Nanoparticle-Mediated Visualization of Endo-Lysosomal Remodeling in Bacterially-Infected Cells

Abstract

Source: Zhang, Y., et al. Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria. J. Vis. Exp. (2015)

This video demonstrates the use of fluorescent nanoparticles to visualize endo-lysosomal remodeling in human epithelial cells infected by intracellular bacteria. Following bacterial invasion, the pathogens manipulate the host cytoskeleton and endo-lysosomal system, forming vacuoles with tubular projections that alter vesicle transport. Fluorescently labeled nanoparticles are then used to trace these changes, allowing for high-resolution imaging of pathogen-induced intracellular trafficking events.

Protocol

1. Culture of HeLa Cells
   1. HeLa cells permanently expressing LAMP1-GFP (Lysosome-associated membrane glycoprotein 1-Green fluorescent protein) are cultured in Dulbecco’s Modified Eagle Medium, or 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 overnight (O/N).
2. 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 the addition of 50 µg/ml carbenicillin (WT) and LB with the 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.
3. Infection of HeLa cells by Salmonella and Gold-BSA-Rhodamine Nanoparticles or 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 Phosphate-buffered saline or 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 (Eagle's 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. (Bovine Serum Albumin, or BSA)
      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.

Results

Figure 1. Scheme for the preparation of HeLa cells for live cell imaging. HeLa cells were seed...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gold chloride	Sigma-Aldrich	520918
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