Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Summary

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Abstract

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C. trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Introduction

Infectious diseases caused by species of the intracellular bacterium Chlamydia elicit a major burden on global health, including sexually transmitted disease, pelvic inflammatory disease, blindness, pneumonia and possibly atherosclerosis^1-4. The ability of Chlamydia to interact with the host cell, from within a vacuole (termed the inclusion), is a critical determinant for their successful infection of cells and the host. The inclusion is a novel pathogenic compartment that enables chlamydial growth and is dynamically modified throughout the entire 2–3 day developmental cycle of Chlamydia⁵. The obligate intracellul....

Protocol

1. Generation of Fluorescent Host Cell Lines

1. Day 1. Plate 293T cells (or other retroviral packaging line) on 6-well plates at 2 x 10⁶ cells/well, to be ~75% confluent the next day. If desired, plate duplicate wells for each retrovirus to be used.
2. Day 2. Aspirate cells and add 2 ml fresh growth medium (DMEM + 10% FBS + 2 mM L-glutamine). Transfect cells with 5–8 µg each of retroviral vector (containing GFP) and packaging vector (e.g., pVSVg) using Lipofectamine 2000 or similar reagent and following the manufacturer’s instructions.
3. Incubate cells with DNA for 4–6 hr in a 37 °C CO₂....

Results

Mammalian cells expressing cytosolic fluorescent proteins (e.g., GFP) can be engineered to enable illumination of Chlamydia inclusions in live, infected cell cultures. Upon infection with Chlamydia, inclusions are readily visible as black spots in the host cells (Figure 1). The clarity of fluorescence-lacking inclusions can be exploited for the automated identification of inclusions across numerous fields of view and/or treatments (Figure 1). Once fluorescent h.......

Discussion

Here we describe the experimental strategy for generating fluorescent host cells for real time visualization and analysis of Chlamydia inclusions. This vacuole visualization approach confers the powerful ability to illuminate, track and quantitatively measure the dynamic properties of chlamydial inclusions across populations of cells or in single cells over time. Chlamydia inclusions in fluorescent protein labeled cells are strikingly well defined, such that they are easily identified without the need f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lipofectamine 2000	Invitrogen	11668
Opti-Mem	Invitrogen	31985
Polybrene	Sigma	H9268
0.45 µm filters	Fisher	09-719D
G418	Invitrogen	10131
HBSS	Invitrogen	14025
DMEM	Invitrogen	11995
RPMI 1640	Invitrogen	11875
RPMI 1640 w/o phenol red	Cellgro	17-105-CV
Penicillin G	Sigma	13752
Cycloheximide	Sigma	C7698
Glass bottom dishes	MatTek	P35G-1.5-14-C
Chamber slides, Lab-Tek II	Nunc	154526, 154534