We thank Dr. Craig Roy and Dr. Dario Zamboni for providing a template for protozoan cell infections. We thank Dr. Jagdeep Obhrai, Dr. Georgiana Purdy, Dr. Fred Heffron and Todd Wisner for equipment and reagents; Dr. Lulu Cambronne for critical review of the manuscript. Flow cytometry was performed at the OHSU Flow Cytometry Shared Resource facility. This work was supported in part by a grant from the Medical Research Foundation of Oregon and an NIH grant R21 AI088275 (E.D.C.).

1. Preparation of Legionella pneumophila Cultures for Priming Stage Infections
<ol>
	<li>Transform all L. pneumophila strains used in the assay with the plasmid pAM239, encoding an isopropyl &beta;-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 &mu;g/ml chloramphenicol (CM) (for plasmid maintenance) and incubate for 72 hr at 37 &deg;C.</li>
	<li>Transfer an inoculum of the bacterial strain into supplemented ACES buffered yeast extract broth (AYE) with 6.25 &mu;g/ml CM and 1 mM IPTG and cultivate to stationary phase (O/N) on an orbital shaker at 37 &deg;C.</li>
	<li>Confirm GFP expression in the in vitro cultures using fluorescence microscopy. Transfer 10 &mu;l of culture onto a glass slide under a cover slip and image using a 60X objective with GFP excitation / emission cube (AMG EVOS fl).</li>
	<li>Dilute a 100 &mu;l aliquot of each in vitro culture to 1:10 using sterile H2O. Make a blank using 100 &mu;l AYE media with 1 mM IPTG and 6.25 &mu;g/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.</li>
</ol>
2. Priming Stage Infection Using Acanthamoeba castellanii
<ol>
	<li>Maintain and cultivate the amoebae in ATCC 712 PYG medium in 175 cm2 flasks at RT.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &mu;g/ml cm to each well. Incubate the plates at RT for 1 hr.</li>
	<li>Infect wells at an MOI = 20. Centrifuge the plate at 400 x g for 5 min (Eppendorf 5810R) and float in a 37 &deg;C water bath for 5 min. Transfer the plate to a 37 &deg;C, 5% CO2 incubator for 18 hr.</li>
	<li>Confirm the infections using live cell imaging on a fluorescence microscope prior to host cell lysis and harvest of bacteria.</li>
</ol>
3. Seeding THP-1 Cells for Target Cell Stage Infection
<ol>
	<li>(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).</li>
	<li>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 &deg;C, 5% CO2.</li>
</ol>
4. Processing of Bacteria for Target Cell Stage Infection after the Priming Stage Infection
<ol>
	<li>Aspirate the media from the primed amoebae.</li>
	<li>Lyse the amoebae using 500 &mu;l of ice cold, sterile ultra-filtered (UF) H2O and incubate at RT for 10 min.</li>
	<li>Pool the lysates according to strain type and take an E512 nm measurement for each of the pooled lysates using a fluorescence plate reader (Molecular Devices Spectramax Gemini EM).</li>
	<li>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 E512 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.</li>
	<li>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 &deg;C, 5% CO2.</li>
	<li>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.</li>
	<li>Centrifuge the plate, float to raise temperature, and incubate as in the priming stage.</li>
	<li>One hour post-infection, remove the media by aspiration and wash wells 3x with PBS to remove extracellular bacteria.</li>
	<li>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 &deg;C, 5% CO2.</li>
</ol>
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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Suspend the pellets in 1 ml PBS and transfer to 1.5 ml microcentrifuge tubes.</li>
	<li>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 &ge; 1.0) they must be diluted in additional PBS (1:3) to prevent clogging of the lines in the flow cytometer.</li>
	<li>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.</li>
	<li>Collect 20,000 total events for each condition using 488 nm laser excitation and the FL1 channel.</li>
	<li>Gate post-capture cell populations to exclude uninfected cells and plot fluorescence intensity in a histogram (FlowJo) (Figure 1E).</li>
</ol>
5.3 CFU plating
<ol>
	<li>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.</li>
	<li>Plate 20 &mu;l of each dilution onto 1/3 of a CYEA plate with 6.25 &mu;g/ml CM.</li>
	<li>Incubate the plates at 37 &deg;C for 72 hr.</li>
	<li>Count the colonies using a toothpick and cell counter.</li>
</ol>

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The authors declare that they have no competing financial interests.

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...

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 factors1-4. 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 organism6-8. 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 practices9,10. Infection of lung alveolar macrophages proceeds in a manner nearly identical to the intracellular life cycle of the bacterium in protozoan host cells11-13.
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 cell14-16. These effector proteins collectively function to subvert cellular processes in order to generate a replication permissive compartment for the bacterium17,18. Deletions in any of the 26 genes that comprise the Dot/Icm transporter result in strains defective for intracellular multiplication19-23. 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 (&Delta;dotA) (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 &Delta;dotA strain during the priming stage sets a threshold for observation of attenuated growth phenotypes associated with isogenic mutant strains that are cultured in vitro.

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

tractable mammalian cell infections with protozoan-primed bacteria

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.

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...

Watch this Scientific Journal Video about Tractable Mammalian Cell Infections with Protozoan-primed Bacteria at JoVE.com

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

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.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

tractable-mammalian-cell-infections-with-protozoan-primed-bacteria

Research

JoVE Journal

Immunology and Infection

10.4K Views.  Oregon Health & Science University. 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.