Stress Granule Formation During Shigella Infection of Cancerous Epithelial Cells

Abstract

Source: Vonaesch, P. et. al., Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells. J. Vis. Exp. (2017)

This video demonstrates the infection of epithelial cells with the pathogenic bacterium Shigella flexneri and the resulting host cellular response. Bacterial effector proteins disrupt host translation, triggering the formation of membrane-less organelles called stress granules, which reflect the cell's adaptive response to infection.

Protocol

1. Preparation of Bacteria and Host Cells

1. Transfer 12 or 14 mm glass cover slips in either 24- or 12-well plates, respectively, with sterile tweezers, counting one well per experimental condition. Sterilize these by ultraviolet (UV) treatment in a tissue culture hood for 15 min.
   NOTE: Include control wells for bacterial challenge and for stress granule (SG) induction by exogenous stresses.
2. For a 24-well plate with 12 mm coverslips, seed 1 x 10⁵ mammalian cells (e.g., HeLa cells) onto coverslips; press the coverslips down with a sterile pipette tip. Let cells adhere overnight at 37 °C in a 5% CO₂ tissue culture incubator in culture medium appropriate for each cell type.
   NOTE: Plate cells so that the confluence of cells is between 40 - 60% the following day.
   1. For HeLa cells, incubate in Dulbecco's modified Eagle medium (DMEM) with non-essential amino acids (NEAA) and 10% decomplemented fetal calf serum (FCS). Decomplement FCS by heating for 30 min in a 56 °C water bath, swirling the serum once after 15 min.
3. To streak out bacteria, use a sterile pipette tip to remove a small amount from a bacterial glycerol stock (20% glycerol) stored in a -80 °C freezer and place it on a labeled plate. Use a sterile loop to spread the bacteria over about 10% of the plate. Use a new sterile loop and drag it through the smear to make 3 parallel lines, half a plate long. Streak through these lines, covering the rest of the plate. Incubate plate O/N at 37 °C.
   1. Select a single bacterial colony (e.g., Shigella flexneri) from the freshly streaked plate. Avoid working with bacterial colonies older than 1 week.
   2. For S. flexneri strain M90T, inoculate a red colony from a freshly streaked out tryptic soy agar-0.1% Congo red agar plate into 8 mL of Tryptic Soy broth in a 15 mL tube; tighten the lid and incubate shaking at 222 rpm O/N at 30 °C.CAUTION: Do not use large white colonies as they have lost the virulence plasmid and are non-infective.

2. Bacterial Challenge of Host Cells

1. Prepare bacteria to maximize infection potential.
   1. For S. flexneri, subculture bacteria by adding 150 µL O/N culture to 8 mL tryptic soy agar and incubate shaking at 222 rpm at 37 °C to the late exponential phase (~2 h) to induce virulence gene expression and enhance infection.
      1. When the culture is at an optical density between 0.6 and 0.9 (measured at 600 nm), transfer 1 mL of culture into a 1.5 mL tube. Pellet bacteria for 2 min at 8,000 x g and resuspend in infection medium (DMEM with NEAA, lacking FCS) at OD₆₀₀ = 1. Thus, if the OD₆₀₀ is 0.85, resuspend in 850 µL.
         NOTE: For S. flexneri, at OD₆₀₀ = 1, there will be 8 x 10⁸ bacteria per mL when cultured in 8 mL medium. A multiplicity of infection (MOI) of 20 is appropriate. For other pathogens, the number of bacteria per mL at an OD₆₀₀ and the appropriate MOI need to be determined empirically.
   2. To enhance bacterial-host interactions, coat bacteria with poly-L-Lysine (PLL).
      NOTE: PLL treatment of S. flexneri had no effect on SG dynamics. Other pathogens need to be assessed for their amenability to PLL treatment.
      1. To coat bacteria with PLL, centrifuge 1 mL of bacterial culture for 2 min at 8,000 x g and then resuspend the pellet in 10 µg/mL PLL in phosphate-buffered saline (PBS) at an OD₆₀₀ = 1.
      2. Add the bacterial solution to a small dish, such as a well within a 12-well plate, and incubate at RT (~ 20 °C) for 10 min with gentle agitation on a plate shaker.
      3. Pellet bacteria for 2 min at 8,000 x g, remove the excess PLL. Resuspend the pellet in 1 mL PBS and repeat this washing step twice. After the final wash, resuspend in infection medium at OD₆₀₀ = 1.
         NOTE: Here, the infection medium for S. flexneri is host cell medium with 20 mM N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) w/o FCS.
2. Prepare cells for bacterial challenge.
   1. Remove medium from mammalian HeLa cells using a suction pump. Add 500 µL RT HeLa cell medium to wash the cells, swirl, remove medium using a suction pump, and replace with 500 µL room temperature (RT) appropriate infection medium (e.g., host cell medium with 20 mM HEPES w/o FCS).
   2. NOTE: For all washing steps, work quickly and process only up to 4 coverslips at a time to avoid drying out the cells.
3. Challenge prepared HeLa cells with bacteria to infect 20 - 50% of the cells.
   NOTE: The appropriate time and MOI need to be determined for each bacterial species. Generally, a good start is 30 min at 37 °C with a MOI of 10.
   1. For S. flexneri, challenge HeLa cells using one of the two methods (step 2.3.1.1 or 2.3.1.2).
      1. Spin non-PLL-treated bacteria (20 µL of OD₆₀₀ = 1/well of 24-well plate in 500 µL medium) onto cells for 10 min at 13000 x g.
      2. Add PLL-coated bacteria (15 µL of OD₆₀₀ = 1/well of 24-well plate in 500 µL medium) for 15 min at RT to allow bacteria to settle onto cells.
   2. Allow the infection to occur by placing cells with settled bacteria into a 37 °C tissue culture incubator for 30 min.
      NOTE: For short time points, synchronize S. flexneri infection by placing plates in a 37 °C water bath for 15 min instead of in the tissue culture incubator.
4. Stop infection by washing cells.
   1. To wash cells and remove excess bacteria, remove the medium using a suction pump, add 1 mL fresh prewarmed (37 °C) HeLa culture medium (DMEM, 10% FCS, NEAA). Swirl the medium, remove and replace with fresh medium. Repeat twice and leave fresh medium on the cells.
      1. For S. flexneri, or other intracellular bacteria, after infection of HeLa cells and the washes, replace the medium with standard tissue culture medium containing 50 µg/mL gentamicin to kill extracellular bacteria and prevent further cell invasion.
         NOTE: Do not add antibiotics to the medium for extracellular bacteria.
5. Incubate bacteria with cells for the desired amount of time in a tissue culture incubator.
   NOTE: For S. flexneri, infection may be as long as 6 h, depending on the cell type. For HeLa cells, let infection proceed for 1.5 - 2 h before adding exogenous stresses to induce SG formation. For Caco-2 infections, in which Shigella spreads cell-to-cell, infect for 30 min to 6 h before adding exogenous stress. Cells may be fixed at this point without adding exogenous stresses to assess the formation of SGs as a result of the bacterial infection.

