Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Summary

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Abstract

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

Introduction

Numerous bacterial pathogens have adapted generalized strategies to exploit host cells for survival and replication in an intracellular compartment. In many instances, pathogenic mechanisms are similar between protozoan and metazoan cells. However, these two microenvironments are very different and can result in differential expression of virulence factors^1-4. The Legionnaires' disease bacterium Legionella pneumophila is ubiquitously associated with freshwater environments worldwide⁵. Importantly, L. pneumophila cultivated in protozoan cells prior to infection of human monocytes gain a pathogenic advantage, suggesting that global gene expression profiles of the bacterium exiting a protozoan cell are different than that of the in vitro cultivated organism^6-8. In nature, freshwater amoebae provide nutrient rich confines for rapid amplification of an invading bacterium. Human acquisition of L. pneumophila is most often attributed to inhalation of contaminated water droplets that contain the bacterium. It is likely that these droplets harbor protozoan cell-associated bacteria; where protozoan cells are more resistant to conventional water treatment practices^9,10. Infection of lung alveolar macrophages proceeds in a manner nearly identical to the intracellular life cycle of the bacterium in protozoan host cells^11-13.

In order to survive and replicate in eukaryotic cells, L. pneumophila uses a specialized type IVb secretion system termed Dot/Icm to deliver nearly 300 'effector' proteins into the cytosol of the host cell^14-16. These effector proteins collectively function to subvert cellular processes in order to generate a replication permissive compartment for the bacterium^17,18. Deletions in any of the 26 genes that comprise the Dot/Icm transporter result in strains defective for intracellular multiplication^19-23. Historically, deletion of individual effector encoding genes rarely resulted in strains attenuated for intracellular growth. This phenomenon has been attributed to several hypotheses including redundant function and paralogous copies of effectors.

Some virulence factors are only expressed in the context of host cell-associated intracellular growth²⁴. We rationalized that if a particular effector was only expressed in the context of protozoan infection, then the contribution of the effector could not be compared with a wild-type strain when both were cultured in vitro. L. pneumophila transitions from a replicative to a transmissive phase as it enters stationary phase in culture²⁵. The phase switching phenotype represents the nutrient depletion encountered during intracellular growth and is exemplified through assembly of flagella for motility²⁶. Because L. pneumophila is more invasive and virulent when harvested from protozoan cells, we sought to develop an assay that more faithfully represented the pathogenic state of the bacterium when it encountered host macrophages.

To this end, we developed a versatile protozoan priming assay that can accommodate any suitable host for both the first (priming cell) and second (target cell) stage infections. The infection process is tractable through use of bacteria stably expressing green fluorescent protein (GFP). The infection model for the protozoan Acanthamoeba castellanii follows a methodology widely used in the field²⁷. For the priming step, L. pneumophila strains are cultivated in vitro to stationary phase in liquid media to produce labeled 'transmissive' bacteria (Figure 1A). Bacteria are next used to infect monolayers of A. castellanii for 18 hr to achieve a late stage of the intracellular life cycle. Large vacuoles containing bacteria can be visualized at this time point using fluorescence microscopy (Figure 1A). Protozoan cells are then lysed and bacteria recovered from the lysate are measured for emission at 512 nm using a fluorescence plate reader. Fluorescence is correlated with optical density to calculate multiplicity-of-infection (MOI) for the infection of target cells (Figure 1, *Correlation Curve). After invasion (T₀) and 18 hr post-invasion (T₁₈), target cells are quantified for fluorescence, representing intracellular bacteria. Fluorescence can be monitored by microscopy and flow cytometry, and viable counts can be measured through colony plating. The priming assay is always accompanied by infections with wild-type L. pneumophila and a strain defective in the Dot/Icm type IV secretion system (ΔdotA) (Figure 1A). This importantly provides internal controls for direct comparisons between wild-type and any isogenic mutant strains used in the infection process. The inclusion of the avirulent ΔdotA strain during the priming stage sets a threshold for observation of attenuated growth phenotypes associated with isogenic mutant strains that are cultured in vitro.

