extracellular bacteria. Transfer the plate in an incubator at 37°C and 5% CO₂.
   7. Preparing fresh buffers with detergents and antibodies. 
      1. Just before the desired time point of analysis, prepare a fresh stock solution of digitonin at 1 mg/ml in KHM Buffer (110 mM potassium acetate, 20 mM HEPES, 2 mM MgCl₂, pH 7.3). Filter the stock and dilute in KHM Buffer to a working concentration of 50 µg/ml of digitonin.
      2. In addition, prepare a solution of 0.1% Saponin in KHM Buffer, anti-Salmonella-FITC (CSA-1, 0.1 µg/ml), anti-calnexin at 1/100 or anti-PDI at 1/100 in KHM Buffer supplemented with 3% BSA (see materials table for antibody concentration).
   8. Cell permeabilization.
      1. Remove plate from incubator and wash wells 3x with 0.5 ml of KHM Buffer (pre-warmed to 37 °C). Remove KHM Buffer and add 0.25 ml plain KHM Buffer to wells A4 and B1/C1, KHM Buffer with digitonin to wells A1 - A3 and B2/C2 or KHM Buffer with saponin to wells A5 and B3/C3 (according to Table 1). Incubate for exactly 1 min at RT and immediately wash all wells 3x with 0.5 ml of KHM Buffer (pre-warmed to 37 °C).
   9. Primary antibody staining.
      1. Remove the KHM Buffer and add the primary antibody solutions (Goat anti Salmonella CSA1-FITC, 250 µl/well, 0.1 µg/ml) to the appropriate wells (Figure 2, Table 1). Incubate the plate for 15 min in an incubator at 37 °C and 5% CO₂. Wash 3x with 1 ml PBS.
   10. Fixation.
       1. Remove PBS and add 250 µl of PFA 4% in PBS. Incubate for 10 min at 37 °C.
       2. Wash wells 2x with 1 ml of PBS and add 250 µl of 0.1 M Glycine in PBS for 10 min to quench the fixative. Wash wells 2x with 1 ml of PBS.
   11. Total Salmonella staining of infected samples.
       1. Wash coverslips 3x with PBS with 0.1% Saponin/3% BSA and incubate the samples with an anti-CSA1 antibody (Goat anti-CSA1, 0.1 µg/ml, mouse anti-LPS works as well, 0.1 µg/ml) for 1 hr at RT in PBS with 0.1% Saponin/3% BSA.
       2. Wash 3x in PBS with 0.1% Saponin/3% BSA. Incubate your samples with a secondary anti-Goat antibody coupled to fluorophore of choice in PBS with 0.1% Saponin/3% BSA for 30 min at RT in the dark.
       3. Wash coverslips 3x with 1 ml of PBS and mount the coverslips for analysis in mounting medium with DAPI (1.5 μg/ml).  
   12. Microscopy analysis.
       1. Acquire data with a confocal microscope by counting cytosolic Salmonella (anti-Salmonella-FITC positive) and total Salmonella (double positive) (Figure 5). Permeabilization controls should show: no staining for non-permeabilized cells, Calnexin staining for digitonin-permeabilized cells and PDI staining for saponin-permeabilized cells (Figure 4). Wells A4 and A5 will serve as internal controls for the Salmonella staining.

Results

Figure 1 and Figure 2 show the schematics of the digitonin assay described in protocol 1 and protocol 2 illustrating the critical steps in the protocol and the results obtained. Figure 3 shows typical FACS results. Positive and negative controls are used to set the gates for mCherry+/FITC- bacteria (vacuolar Salmonella) and mCherry+/FITC+ bacteria (cytosolic Salmonella). Based on these gates the percentage of cytosolic and vacuolar bacteria can be determined in the experime...

Discussion

Differential permeabilization is an easy and robust method to analyze and quantify the subcellular distribution of bacterial pathogens between vacuolar compartments and the cytosol. The same assay has been used successfully with bacteria such as Francisella novicida^2,4 and Shigella flexneri¹². However, since many intracellular pathogen alter or modify the host endomembrane structures, the robustness of the digitonin permeabilization will have to be determined on an individual basis...

Materials

Name	Company	Catalog Number	Comments
Digitonin	Sigma	D5628	50 μg/ml
PFA	mpbio	219998380
HEPES	Life Technologies	15630
Potassium Acetate	Sigma	791733
MgCl2	Sigma	M8266
BSA	Sigma	A2153
anti-Salmonella CSA-1-FITC	KPL	01-91-99-MG	1/500
anti-Salmonella CSA-1	KPL	02-91-99-MG	1/500
anti-Calnexin	Enzo Lifesciences	SPA-860D	1/100
anti-PDI	Enzo Lifesciences	SPA-890	1/100
Saponin	Sigma	47036
Vectashield mounting medium	Vectorlabs	H-1200
Anti Rabbit antibody-488	Molecular Probes	A-11070	1/500
Glycine	Sigma	G8898
Gentamicin	Life Technologies	15710-49	100 μg/ml and 10 μg/ml
Triton X-100	Promega	H5141
PBS	Gibco	20012-019
DMEM	Sigma	D6429
NEAA	Amimed	5-13K00-H
LSM700 Confocal microscope	Zeiss		imaging done at 63X
FACS Fortessa	BD technologies		detection mCherry 610 nm
FACS Fortessa	BD technologies		detection FITC 530 nm