Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

Summary

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Abstract

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

Introduction

Infection-based experimental models are inherently challenging due to their dependence on the starting state conditions of the host and pathogen, the various pathogen infection strategies, and the difficulty of attributing pathogen- and host-driven processes based on outcomes. The bacterium Listeria monocytogenes (Lm) has become an ideal pathogen to probe host defense responses because of its genetic and microbiological tractability, its rapid and processive cellular infection strategy, and the relatively clear relationship between its cellular- and organismic-level infection phenotypes. The cellular infection of Lm proceeds through four distinct phases¹: (i) cellular invasion that concludes with Lm being enclosed within a vacuole; (ii) Lm-directed dissolution of the vacuole membrane and release of Lm into the cytosol; (iii) intracytosolic replication; and (iv) physical penetration of the plasma membrane that results in either the infection of directly adjacent cells (such as in an epithelial sheet) or, in solitary cells, release of Lm into the extracellular milieu. Each of these phases are promoted by specific Lm-encoded factors (referred to as 'virulence factors') that, when deleted, cause infection defects in both cellular and animal models. This general infection strategy has been independently evolved by a number of the so-called 'cytosolic' pathogens².

Colony-forming unit (CFU) assays are widely employed to evaluate both in vitro (i.e., cellular) as well as in vivo (i.e., organismic) infection outcomes. In addition to their high sensitivity, particularly for in vivo infections, CFU assays provide an unambiguous readout for pathogen invasion and intracellular survival/proliferation. CFU assays have been extensively used to analyze both Lm and host cell determinants that impact infection. As informative as these prior studies have been to analyze cellular invasion and intracytosolic replication, CFU assays have not, to the best of our knowledge, been used to track the fourth phase of the Lm infection process: cellular escape. Here, we describe relatively simple means of how cellular escape (hereafter referred to as 'emergence') can be monitored by CFU assay (as well as by flow cytometry) and show examples of how both pathogen- and host-encoded factors regulate this phase of the Lm infection cycle. The analysis of the terminal phase of the cellular Lm infection cycle may make it possible to identify additional pathogen and host cell infection-specific factors and activities.

Protocol

Mice were treated humanely in accordance with all appropriate government guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and their use was approved for this entire study by the University of Miami Institutional Animal Care and Use Committee (protocol 16-053).

1. Preparing Cells for Infection

1. Propagate mouse macrophage-like cell line RAW 264.7 in DMEM tissue culture media supplemented with 10% fetal calf serum (henceforth referred to as DMEM/FCS) using a 125 mL flask and a tissue culture incubator maintained at 37 °C/5% CO₂.
2. The day before the infection, use a cell scraper to remove cells from the expansion flask and collect in a 15 mL screw cap conical tube. Centrifuge cells at 800 x g for 10 min, decant supernatant and suspend the cell pellet in 1 mL of DMEM/FCS (without antibiotics) and determine the titer using a hemocytometer or an automated cell counter.
3. Adjust titer to 2 x 10⁶ cells/mL and add 0.10 mL (2 x 10⁵ cells) to wells of a 48-well tissue culture dish. Ensure that the cell suspension covers the entire surface of the well.
   1. Plate cells in additional wells for a later source of conditioned media. Also place 0.10 mL of DMEM/FCS in three additional wells to serve as a 'no cell control'.
4. Incubate overnight in a tissue culture incubator at 37 °C/5% CO₂. The next day, do not remove from incubator until the bacterium inoculum is prepared and ready to use.

2. Preparing Listeria monocytogenes (Lm) for infection

1. Inoculate 2 mL of brain-heart infusion (BHI) with a single colony from a freshly-streaked agar plate (<2 days) of Lm and incubate overnight at 37 °C with shaking at 130 rotations/min.
2. The next day, add 0.10 mL of overnight culture to 2 mL pre-warmed BHI and continue incubating at 37 °C with shaking until the optical density (OD₆₀₀) is between 0.4 and 0.6.
3. Determine approximate bacterial titer using an empirically-derived conversion formula. In our lab, an exponentially growing Lm culture in BHI is approximately 1.3 x 10⁹ colony forming units (CFU)/OD₆₀₀. Minimize the amount of time bacterial cultures are exposed to room temperature.
4. Just prior to infection adjust the titer to 5 x 10⁷ CFU/mL using pre-warmed DMEM/FCS as diluent.

3. Infection

1. Working quickly and precisely, add 20 µL (1 x 10⁶ CFU; multiplicity of infection, MOI = 5) of the freshly-prepared Lm inoculum (step 2.4 above) to wells by slightly tilting the dish and carefully placing the pipet tip in the overlying media;
   NOTE: avoid touching the walls of the well with the pipet tip.
2. Gently pipet the contents of each well to ensure complete mixing; do not shake or significantly tilt the plate as this will spread Lm over the walls of the well. Return cell culture dish to 37 °C/5% CO₂ incubator.
3. Prepare a 10 µg/mL gentamicin solution using conditioned media from uninfected wells as diluent.
4. At 30 minutes post infection (mpi), carefully add 30 µL of the gentamicin solution (final concentration 2.5 µg/mL) and gently pipet the contents of the well as before and return cell culture dish to 37 °C/5% CO₂ incubator.
5. At 60 mpi, harvest appropriate wells for either CFU assay (60 mpi time point) or FCM analysis (as described below in sections 4 and 5, respectively).
6. For remaining wells, at various times thereafter either harvest cells as before or, for the Lm emergence assay, entirely remove media from well and replace with 150 µL of gentamicin-free conditioned media.

