Quantitative Dextran Trafficking to the Coxiella burnetii Parasitophorous Vacuole

Seth Winfree1, Stacey D. Gilk2

1 Indiana University School of Medicine, Indianapolis, Indiana, 2 Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6C.2
DOI:  10.1002/cpmc.34
Online Posting Date:  August, 2017
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Abstract

The gram‐negative bacterium Coxiella burnetii causes human Q fever, a disease characterized by a debilitating flu‐like illness in acute cases and endocarditis in chronic patients. An obligate intracellular pathogen, Coxiella burnetii survives within a large, lysosome‐like vacuole inside the host cell. A unique feature of the Coxiella parasitophorous vacuole (PV) is high levels of fusion with the host endocytic pathway, with PV‐endosome fusion critical for Coxiella survival within the host cell. This unit describes quantitating PV‐endosome fusion by measuring delivery of the fluid phase endosome marker dextran to the PV using live cell imaging. To study the effect of host cell proteins involved in PV‐endosome fusion, details are provided for using siRNA knockdown host cells. This method is a powerful tool for understanding mechanisms underlying Coxiella’s ability to manipulate host cell trafficking pathways. © 2017 by John Wiley & Sons, Inc.

Keywords: Coxiella burnetii; endocytic trafficking; parasitophorous vacuole; quantitative microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Trafficking of Fluid Phase Marker to the Coxiella PV
  • Support Protocol 1: Infection of HeLa Cells with Coxiella
  • Support Protocol 2: Transfection of HeLa Cells with siRNA
  • Support Protocol 3: Replating Infected Cells from 24‐Well Plate onto IBIDI μ‐Slides
  • Support Protocol 4: Setup of Live Cell Imaging Chamber
  • Support Protocol 5: Analysis of Trafficking Time Course
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Trafficking of Fluid Phase Marker to the Coxiella PV

  Materials
  • Complete culture medium (see recipe)
  • 2 mg/ml Alexa 488–labeled dextran (see recipe)
  • HeLa cells infected with mCherry‐Coxiella (see protocol 2) and plated on Ibidi μ‐Slides (see protocol 4)
  • 37°C incubator
  • Live cell chamber, humidified and equilibrated to 37.1°C, 5% CO 2 (see protocol 5)
  • Motorized and fully automated inverted microscope (e.g., Nikon Ti‐E) with a 60× 1.4 NA oil immersion objective
  • Spinning disk confocal system (e.g., Yokogawa CSU‐X1) equipped with:
    • Camera (e.g., Andor iXon 887 EM‐CCD)
    • Laser combiner (e.g., Agilent MLC 400) with 488 and 561 nm lasers
    • Filter wheel (e.g., Lambda 10‐3 high‐speed filter wheel) fitted with ET525/36m and ET605/70m emission filters

Support Protocol 1: Infection of HeLa Cells with Coxiella

  Materials
  • Trypsinized HeLa cells
  • Complete culture medium (see recipe)
  • Coxiella burnetii stock expressing mCherry (see Justis et al., )
  • Phosphate‐buffered saline (PBS; see appendix 2A)
  • Hemocytometer
  • 24‐well tissue culture plate
  • 15‐ml conical tube
  • Cell culture incubator, 37°C, 5% CO 2

Support Protocol 2: Transfection of HeLa Cells with siRNA

  Materials
  • Dharmafect 1 (e.g., GE Healthcare)
  • Serum‐free RPMI
  • 20 µM siRNA stock solutions: control (e.g., non‐targeting) and experimental
  • Trypsin (see appendix 4E)
  • HeLa cells
  • Complete culture medium (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • 24‐well tissue culture plate
  • Vortex
  • Hemocytometer
  • Cell culture incubator, 37°C, 5% CO 2

Support Protocol 3: Replating Infected Cells from 24‐Well Plate onto IBIDI μ‐Slides

  Materials
  • Complete culture medium (see recipe)
  • HeLa cells infected with Coxiella burnetii in a 24‐well plate (see protocol 2)
  • Trypsin (see appendix 4E)
  • 15‐ml conical tubes
  • Swinging bucket centrifuge
  • Hemocytometer
  • µ‐Slide VI0.4 (e.g., Ibidi, cat. no. 80606)
  • Cell culture incubator, 37°C, 5% CO 2

Support Protocol 4: Setup of Live Cell Imaging Chamber

  Materials
  • Bold Line universal stage top incubator (Okolab H301 Bold Line) with:
    • Incubating chamber
    • Humidity controller
    • Two‐position slide adapter
    • Gas controller (CO 2 module)
    • Temperature controller
    • Oko‐Touch controller
  • Microscope with 60× 1.4 NA oil immersion objective
  • Objective heater
  • Type 37 immersion oil
  • µ‐Slide 2 well Ph+ imaging dish (e.g., Ibidi, cat. no. 80296)
  • µ‐Slide VI0.4 (e.g., Ibidi, cat. no. 80606)

Support Protocol 5: Analysis of Trafficking Time Course

  Materials
  • Computer running the following software:
    • FIJI Version 2.0.0‐rc‐59/1.51j with Java 8 (available at http://imagej.net/Downloads)
    • Excel or other spreadsheet software
    • Prism or other graphing software
  • Digital datasets from microscope
NOTE: This protocol is demonstrated in Video 1.
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Figures

Videos

Literature Cited

Literature Cited
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