Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Podsumowanie

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Streszczenie

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

Wprowadzenie

Fluorescent nanoparticles (NPs), including metal NPs, quantum dots, polymer NPs, silica NPs, carbon dots, etc., have attracted considerable attention during the past decades^1,2. Compared to traditional organic dyes, fluorescent NPs show desirable chemical, optical and mechanical properties, such as strong signal strength, resistance to photobleaching and high biocompatibility^3,4. These advantages make them the method of choice for intracellular sensing and live cell imaging. Furthermore, a variety of electron-dense NPs are visible by electron microscopy (EM), facilitating their use for correlated microscopic analysis, which allows combination of live cell tracking with light microscopy (LM) and higher resolution at ultrastructural level with EM⁵. For example, gold NPs have been long time efficiently used as biosensors in living cells for sensitive diagnosis as well as in the field of immuno-labeling⁶. Recent studies indicate that gold NPs with different size and shape can be readily uptake by a large variety of cell lines and routinely transport through the endosomal pathway, therefore have great potential being applied for intracellular vesicle transportation tracking and the endo-lysosomal system labeling^7,8.

Microbial pathogens, such as Salmonella enterica, Shigella flexneri and Listeria monocytogenes, have developed different mechanisms to invade non-phagocytic host cells⁹. After being internalized, the pathogens, either localized in the cytosol or sequestered in membrane-bound compartments, interact extensively with their host environments and modulate these to favor their own survival¹⁰. For instance, Salmonella enterica resides and replicates within an intracellular phagosomal compartment termed the Salmonella-containing vacuole (SCV) upon infection¹¹. The maturing SCV traffics towards the Golgi apparatus, undergoing continuous interactions with the endocytic pathway, and induces formation of extensive tubular structures, such as Salmonella-induced filaments (SIF), sorting nexin tubules, Salmonella-induced secretory carrier membrane protein 3 (SCAMP3) tubules, etc.^12-14. Studying how these bacterial pathogens manipulate host-cell pathways is essential to understanding infectious disease.

Here, gold-BSA-rhodamine NPs were used as fluid tracers to label the host cellular endo-lysosomal system, and the human gastrointestinal pathogen Salmonella enterica serovar Typhimurium (Salmonella) was used as a model bacterium to study the interactions of the pathogen with the host endocytic pathway. Intracellular gold-BSA-rhodamine NPs in non-infected cells and cells infected with WT Salmonella or mutant strains were imaged by a confocal laser-scanning microscope (CLSM). Then Imaris software was used to quantify the distribution of NPs, indicating that Salmonella infection induced extreme rearrangement of the endosomes/lysosomes. Following the description of this method, analogous experiments can be designed to track long-term fate of the internalized NPs and to investigate the influence of various exogenous substances or endogenous factors on the endocytic pathway of eukaryotic cells.

Protokół

1. Synthesis of 10 nm Gold Nanoparticles (Gold NPs)¹⁵

1. Prepare solution A: add 2 ml 1% aqueous gold chloride into 160 ml Milli-Q, or double distilled, water.
2. Prepare solution B: add 8 ml 1% tri-sodium citrate x 2 H₂O and 160 µl 1% Tannic acid into 32 ml Milli-Q, or double distilled, water.
3. Warm up solution A and B to 60 °C and mix them while stirring. Observe a dark blue color immediately. Observe red color after about 15 min. Then heat up to 95°C, keep 5 min and cool the solution to RT.
4. Add a drop of the NP suspension onto a carbon coated grid and allow to dry in air. Check the size and morphology of NPs by transmission electron microscopy.