4. Sampling Processing and Analysis of Intracellular and Emergent Lm by CFU Assay

1. To harvest, slightly tilt the dish and carefully remove the entire media from the well. Add 0.50 mL of distilled sterile water (dsH₂O) to the well. After 30 s, transfer the resulting water lysate (10⁰) to a 1.5 mL microcentrifuge tube and vortex vigorously for 10 s.
2. Prepare 10^-1 and 10^-2 dilutions by adding either 4.5 µL or 50 µL of the water lysate to 450 µL of dsH₂O and briefly vortex to ensure thorough mixing.
3. Spread 50 µL of the 10⁰, 10^-1 and/or 10^-2 samples onto lysogeny broth (LB) agar plates.
4. To assay for emergent Lm (step 3.6 above), remove 10 µL of the overlaying media and divide it into two 5 µL aliquots: to one aliquot add 5 µL of DMEM/FCS and to the second aliquot add 5 µL of DMEM/FCS containing 5 µg/mL gentamicin. The latter control distinguishes between extracellular Lm (gentamicin sensitive) and intracellular Lm (gentamicin resistant) in the subsequent CFU assay.
5. After 5 min at room temperature, add 90 µL of dsH₂O, vortex vigorously for 10 s, and spread 50 µL on LB agar plates.
6. Enumerate Lm colonies on LB agar plates following 2 days of incubation at 37 °C.

5. Sampling Processing and Analysis of Intracellular and Emergent Lm by Flow Cytometry (FCM)

1. Prepare RAW 264.7 cells as described in steps 1.1-4. Adjust cells to 1 x 10⁶ cells/mL and add 1 mL (1 x 10⁶ cells) to wells of a 6-well tissue culture dish.
2. Prepare bacterium inoculum as described in step 2.1-2.3 and infect cells with volume of inoculum corresponding to a MOI of 50 (5 x 10⁷ CFU).
3. At 1.5 hours post infection (hpi), collect media from first set of wells and place in a 1.5 mL microcentrifuge tube with 500 µL of 4% paraformaldehyde (PFA) and place on ice until all wells have been processed.
4. To collect intracellular Lm, add 1 mL of dsH₂O to the well and after 60 s, transfer the resulting water lysate to a 1.5 mL microcentrifuge tube with 500 µL of 4% PFA. Vortex vigorously for 10 s and place on ice until all wells have been processed. (Sample processing is described in step 5.8 below.)
5. For remaining wells, remove media and replace with 1 mL of DMEM/FCS with 5 µg/mL gentamicin to kill extracellular Lm and promptly return cells to incubator.
6. After 30 min (2 hpi) remove media and replace with DMEM/FCS without gentamicin.
7. After 2 and 4 h (4 and 6 hpi) take second and third time points by collecting media and lysates as steps 5.3 and 5.4.
8. Centrifuge tubes at 10,000 x g for 7 min. Remove supernatant without disturbing pellet.
9. Suspend each pellet in staining cocktail of 50 µL of 4% PFA and 15 µL of phalloidin. Incubate tubes in the dark at 4 °C for 20 min.
10. After incubation add 1 mL of FACS buffer to each tube and pipette up and down to mix.
11. Spin tubes at 10,000 x g for 7 min. Remove supernatant without disturbing pellet.
12. Suspend each pellet in 400 µL of FACS buffer for immediate use, or 200 µL of FACS buffer and 200 µL of 4% PFA if storing for later analysis.
13. Run samples on a flow cytometry and analyze the collected data.

