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Abstract

Introduction

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Representative Results

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Acknowledgements

Materials

References

Immunology and Infection

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Published: April 2nd, 2013

DOI:

10.3791/50300

1Department of Molecular Microbiology & Immunology, 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.

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.

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 globa....

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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 (C.......

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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.......

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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.......

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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.).

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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

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