Isolation of Intracellular Pathogenic Bacteria from Infected Host Cells

Abstract

Source: Sigal, N., et al. RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells. J. Vis. Exp. (2016).

This video demonstrates the isolation of internalized pathogenic bacteria from infected bone marrow-derived macrophages (BMDMs). Bacteria are allowed to attach to host cells, and unattached bacteria are washed away. After internalization, an antibiotic is added to eliminate non-internalized bacteria. The host cells are lysed, centrifuged, and the supernatant is filtered to capture the bacteria on filter paper, which is flash-frozen and stored for further use.

Protocol

NOTE: During the entire experiment, macrophage cells are incubated at 37 °C in a 5% CO₂ forced-air incubator and taken out of the incubator only for experimental manipulations, which are performed in a Class II biological safety cabinet. Working with Listeria monocytogenes bacteria is according to biological safety level 2 regulations.

1. Cell Preparation and Bacterial Infection (Days 1 and 2)

1. Day 1
   1. Seed 2.0 x 10⁷ bone marrow-derived macrophage cells (BMDM) on a 145 mm dish in 30 ml BMDM + Pen-Strep media (Table 2). Seed 3 plates for each bacterial strain to be analyzed. Incubate overnight at 37 °C in a 5% CO₂ forced-air incubator.
   2. Start an overnight bacterial culture of wild-type (WT) L. monocytogenes strain 10403S in 10 ml of brain heart infusion (BHI) medium, at 30 °C in a standard incubator. Place culture tubes slanted without shaking. 
      NOTE: These growth conditions upregulate the expression of the flagella genes, which promotes efficient infection.
2. Day 2
   1. Pre-warm BMDM medium (without Pen-Strep antibiotics) (Table 2) and phosphate buffered saline (PBS) at 37 °C.
   2. Wash macrophage monolayer twice with 25 ml of pre-warmed PBS to remove antibiotics. Add 30 ml fresh BMDM medium.
   3. Wash 1.5 ml of overnight bacterial culture with PBS by centrifuging bacteria at > 14,000 x g for 1 min, discarding the supernatant, and resuspending bacteria gently in 1.5 ml of PBS by pipetting. Repeat twice.
   4. Infect each macrophage plate with 0.5 ml of a washed overnight culture of wild-type L. monocytogenes. If using multiple plates, infect each plate 15 min apart. This time interval will enable harvesting each plate individually at the end of the infection.
      NOTE: The multiplicity of infection (MOI) is assayed by plating serial dilutions of bacterial culture used for infection on BHI agar plates and counting colony forming units (CFU) after 24 hr incubation of plates at 37 °C. Using 0.5 ml of WT L. monocytogenes strain 10403S results in a MOI of ~ 100 (100 bacteria CFU per macrophage cell). When analyzing bacterial mutants defective in intracellular growth, a higher MOI should be considered.
   5. Following 0.5 hr incubation at 37 °C, wash the infected cells twice with PBS to remove unattached bacteria, and add 30 ml pre-warmed BMDM medium. Attached bacteria will internalize during the next 0.5 hr.
   6. At 1 hr post-infection, add 30 µl of gentamicin (1:1,000 to reach a final concentration of 50 µg/ml) to kill extracellular bacteria.
   7. Assemble the filter apparatus by placing a filter head on a collecting liquid flask with a vacuum outlet port. Then place a filter (0.45 µm) on a filter head, followed by a cylinder funnel, and secure the different parts with a metal clamp. Prepare ice-cold RNase-free water.
   8. At 6 hr post-infection, harvest bacteria from infected cells as follows. Treat each plate individually.
      NOTE: Usually, L. monocytogenes completes 1-log of growth during 6 hr of infection in macrophage cells. Longer incubations could result in bacterial overgrowth and cell death of the macrophages, while shorter incubation periods could result in considerably lower bacterial loads. The MOI and the time of infection can be modified to reach optimal conditions.
      1. Wash infected cells with PBS once. Add 20 ml of ice-cold RNase-free water to lyse macrophage cells. Using a cell scraper, scrape the cells off the plate quickly but carefully.
      2. Collect lysed cells into a 50 ml conical tube. Vortex for 30 sec. Centrifuge at 800 x g for 3 min at 4 °C.
      3. Pass the supernatant through the filter apparatus using a vacuum system. Using tweezers, roll the filter and quickly transfer it to a 15 ml conical tube. Snap-freeze the tubes with the filters in liquid nitrogen.
      4. Assemble the filtration apparatus with a new filter for the next sample, as described in 1.2.7.
      5. Store the frozen filters at -80 °C for the next day. Alternatively, proceed directly to nucleic acid extraction.

Table 1: DNase Reaction Protocol.

RNA	up to 2 µg (in max volume of 44 µl)
10x DNase buffer	5 µl
RNase-free water	complete to 50 µl total volume
DNAse	1 µl (1 unit)

Table 2: Recipes of Media and Buffers.

1. 500 ml BMDM+Pen-Strep media (Filter sterilized):
Dulbecco’s Modified Eagle Medium (DMEM)	235 ml
Fetal bovine serum (FBS) (inactivated 30 min at 54 °C)	100 ml
Macrophage colony-stimulating factor or M-CSF (L-929 conditioned medium)	150 ml
Glutamine	5 ml
Sodium pyruvate	5 ml
β-Mercaptoethanol	0.5 ml
Penicillin/Streptomycin	5 ml
Total	500 ml
2. 500 ml BMDM (Filter sterilized):
DMEM	235 ml
FBS (inactivated 30 min at 54 °C)	100 ml
M-CSF (L-929 conditioned medium)	150 ml
Glutamine	5 ml
Sodium pyruvate	5 ml
β-Mercaptoethanol	0.5 ml
Total	500 ml
3. AE buffer
Sodium acetate (NaOAc) pH 5.2	50 mM
Ethylenediaminetetraacetic acid (EDTA)	10 mM
RNase-free water
4. phenol-chloroform-IAA
Phenol	25 ml
Chloroform	24 ml
Iso-amyl alcohol	1 ml
5. chloroform-IAA
Chloroform	24 ml
Iso-amyl alcohol	1 ml

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Listeria monocytogenes 10403S
Bone marrow derived macrophages prepared from C57B/6 female mice
H₂O, RNAse free	Thermo Scientific	10977-015	DEPC-treated water can be used
DMEM	Gibco	41965039
Glutamine	Gibco	25030081
Sodium pyruvate	Gibco	11360-088
β-Mercaptoethanol	Gibco	31350010
Pen/Strep	Gibco	15140-122
Gentamicin	Sigma-Aldrich	G1397
FBS	Gibco	10270106
Dulbecco’s Phosphate Buffered Saline-PBS	Sigma-Aldrich	D8537
Brain heart infusion (BHI)	Merckmillipore	1104930500
Sodium acetate	Sigma-Aldrich	W302406
EDTA	Sigma-Aldrich	EDS
Ethanol absolute	Merck Millipore	1070174000
37 °C, 5% CO₂ forced-air incubator	Thermo Scientific	Model 3111
Cell scrapers	Nunc	179693
Kontes glass holder for 45 mm filters	Fisher	K953755-0045
MF-Millipore filters 45 mm, 0.45 µm	Merck Millipore	HAWP04700
SpeedVac system	Thermo Scientific	SPD131DDA
Vortex-Genie 2	Scientific Industries	Model G560E
145 mm cell culture dishes	Greiner	639 160
1.7 ml tubes, RNase-free	Axygen	MCT-175-C
4 °C table centrifuge	Eppendorf	5417R
Sterile pipettes, 25 ml	Greiner
Falcon tubes, 50 ml	Greiner