Measuring pH of the Coxiella burnetii Parasitophorous Vacuole

Dhritiman Samanta1, Stacey D. Gilk1

1 Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6C.3
DOI:  10.1002/cpmc.38
Online Posting Date:  November, 2017
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Abstract

Coxiella burnetii is the causative agent of human Q fever, a zoonotic disease that can cause a debilitating, flu‐like illness in acute cases, or a life‐threatening endocarditis in chronic patients. An obligate intracellular bacterial pathogen, Coxiella survives and multiplies in a large lysosome‐like vacuole known as the Coxiella parasitophorous vacuole (CPV). A unique characteristic of the CPV is the acidic environment (pH ∼5.0), which is required to activate Coxiella metabolism and the Coxiella type 4 secretion system (T4SS), a major virulence factor required for intracellular survival. Further, inhibiting or depleting vacuolar ATPase, a host cell protein that regulates lysosomal pH, inhibits intracellular Coxiella growth. Together, these data suggest that CPV pH is an important limiting factor for Coxiella growth and virulence. This unit describes a method to determine CPV pH using live cell microscopy of a pH–sensitive fluorophore conjugated to dextran. This technique is useful to measure changes in CPV pH during infection or in response to drug treatment. © 2017 by John Wiley & Sons, Inc.

Keywords: Coxiella burnetii; parasitophorous vacuole; pH; quantitative microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Imaging of Fluorescent Dextran in the CPV
  • Support Protocol 1: Set Up of TOKAI HIT INU Stage Top Incubator for Live‐Cell Microscopy
  • Support Protocol 2: Analysis of the Image Datasets
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Imaging of Fluorescent Dextran in the CPV

  Materials
  • Dextran working solution (see recipe)
  • Complete RPMI‐1640 culture medium (with 10% fetal bovine serum)
  • HeLa cells infected with mCherry‐Coxiella and plated on ibidi µ‐Slides (Support Protocols protocol 21 and protocol 33, Winfree & Gilk, , unit 6.2)
  • Phosphate‐buffered saline (PBS; HyClone, GE Healthcare Life Sciences, cat. no. SH3025601), sterile
  • Ionophore working solution (see recipe)
  • pH standards (see recipe)
  • Water bath
  • Sterile biosafety cabinet
  • CO 2 incubator for tissue culture
  • 200‐µl pipette and sterile tips
  • TOKAI HIT INU series stage top incubator (see protocol 2)
  • Leica DMI 6000B inverted microscope with 63X oil immersion objective lens

Support Protocol 1: Set Up of TOKAI HIT INU Stage Top Incubator for Live‐Cell Microscopy

  Materials
  • Sterile deionized water
  • 70% ethanol
  • Leica Microsystems 3P immersion oil Type F
  • TOKAI HIT INU series stage top incubator and TOKAI HIT INU temperature and gas‐mixer control unit (Fujinomiya, Japan)
  • µ‐Slide VI0.1 (Ibidi, cat. no. 80666)
  • TOKAI HIT CSG1‐200F sensor lid for chamber slides (Fujinomiya, Japan)

Support Protocol 2: Analysis of the Image Datasets

  Materials
  • FIJI, Version 2.0.0‐rc‐59/1.51j with Java 8 installed for your computer platform (http://imagej.net/Downloads)
  • Digital datasets from microscope
  • Excel (Microsoft) or another spreadsheet software
  • Prism (GraphPad) or other graphing software
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Figures

Videos

Literature Cited

Literature Cited
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