2. Coating of Gold NPs with BSA and Labeling with Rhodamine N-hydroxysuccinimidyl Ester (NHS)¹⁶

1. Add 900 μl gold NPs into a 1.5 ml Eppendorf tube, centrifuge at 15,000 x g for 30 min. Optionally, prepare multiple tubes may at once.
2. Discard the supernatant, re-suspend the pellet in 900 μl sterilized Milli-Q water and add 100 μl 2 mg/ml BSA/PBS, mix on a Vortex at 800 rpm for 30 min.
3. To remove excess BSA, centrifuge the preparation at 15,000 x g for 60 min.
4. Discard the supernatant, and re-suspend the gold-BSA NPs in 125 μl PBS, add 12.5 µl of 1 M bicarbonate.
5. Immediately before use, prepare a 10 mg/ml solution of rhodamine NHS/DMSO, add 30 µl of rhodamine NHS/DMSO solution to 1ml of the gold-BSA NPs suspension. Incubate the reaction for 2 hr (or O/N) at RT during 800 rpm mixing, avoiding exposure to light.
6. Purify the gold-BSA-rhodamine NPs through dialysis against PBS at 4 ºC with 5 buffer changes over the period of 36 - 48 hr.
7. To stabilize NPs, add 10 μl of 200 mg/ml BSA/PBS into each 1 ml gold-BSA-rhodamine NPs. To remove the free or released BSA-rhodamine, centrifuge at 15,000 x g for 60 min. Resuspend the pellet in 2 mg/ml BSA/PBS, measure OD₅₂₀, and store at 4 °C, avoiding exposure to light.
8. Dilute the NPs 10 times with Milli-Q or double distilled water, add a drop onto a carbon-coated grid, and allow to dry in air. Check size and morphology of NPs by transmission electron microscopy (TEM).
   NOTE: All materials used for NP preparation were sterilized and the operation was conducted in a cell culture hood or on a bench beside a flame.

3. Culture of HeLa Cells

1. HeLa cells permanently expressing LAMP1-GFP are cultured in DMEM with 10% fetal calf serum (FCS) and grown at 37 °C in an atmosphere containing 5% CO₂.
2. Seed the cells at a density of 50,000 per well in an 8-well chamber slide (Ibidi) and incubate O/N.

4. Culture of Bacteria

1. Use Salmonella enterica serovar Typhimurium NCTC 12023 wild-type (WT) strain. For comparison, use mutant strains ΔssaV defective in the SPI2-T3SS and ΔsifA lacking key effector SifA for SIF. Use strains harboring plasmid pFPV25.1 for constitutive expression of enhanced GFP.
   NOTE: Bacterial strains are routinely cultured in Luria-Bertani broth (LB) with addition of 50 µg/ml carbenicillin (WT) and LB with addition of 50 µg/ml carbenicillin and/or 50 µg/ml kanamycin (ΔssaV, ΔsifA) of required to maintain plasmids.
2. Inoculate a single colony of bacteria in 3 ml LB with appropriate antibiotics and grow O/N at 37 ºC under shaking conditions for aeration, then dilute 1:31 in fresh LB and continue growth for 3.5 hr. At this time-point, the cultures reach the late log phase and bacteria are highly invasive. A ‘roller drum’ is convenient to incubate test tube cultures with aeration.

5. Infection of HeLa cells by Salmonella and Gold-BSA-Rhodamine NPs Pulse Chase-labeling (see Figure 1 for scheme)

1. Measure OD₆₀₀ of the sub-cultured bacteria and dilute to OD₆₀₀ = 0.2 in 1 ml PBS (~3 × 10⁸ cfu/ml), add appropriate amounts of bacteria to HeLa cells in 8-well chamber slides to achieve a multiplicity of infection (MOI) of 100.
2. Incubate for 30 min in the cell incubator, wash 3 times with PBS to remove non-internalized bacteria (this time point was set as 0 hr post-infection, or 0 hr p.i.). Add 300 µl of fresh culture medium containing 100 µg/ml gentamicin and maintain for 1 hr. Then replace the medium with fresh medium containing 10 µg/ml gentamicin for the rest of the incubation time.
3. After incubation with culture medium containing 100 µg/ml gentamicin for 1 hr, the medium is replaced by imaging medium (Eagles MEM without FCS, L-glutamine, phenol red and sodium bicarbonate, with 30 mM HEPES, pH 7.4) containing 10 µg/ml gentamicin. Gold-BSA-rhodamine NPs are added to HeLa cells to obtain a final concentration of OD₅₂₀ = 0.1.
   NOTE: Gold-BSA-rhodamine NPs may also be added to cells before infection, or at various time-points p.i.
4. After 1 hr incubation, remove the medium, wash 3 times with PBS, and add 300 µl of fresh imaging medium containing 10% FCS and 10 µg/ml gentamicin for the rest of the incubation time.
   NOTE: Duration of incubation may vary depending on concentration of NPs and cell lines used. For RAW264.7 macrophages, we observed that 30 min incubation with NPs at a concentration of OD₅₂₀ = 0.05 allowed sufficient internalization.

