Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Summary

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Abstract

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Introduction

The Gram positive bacterium Listeria monocytogenes is a food-borne pathogen that invades host cells, disrupts its internalization vacuole and replicates in the cytoplasm of host cells¹. L. monocytogenes ease of manipulation in the laboratory context (rapid growth, low toxicity for healthy individuals) associated with the persistence of bacterial virulence traits observed in cellular and animal models (hemolytic activity, leukocytosis) allowed its initial use in the 1960s as a major model for the study of intracellular parasitism and for the establishment of the theoretical foundations of cellular immunity against infection². In ....

Protocol

1. Preparation of Cellular and Bacterial Cultures, Transfection Tools and Primary Antibodies

1. Prepare a fresh agar plate to isolate individual L. monocytogenes colonies from a bacterial glycerol stock (50% glycerol/50% saturated bacterial liquid overnight culture) kept at -80 °C.
   1. Using an aluminum rack (kept at -80 °C) to transport a frozen bacterial glycerol stock, streak bacteria on a Brain Heart Infusion (BHI) agar plate.
   2. Incubate the plate at 37 °C during 48 hr (or until individual bacterial colonies can be isolated).
   3. Keep this working plate afterwards at 4 °C during a maximum time of 1 mont....

Results

Fluorescent labeling of cytoplasmic InlC provides a robust readout for cell infection by L. monocytogenes, as illustrated in Figure 1: the central cell in the micrograph is highly infected by the strain P14.PrfA*²³ as it can be observed in the phase contrast image (arrowheads, Figure 1A) and it is confirmed by the DAPI signal where individual bacteria can be clearly distinguished (Figure 1B). The InlC staining (superposed to the DAPI staining in Figur.......

Discussion

Several parameters are critical for the success of our InlC-detection protocol, including the use of healthy cell lines displaying a sufficiently large cytoplasm to allow an unambiguous detection of the InlC signal. In the assay we present in this article we propose the use of HeLa CCL2 cells, which are particularly well suited for our assay due to the extension of their cytosolic space; other HeLa clones such as HeLa Kyoto cells display a smaller cytoplasm but can be used with our infection protocol (HeLa Kyoto cells ar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacto Brain Heart Infusion	BD	237500	For liquid BHI preparation
Bacto Agar	BD	214010	Supplement to liquid BHI for BHI agar plates
Heat-Inactivated Fetal Bovine Serum	Biowest	51830-500
DMEM	Invitrogen	61965-026
Lipofectamine RNAiMax	Invitrogen	13778-100
Gentamicin	Sigma	G1397-10ML
Formaldehyde (16%)	EMS	15710	Prepare fresh before each experiment
Anti-Rabbit Alexa Fluor 546	Invitrogen	A-11035
DAPI	Invitrogen	D-1306
Phalloidin Dy647	Dyomics	647-33
siRNA Scramble	Dharmacon	D-001810-10
siRNA Met	Dharmacon	L-003156-00-0005
Black 384-well microscopy cell culture plate	Corning	3985
AxioObserver Z1 microscope	Zeiss	431007 9901
sCMOS camera	Andor	Neo
Metamorph analysis software	Molecular Devices	4000
CellProfiler analysis software	Broad Institute	Public software available at http://www.cellprofiler.org/