Protocol

1. Preparation of Legionella pneumophila Cultures for Priming Stage Infections

1. Transform all L. pneumophila strains used in the assay with the plasmid pAM239, encoding an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible green fluorescent protein (GFP)²⁸. Streak the bacterial strains onto iron and cysteine supplemented N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffered charcoal yeast extract agar (CYEA) containing 6.25 μg/ml chloramphenicol (CM) (for plasmid maintenance) and incubate for 72 hr at 37 °C.
2. Transfer an inoculum of the bacterial strain into supplemented ACES buffered yeast extract broth (AYE) with 6.25 μg/ml CM and 1 mM IPTG and cultivate to stationary phase (O/N) on an orbital shaker at 37 °C.
3. Confirm GFP expression in the in vitro cultures using fluorescence microscopy. Transfer 10 μl of culture onto a glass slide under a cover slip and image using a 60X objective with GFP excitation / emission cube (AMG EVOS fl).
4. Dilute a 100 μl aliquot of each in vitro culture to 1:10 using sterile H₂O. Make a blank using 100 μl AYE media with 1 mM IPTG and 6.25 μg/ml cm and water. Take OD600 measurements of the dilutions using a spectrophotometer (Bio-Rad Smart Spec Plus). Calculate the volume necessary to infect a well at an MOI = 20 for each in vitro culture: V = [(amoeba seeded) x MOI]/[OD₆₀₀ x (dilution factor) x (constant)] = [(1 X 10⁶) x (20)]/[OD₆₀₀ x (10) x (1 x 10⁶)] = 2/OD₆₀₀. Concentration of bacteria is determined as OD₆₀₀ = 1.0 = 1 x 10⁹ CFU/ml.

2. Priming Stage Infection Using Acanthamoeba castellanii

1. Maintain and cultivate the amoebae in ATCC 712 PYG medium in 175 cm² flasks at RT.
2. Replace media in amoebae cultures 24 hr before starting the bacterial liquid cultures. On the same day liquid bacterial cultures are started, collect and count cells on a light microscope using a hemocytometer.
3. Dilute the amoebae with fresh media to a final concentration of 1 x 10⁶ cells/ml. Seed 12-well cell culture plates with 1 ml aliquots of the amoebae using a repeat pipettor and incubate at RT O/N.
4. After incubation, wash the wells of the 12-well cell culture plates 3x with 1 ml sterile PBS using a 10 ml serological pipette and a manual pipette aid.
5. After aspiration of PBS, add 1ml of infection media (ATCC 712 PYG media minus glucose, peptone, and yeast extract) with 1 mM IPTG and 6.25 μg/ml cm to each well. Incubate the plates at RT for 1 hr.
6. Infect wells at an MOI = 20. Centrifuge the plate at 400 x g for 5 min (Eppendorf 5810R) and float in a 37 °C water bath for 5 min. Transfer the plate to a 37 °C, 5% CO₂ incubator for 18 hr.
7. Confirm the infections using live cell imaging on a fluorescence microscope prior to host cell lysis and harvest of bacteria.

3. Seeding THP-1 Cells for Target Cell Stage Infection

1. (Begin this process 24 hr prior to setting up liquid bacterial cultures). Cultivate THP-1 cells in a 75 cm² culture flasks to near-confluency in RPMI 1640 media with 10% heat-inactivated fetal bovine serum (FBS).
2. Count the suspension cells using a hemocytometer, dilute the THP-1 cells in RPMI 1640 media with 10% FBS and 100 ng/ml phorbol-12-myristate-13-acetate (PMA) to a concentration of 1 x 10⁶ cells/ml, and plate in 1 ml aliquots on 12-well cell culture plates. Incubate the plates for 48 hr at 37 °C, 5% CO₂.

4. Processing of Bacteria for Target Cell Stage Infection after the Priming Stage Infection

1. Aspirate the media from the primed amoebae.
2. Lyse the amoebae using 500 μl of ice cold, sterile ultra-filtered (UF) H₂O and incubate at RT for 10 min.
3. Pool the lysates according to strain type and take an E_{512 nm} measurement for each of the pooled lysates using a fluorescence plate reader (Molecular Devices Spectramax Gemini EM).
4. Calculate an OD₆₀₀ measurement for the pooled lysates: calcOD₆₀₀ = 0.0008(E₅₁₂ - lysate background) + 0.0019. The formula was previously determined by graphing a direct comparison of both the OD₆₀₀ and the E_{512 nm} measurements of dilutions of L. pneumophila wild-type expressing GFP from stationary phase in vitro culture (Figure 1*). The lysates of uninfected amoeba, subject to the same experimental conditions as the infected amoeba, are used as a blank and provided a correction value (e.g. lysate background), which was incorporated into the equation. The volume necessary to infect a well at an MOI = 20 is calculated for each lysate pool: V = [(THP-1 seeded) x MOI] / [calcOD₆₀₀ x (constant)] = [(1 X 10⁶) x (20)] / [calcOD₆₀₀ x (1 x 10⁶)] = 20 / calcOD₆₀₀.
5. Wash the THP-1 cells 3x with PBS and add 1 ml fresh RPMI 1640 (10% FBS) to each well. Incubate THP-1 cells for 1 hr at 37 °C, 5% CO₂.
6. Infect the THP-1 cells at the calculated MOI using the pooled lysates. Allow a set of wells to remain uninfected, serving as a negative control for flow cytometric analysis. Processing of the priming stage should be completed in less than 30 min. The lysates are kept on ice to limit any changes in bacterial gene expression before infection.
7. Centrifuge the plate, float to raise temperature, and incubate as in the priming stage.
8. One hour post-infection, remove the media by aspiration and wash wells 3x with PBS to remove extracellular bacteria.
9. Add 1 ml fresh RPMI 1640 (10% FBS) to wells and return the plate to the incubator. The time immediately after media replacement serves as time zero (T₀). Incubate the THP-1 cells for 14-16 hr at 37 °C, 5% CO₂.