6. Sampling Processing and Analysis of Lm Colonization and Host Responses in the Liver

1. Prepare Lm for infection as described in step 2.1-2.3.
2. Calculate the necessary amount of subculture for desired titer of 3 x 10⁶ CFU/mL.
3. Just prior to infection dilute the amount of calculated bacteria with pre-warmed Hank's Balanced Salt Solution (HBSS) in a final volume of 1 mL. This is the input inoculum that is injected into the mouse.
4. Using a 28 G½ 100U insulin syringe, draw 100 µL (3 x 10⁵ CFU) of input inoculum and remove any visible air bubbles.
5. Place the mouse (strain C57BL/6; males between 6 and 12 weeks old) into the holding restraint and use warm water (no more than 45 °C) to dilate the tail vein, then inject the input inoculum into the vein.
6. Plate 10^-2 and 10^-3 dilutions of the input inoculum onto LB agar 10bcm plates and incubate overnight at 37 °C to determine the actual input dosage.
7. At 18 hpi, humanely euthanize mouse (CO₂ asphyxiation followed by cervical dislocation) and using sterile scissors cut open mouse peritoneum to collect frontal lobe of the liver using separate pairs of tweezers and scissors for the outside and inside of the mouse. Place the liver in a 6 cm Petri dish on ice.
8. Cut the liver into small cubes and place into a glass vial with a magnetic stir bar. Add 2 mL of 2 mg/mL collagenase D reconstituted in DMEM without FCS. Incubate vials at 37 °C on a magnetic stirrer for 30−45 min. Once the tissue is homogenized, add 3 mL of DMEM/FCS to inactivate the collagenase.
9. Filter the cells through a 70 µm cell strainer into a conical tube, adding more DMEM/FCS if necessary.
10. Centrifuge cells at 800 x g to pellet at 4 °C for 10 min, remove supernatant and suspend cell pellet in 500 µL of ACK buffer to lyse red blood cells.
11. Incubate cells at room temperature for 5 min in ACK buffer. Suspend cells in 1 mL of DMEM/FCS.
12. Centrifuge cells at 800 x g to pellet at 4 °C for 10 min, remove supernatant and suspend in 1 mL of DMEM/FCS. Then, count the cells using a hemocytometer or an automated cell counter.
13. Transfer 1 x 10⁶ cells to 1.5 mL microcentrifuge tube and spin to pellet. Then suspend in 1 mL of dsH₂O and vortex to lyse the cells. Plate 50 µL on LB plates at undiluted and 10^-2 dilutions and incubate at 37 °C to enumerate Lm for CFU analysis.
14. Transfer 1 x 10⁶ cells into FACS tubes for each tissue sample according to the cell counts and wash with 1 mL of FACS buffer.
15. Centrifuge cells at 800 x g to pellet at 4 °C for 10 min and remove supernatant.
16. Prepare an antibody cocktail containing the antibodies of interest based on the recommended concentration for each antibody and FACS buffer. See Table of Materials for recommended amounts used in the analysis.
17. Suspend pellet in 100 µL of antibody cocktail and incubate tubes in the dark at 4 °C for 30 min.
18. After incubation, add 2 mL of FACS buffer to each tube and suspend cells.
19. Centrifuge cells at 800 x g to pellet at 4 °C and discard the supernatant. Then suspend the pellet in 400 µL of FACS buffer for immediate use, or 200 µL of FACS buffer and 200 µL of 4% PFA if storing for later.
20. Analyze cells by flow cytometry (see step 5.13).

Results

Assessing the role of pathogen and host-encoded factors impacting cellular infection
Using the infection conditions described above, 0.15% of the input wild-type Lm is recovered after 1.5 h of co-incubation with cultured macrophages (Figure 1A). In the subsequent 1.5 h of co-incubation (3 h post infection, hpi), there was a 4-fold increase in recovery of viable Lm and from 3 to 6 hpi there was an additio...

Discussion

Due to its rapid and processive cellular infection program, Lm is an ideal pathogen to probe cellular activities that impact infection. A number of host factors have been identified that either positively or negatively affect Lm cellular infection^11,12,13,14. Two such host factors characterized in our laboratory, Perforin-2 and the Heme Regulated Inhibitor (P2 and hI), regulat...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ACK Lysing Buffer	Gibco	A1049201
ArC Amine Reactive Compensation Beads	Life Technologies	A10346
BHI (Brain Heart Infusion) broth	EMD Milipore	110493
Cell Strainer, 70 µm	VWR	10199-656
Collagenase D	Roche	11088858001
DMEM media	Gibco	11965-092
FACS tubes	BD Falcon	352054
FBS - Heat Inactivated	Sigma-Aldrich	F4135-500ML
Hanks’ Balanced Salt solution	Sigma-Aldrich	H6648-6X500ML
LB agar	Grow Cells MS	MBPE-4040
LIVE/DEAD Fixable Yellow Dead Cell Stain kit	Life Technologies	L349S9
Rhodamine Phalloidin	Thermo Fischer	R415
SP6800 Spectral Analyzer	Sony
Syringe 28 G 1/2" 1cc	BD	329461
TPP Tissue Culture 48 Well Plates	MIDSCI	TP92048
TPP Tissue Culture 6 Well Plates	MIDSCI	TP92406
UltraComp eBeads	eBioscience	01-2222-42
Antigen
CD11b	Biolegend		Flurochrome = PE Cy5, Dilution = 1/100, Clone = M1/70
CD11c	Biolegend		Flurochrome = AF 647, Dilution = 1/100, Clone = N418
CD45	Biolegend		Flurochrome = APC Cy7, Dilution = 1/100, Clone = 30-F11
F4/80	Biolegend		Flurochrome = PE, Dilution = 1/100, Clone = BM8
Live/Dead	Invitrogen		Flurochrome = AmCyN, Dilution = 1/100
Ly6C	Biolegend		Flurochrome = PacBlue, Dilution = 1/200, Clone = HK1.4
MHC II	Biolegend		Flurochrome = AF 700, Dilution = 1/200, Clone = M5/114.15.2
NK 1.1	Biolegend		Flurochrome = BV 605, Dilution = 1/100, Clone = PK136