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. Этот метод предоставляет метод для сбора, нормализации и количественного роста внутриклеточных бактериальных патогенов, которые предварительно выращивают в естественных клеток-хозяев простейших до инфекциями клеток млекопитающих. Этот метод может быть мо...

Video: Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...