5. Experimental Analysis

5.1 Live cell imaging

Image the infected wells using a fluorescence microscope (AMG EVOS fl) at 10X or 20X magnification (Figures 1A-D). The images can be compared either qualitatively or quantitatively to determine levels of infection in both the priming stage and target cell stage infections.

5.2 Flow cytometry

1. The emission peaks of different infections can be compared by flow cytometry (BD FACS Calibur). Trypsinize the infected THP-1 cells and gently wash them from the wells by mixing in PBS with a pipette.
2. Pool the cells by strain type into 15 ml Falcon tubes and pellet at 1,800 x g for 2 min in the table top centrifuge.
3. Suspend the pellets in 1 ml PBS and transfer to 1.5 ml microcentrifuge tubes.
4. Centrifuge the suspensions at 1,800 x g (Eppendorf 5424) and re-suspend the pellets in 1 ml PBS. If the resulting suspensions are highly turbid (OD₆₀₀ ≥ 1.0) they must be diluted in additional PBS (1:3) to prevent clogging of the lines in the flow cytometer.
5. Use sterile filtered PBS for equilibration of the flow cytometer and for wash steps between sample injections. Plot the forward / side scatter of uninfected target cells prior to injection of target cell infection samples.
6. Collect 20,000 total events for each condition using 488 nm laser excitation and the FL1 channel.
7. Gate post-capture cell populations to exclude uninfected cells and plot fluorescence intensity in a histogram (FlowJo) (Figure 1E).

5.3 CFU plating

1. Examine the efficiency of infection by CFU plating of the cells harvested for flow cytometry. Prepare serial dilutions of the samples in ultra-filtered H₂O at 10^-1, 10^-2, and 10^-3.
2. Plate 20 μl of each dilution onto 1/3 of a CYEA plate with 6.25 μg/ml CM.
3. Incubate the plates at 37 °C for 72 hr.
4. Count the colonies using a toothpick and cell counter.

Results

A typical result for the entire infection process is outlined in Figure 1. Live cell fluorescence micrographs depicting monolayers of A.castellanii infected with wild-type L. pneumophila during the priming stage is shown in Figure 1A. A successful measure of the priming step would lead to a population of approximately 90% of the host cells containing large vacuoles populated with GFP labeled bacteria at this MOI. At 18 hr post-infection, most A. castellanii cel...

Discussion

Bacterial gene expression is tightly controlled through a combination of life cycle progression and response to signals in the surrounding microenvironment. Vacuolar pathogens such as L. pneumophila respond to a multitude of host cell-derived cues when compartmentalized in a phagosome. As a collective result of nutrient exhaustion in the host cell, the bacterium compensates by expressing factors required for successful dissemination to a subsequent host cell²⁵. L. pneumophila adapted to effic...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
chloramphenicol	Fisher Scientific	BP904-100	antibiotic
IPTG	Fisher Scientific	BP1755-10
ACES	Sigma	A9758-1KG	media component
ATCC medium: 712 PYG	ATCC		growth media for protozoans
1X PBS	Fisher Scientific	SH30256FS	phosphate buffered saline
activated charcoal	Fisher Scientific	C272-212	media component
yeast extract	Fisher Scientific	BP1422-500	media component
peptone	BD Diagnostics	211677	media component
agar	Fisher Scientific	BP1423-2	media component
L-cysteine, 99%+	Acros organics	173601000	media supplement
Ferric nitrate nonahydrate	Fisher Scientific	I110-100	media supplement
Equipment
EVOS fl	AMG	EVOS fl	fluorescence microscope
Smart Spec Plus	Bio-Rad	170-2525	spectrophotometer
5810 R centrifuge	Eppendorf	22627023	bench top centrifuge
Repeater plus	Eppendorf	22230201	repeating pipette
SpectraMax Gemini EM	Molecular Devices		microplate reader
Softmax Pro 5.3	Molecular Devices	0200-310	microplate reader software
5424 microfuge	Eppendorf	22620401	table top microcentrifuge
Fast-release pipette pump II	Scienceware	379111010	pipette aid
FACS Calibur	BD Bioscience		flow cytometer
FlowJo 7.6.1	FlowJo	license	flow cytometery software
15 ml tube	BD Falcon	352096	polypropylene conical tube
1.6 ml microfuge tube	Neptune	3745.X	microcentrifuge tubes