6. Imaging

1. Use a confocal imaging system such as a confocal laser-scanning (CLSM) or spinning disc (SD) microscope with a humidified environment chamber for high resolution imaging at different time-points.
2. Switch on the temperature control system and wait until it is stable. Optimize the imaging settings such as magnification, scanning speed, resolution, Z-stack etc. Use appropriate excitation/emission settings for GFP and gold-BSA-rhodamine NPs. For this protocol, use a 400 Hz scanning speed, resolution of 512 x 512 pixels and Z-step size of 0.25 µm. Excite GFP and gold-BSA-rhodamine using an Ar laser (488 nm) and a HeNe laser (543 nm), respectively. Include a bright-field (BF) channel for observing the shape of the cells. For other microscopy systems and infection conditions, the setting have to be adjusted accordingly.
   NOTE: Use the same Ar laser as the light source of BF channel and signal was detected with a photo multiplier (PMT) detector.
3. At indicated time-points p.i., mount the 8-well chamber slides containing infected cells on the microscope stage and record images.

7. Analysis of Images

1. Use microscopy image analysis software (see Materials and Equipment’s Table) to analyze the remodeling of the endo-lysosomal system by intracellular Salmonella. Open the data by clicking ‘Open’ and choosing the file.
   NOTE: Alternatively, open source software packages such as ImageJ or FIJI may be used to quantitative image analyses.
2. In the objects toolbar of the Surpass view click on the icon to add a new surface item. Click on (Next).
3. To analyze a Region of Interest (ROI), select segment a ROI. In a viewing area, a rectangle-bordered section overlaid on image is representing the ROI. Enter the values in the corresponding x-, y-, and z- fields, or directly click on the arrows in the preview rectangle to modify size and position of the ROI. Then click (Next).
4. As a source channel, select Channel 3 (signal for Gold-BSA-Rhodamine NPs). Check the smooth option to set up the smoothness of the resulting area. Define a value manually or accept the automatically generated value. For Threshold, select the Absolute Intensity option.
5. For the threshold adjustment, select the manual option and set a value. In the viewing area, a surface threshold preview is displayed in gray.
6. On the tab Classify Surfaces the resulting surface can be filtered by various criteria. A default filter is ‘number of voxels >10’, and other filters can also be included by clicking ‘add’. If a filtering is not necessary, then delete all filters by clicking on the delete button. Click (Next).
7. To complete Surface creation, click on (Finish). In the Object list, now un-check the box for the item Volume and new created surface is displayed in viewing area.
8. Export the statistics. Now, in the Surpass view the color of the subjects vary from purple to red according to their area. And the ‘Plot Numbers Area’ table shows the statistics information (e.g., ID number and area of objects). Export all statistics of surface 1 to an Excel file by click (Save).

Wyniki

Gold NPs were generated through a well-established method via reduction of chloroauric acid by citrate and tannic acid. As shown in Figure 2A, the synthesized gold NPs were quasi-spherical in shape with a size of approximately 10 nm. BSA-coating and rhodamine-labeling did not influence their morphology or size (Figure 2B).

It has been reported that gold NPs can be readily taken up by various mammalian cells and ended up in the endocytic systems⁷. In...

Dyskusje

The endo-lysosomal system of mammalian cells controls important physiologic processes, including nutrient absorption, hormone-mediated signal transduction, immune surveillance, and antigen presentation¹⁷. Up to now, a variety of markers have been used for labeling of the endocytic pathway and tracking studies. For example, LysoTracker probes are fluorescent acidotropic probes developed by Molecular Probes (Life Technologies, USA) for lysosome labeling, which can selectively accumulate in cellular compartments ...

Materiały

Name	Company	Catalog Number	Comments
Gold chloride	Sigma-Aldrich	520918
Tannic acid	Sigma-Aldrich	403040
Tri-sodium citrate	Sigma	C8532
Bovine serum albumin	Sigma	A2153
NHS-Rhodamine	Pierce	46406
DMSO	Sigma	D8418
HEPES	Sigma	H3375
Gentamicin	Applichem	A1492
Kanamcyin	Roth	T832
Carbenicillin	Roth	6344
8-well chamber slides	Ibidi	80826	tissue culture treated, sterile
Imaris Software	Bitplane	version 7.6	various configurations available