Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Summary

Here, we present protocols to visualize calcium (Ca²⁺) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca²⁺ fluorescent probes, atypical global and local Ca²⁺ signals induced by bacteria over a large range of infection kinetics are characterized.

Abstract

Ca²⁺ is a ubiquitous ion involved in all known cellular processes. While global Ca²⁺ responses may affect cell fate, local variations in free Ca²⁺ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca²⁺ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca²⁺ signals induced by pathogens raises major technical challenges that need to be addressed.

Here, we report protocols for the detection of global and local Ca²⁺ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca²⁺ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca²⁺ signals during extended infection kinetics using the chemical probe Fluo-4.

Introduction

Ca²⁺ regulates all known cell processes, including cytoskeletal reorganization, inflammatory responses, and cell death pathways related to host-pathogen interactions^1,2,3. Under physiological conditions, basal cytosolic Ca²⁺ concentrations are low, in the hundreds of nM range, but can be subjected to transient increases upon agonist stimulation. These variations often show oscillatory behavior through the action of pumps and channels at the plasma and endoplasmic reticulum membranes. The pattern of these oscillations is characterized by the period, durat....

Protocol

1. Preparations

1. Bacteria preparation
   1. Plate the bacteria—Shigella wild-type strain expressing the AfaE adhesin (M90T-AfaE)—onto a trypticase soy (TCS) agar plate containing 0.01% Congo red (CR) and incubate them for 18 h at 37 °C.
      Note: To increase their reproducibility, the Shigella plates obtained in step 1.1.1 are stored at 4 °C and should be used within a week, because all colonies on CR medium will eventually turn red over time.
   2. Inoculate TCS broth pre-culture by picking 3 red colonies from the streak plates.
      Note: An inoculation with 3 colonies is performed....

Results

Shigella invasion is associated with atypical long-lasting local Ca²⁺ responses:

Following the protocol mentioned above, Fluo-4-loaded HeLa cells were challenged with WT Shigella and stream acquisitions were performed to analyze Ca²⁺ signals. A representative experiment is shown in Figure 1, with time-lapse images series of the fluorescence intensity of the Fluo-.......

Discussion

This manuscript describes the protocol that we engineered to follow local Ca²⁺ signals during the relatively short kinetics of a Shigella invasion, as well as global Ca²⁺ responses during the extended kinetics of Shigella. Below, key issues can be found that need to be addressed to optimize the detection of Ca²⁺ signals while minimizing any interference with the biological processes.

Chemical vs. genetically encoded Ca²⁺ probe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluo-4 AM	Invitrogen	F14201
Metamorph version 7.7	Universal Imaging
CoolLED illumination system pE-2	Roper Scientific
micro-dish 35 mm, high	IBIDI	81156
Trypticase Soy (TCS) broth	Thermofisher		B11768
TCS agar	Thermofisher		B11043
Congo red	Sigma-Aldrich		75768
M90T-AfaE	Sun et al. 2017	Shigella flexneri serotype V. expressing the AfaE adhesin
ipgD-AfaE	Sun et al. 2017	isogenic ipgD mutant strain expressing the AfaE adhesin