Name: imaging ca2+ responses during shigella infection of epithelial cells
Uploaded: 2018-05-24T14:45:00
Description: Watch this Scientific Journal Video about Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells at JoVE.com

We thank Jenny-Lee Thomassin for her help in editing the manuscript. The work was supported by the ANR grants MITOPATHO and PATHIMMUN, grants from the Labex Memolife and PSL IDEX Shigaforce. Chunhui Sun is a recipient of a Ph.D. grant from the China Scholarship Council. Laurent Combettes and Guy Tran Van Nhieu are recipients of a WBI-France exchange Tournesol program N&#176;31268YG (Wallonie-Bruxelles International, Fonds de la Recherche Scientifique, Minist&#232;re Fran&#231;ais des Affaires &#233;trang&#232;res et europ&#233;ennes, Minist&#232;re de l&#39;Enseignement sup&#233;rieur et de la Recherche dans le cadre des Partenariats Hubert Curien).

1. Preparations
<ol>
	<li>Bacteria preparation
	<ol>
		<li>Plate the bacteria&#8212;Shigella wild-type strain expressing the AfaE adhesin (M90T-AfaE)&#8212;onto a trypticase soy (TCS) agar plate containing 0.01% Congo red (CR) and incubate them for 18 h at 37 &#176;C. 
		Note: To increase their reproducibility, the Shigella plates obtained in step 1.1.1 are stored at 4 &#176;C and should be used within a week, because all colonies on CR medium will eventually turn red over time.<...

<ol>
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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...

Ca2+ regulates all known cell processes, including cytoskeletal reorganization, inflammatory responses, and cell death pathways related to host-pathogen interactions1,2,3. 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, duration, and amplitude of Ca2+ increases, and is decrypted by cells which, in turn, trigger specific responses in what is known as the Ca2+ code4,5. A sustained increase in the cytosolic Ca2+ concentration under pathological conditions may lead to cell death associated with the permeabilization of mitochondrial membranes and the release of pro-apoptotic or necrotic factors6,7.
Shigella, the causative agent of bacillary dysentery, invades epithelial cells by injecting effectors into host cells using a type III secretion system (T3SS)8,9. A Shigella invasion of host cells is associated with local and global Ca2+ signals elicited by the T3SS. As for pore-forming toxins, the T3SS translocon that inserts into host cell membranes and is required for the injection of T3SS effectors is likely responsible for the activation of PLC and the inositol (1, 4, 5) trisphosphate (InsP3)-dependent Ca2+ release. The combination of the localized PLC stimulation and the accumulation of polymerized actin at sites of a Shigella invasion result in an atypically long-lasting InsP3-dependent Ca2+ release10. The type III effector IpgD, a phosphatidyl 4,5 bisphosphate (PIP2)-4-phosphatase, limits the local amount of PIP2, thereby controlling the quantity of available substrate for PLC to generate InsP3, which contributes to the confinement of local Ca2+ responses at bacterial invasion sites11,12. These local Ca2+ responses likely contribute to the actin polymerization at Shigella invasion sites10. Global Ca2+ responses that are also elicited by Shigella, however, are dispensable for the bacterial invasion process but trigger the opening of connexin hemichannels at the plasma membrane and the release of ATP in the extracellular compartment. Released ATP acting in a paracrine manner, in turn, stimulates Ca2+ oscillatory responses in cells next to the infected cell. IpgD is also responsible for shaping global Ca2+ responses into erratic isolated responses with slow dynamics. Eventually, upon a prolonged bacterial infection, IpgD leads to the inhibition of InsP3-mediated Ca2+ signals. Through its interference with Ca2+ signaling, IpgD delays a Ca2+-dependent calpain activation leading to the disassembly of focal adhesion structures and the premature detachment of infected cells13.
While Ca2+ signals are involved in critical aspects of pathogenesis, the use of a microorganism raises a number of technical challenges that are not encountered in classical agonist studies. The protocols described here use the commonly used fluorescent Ca2+ chemical indicator Fluo-4 that we engineered to characterize local Ca2+ signals during a Shigella infection. Steps critical for the detection of these signals are discussed, as well as procedures implemented for their quantitative analysis that is required to characterize the role of bacterial effectors in Ca2+ signaling.

<table border="0" cellpadding="0" cellspacing="0" style="width:693px;" width="694">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fluo-4 AM</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:136px;">F14201</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Metamorph version 7.7</td>
			<td style="width:120px;">Universal Imaging</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CoolLED illumination system pE-2</td>
			<td style="width:120px;">Roper Scientific</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">micro-dish 35 mm, high&#160;</td>
			<td style="width:120px;">IBIDI</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Trypticase Soy (TCS) broth</td>
			<td style="width:120px;">Thermofisher</td>
			<td style="width:136px;"></td>
			<td>B11768</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">TCS agar</td>
			<td style="width:120px;">Thermofisher</td>
			<td style="width:136px;"></td>
			<td>B11043</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Congo red</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:136px;"></td>
			<td>75768</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">M90T-AfaE</td>
			<td style="width:120px;">Sun et al. 2017</td>
			<td style="width:136px;">Shigella flexneri serotype V. expressing the AfaE adhesin</td>
			<td></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">ipgD-AfaE</td>
			<td style="width:120px;">Sun et al. 2017</td>
			<td style="width:136px;">isogenic ipgD mutant strain expressing the AfaE adhesin</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging ca2+ responses during shigella infection of epithelial cells

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.

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-...

Watch this Scientific Journal Video about Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells at JoVE.com

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

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.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

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