3. Fixation and Immunofluorescence Analysis of Stress Granule Formation

NOTE: Process the control and experimental coverslips at the same time to avoid staining differences that may impact image analysis in subsequent steps. The control samples include no infection with and without SG inducing treatment, and infected samples with and without SG inducing treatment.

1. Remove medium completely using a suction pump and fix samples by adding 0.5 mL RT 4% paraformaldehyde (PFA) in PBS. Incubate for 30 min at RT.
   CAUTION: PFA is harmful to the handler and the environment. Take proper measures to protect the handler and dispose of chemical waste.
   NOTE: Alternatively, fix for 15 min and then replace with -20 °C prechilled methanol for 10 min to retain more cytoplasmic localization of SG markers.
2. Remove the PFA with a pipette and dispose of the liquid in an appropriate waste container. Wash the coverslip with 1 mL tris-buffered saline (TBS: 25 mM Tris, pH 7.3; 15 mM sodium chloride, NaCl). Incubate the coverslip for 2 min with gentle shaking on a plate shaker during washing.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Shigella flexneri			Available from various laboratories by request
Tryptone casein soya (TCS) broth	BD Biosciences	211825	Standard growth medium for Shigella, application - bacterial growth
TCS agar	BD Biosciences	236950	Standard growth agar for Shigella, application - bacterial growth
Congo red	SERVA Electrophoresis GmbH	27215.01	Discrimination tool for Shigella that has lost the virulence plasmid, application - bacterial growth
Poly-L-Lysine (PLL)	Sigma-Aldrich	P1274	Useful to coating bacteria to increase infection, application - infection
Gentamicin	Sigma-Aldrich	G1397	Selective killing of extracellular but not cytosolic bacteria, application - infection
HEPES	Life Technologies	15630-056	pH buffer is useful when cells are incubated at RT, application - cell culture
Dulbecco's modified Eagle medium (DMEM)	Life Technologies	31885	Standard culture medium for HeLa cells, application - cell culture
Fetal calf serum (FCS)	Biowest	S1810-100	5% supplementation used for HeLa cell culture medium, application - cell culture
Non-essential amino acids (NEAA)	Life Technologies	11140	1:100 dilution used for HeLa cell culture medium, application - cell culture
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650	Reagent diluent, application - cell culture
Paraformaldehyde (PFA)	Electron Microscopy Scences	15714	4% PFA is used for standard fixation of cells, application - fixation
24-well cell culture plate	Sigma-Aldrich	CLS3527	Standard tissue culture plates, application - cell culture
12 mm glass coverslips	NeuVitro	1001/12	Cell culture support for immunofluorescent applications, application - cell support
Forceps	Sigma-Aldrich	81381	Cheap and robust mounting medium that works well for most fluorophores, application - mounting