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 subse....

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. ....

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

List of materials used in this article

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