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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 the late 1980s and early 1990s, the dissection of the bacterial intracellular cycle³ as well as the molecular characterization of the most important bacterial virulence factors^4-7 favored the use of L. monocytogenes as a key molecular tool for the manipulation and study of host cell functions. The presence of avirulent (L. innocua) and virulent (L. monocytogenes) species in the Listeria genus paved the way for comparative genomic studies⁸ that, together with the recent establishment of the complete L. monocytogenes transcriptome⁹, have increased our understanding of the evolution of L. monocytogenes as a human pathogen and as a model system for infection studies¹⁰.

L. monocytogenes induces its internalization into host cells upon interaction of the bacterial surface proteins InlA and InlB with their host cell receptors E-cadherin and Met, respectively^11-12. Initial candidate-based studies led to the identification of the α/β catenins-actin link as a significant component of the InlA-invasion pathway¹³ and of the phosphoinositide 3-kinase (PI 3-K) as a critical effector of the InlB-dependent invasion cascade^14-15. Proteomic and functional-based assays subsequently allowed the identification of novel cytoskeletal elements¹⁶ and lipid second messengers¹⁷ required for host cell invasion. Transcriptional studies¹⁸ and mass spectrometry-based quantitative proteomics¹⁹ have recently shed new light concerning the activation of host signaling cascades and the repression of immune responses during L. monocytogenes infection. Systems biology approaches based on the inactivation of large sets of genes (kinomes, complete genomes) by small interfering RNA (siRNA) silencing have recently opened new avenues for the analysis of global host signaling cascades in the context of specific cellular functions, including phagocytosis and pathogen internalization²⁰. Genome-wide siRNA screens have been previously performed to investigate cellular cascades required for infection of L. monocytogenes in phagocytic Drosophila S2 cells^21-22, but this type of analysis has not been performed in non-phagocytic cells, which represent critical targets for infection in vivo.

We have optimized a protocol for the microscopical detection of late stages of infection by L. monocytogenes that is suited for high-throughput siRNA studies of bacterial entry within epithelial cells. Our assay takes advantage of a highly invasive L. monocytogenes strain that presents a point mutation in PrfA, the major transcriptional regulator of L. monocytogenes virulence factors⁶: this mutation (named PrfA*) renders PrfA constitutively active²³ and leads to an increased expression of the invasion proteins InlA and InlB, therefore favoring bacterial entry in otherwise poorly infected non-phagocytic cells. Our readout for infection is based on the detection of the cytosolic accumulation of the secreted bacterial protein InlC: this molecule is a pleiotropic effector that is expressed preferentially by intra-cytoplasmic L. monocytogenes⁹ and which participates not only in the bacterial cell-to-cell spread²⁴ but which also modulates host immune responses²⁵. The fluorescent labeling of InlC secretion by intracellular bacteria not only allows to clearly distinguish infected from non-infected cells, but also represents an end-point readout that can be used to subsequently dissect infection in its different steps: entry, vacuolar escape, cytosolic bacterial proliferation and cell-to-cell spread. This microscopy-based protocol can be coupled to siRNA screens therefore to study cellular pathways involved in the infection of host cells by L. monocytogenes.

Protokół

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 month (it will be used to seed liquid cultures).
   4. In our protocol, we use the L. monocytogenes serotype 1/2a EGDe.PrfA* strain, but as illustrated in Figure 1, the L. monocytogenes serotype 4b P14.PrfA* strain gives similar results.
2. HeLa ATCC CCL2 cells are grown in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in the absence of antibiotics.
3. Independent pools of scrambled (control) and anti-Met siRNAs are loaded in black 384-well microscopy cell culture plates.
   1. Dilute 160 pmol of siRNA in 500 μl of RNase-free water and add 5 μl of this solution per well (final concentration: 1.6 pmol).
   2. Keep this plate at -20 °C for a maximum period of 1 year.
4. Polyclonal rabbit antibodies against the bacterial protein InlC have been obtained by immunization using an InlC-GST recombinant protein as described previously²⁵.

2. Reverse siRNA Cell Transfection

1. 72 hr before infection bring to room temperature a black 384-well microscopy cell culture plate containing 1.6 pmol siRNA in 5 μl of RNase-free water in each well.
2. Centrifuge the plate for 3 min at 300 rcf at room temperature to bring down siRNA which could have been deposited in the walls of the wells.
3. Prepare room temperature DMEM supplemented with 0.4% Lipofectamine RNAiMAX.
4. Add 25 μl of the DMEM/RNAiMAX transfection medium to each well (do not incubate the transfection medium longer than 20 min before adding it to the siRNA).
5. Move the plate back and forth to mix the siRNA with the transfection solution, then keep the plate for 1 hr at room temperature to allow siRNA-Lipofectamine complexes to form.
6. Wash HeLa cells from a confluent- or sub-confluent cell culture flask once with 10 ml 37 °C pre-warmed PBS.
7. Detach HeLa cells by adding 1 ml 37 °C prewarmed trypsin to the cell culture flask and incubate for 3 to 5 min at 37 °C.
8. Re-suspend cells in 10 ml 37 °C prewarmed DMEM supplemented with 16% FBS.
9. Count cells and prepare a cell suspension of 12,000 cells per ml in DMEM supplemented with 16% FBS.
10. Add 50 μl of the cell suspension to each well in the 384-well plate.
11. Move the plate quickly back and forth to distribute cells and let cells settle down for 10 min at room temperature.
12. Seal the plate with Parafilm and keep it for 72 hr in a humidified 5% CO₂-containing atmosphere at 37 °C.

3. Cellular Infection and Staining

1. The day before infection, take a single colony of L. monocytogenes from the BHI agar plate and resuspend it in 5 ml of liquid BHI medium in a 15 ml polystyrene tube.
2. Incubate overnight at 37 °C in a shacking device to allow for bacterial growth.
3. The day of the infection, wash 1 ml of the overnight L. monocytogenes culture by centrifuging 2 min at 10,600 rcf on a table-top centrifuge.
4. Discard the supernatant (which contains the secreted cytotoxin listeriolysin O) and re-suspend the pellet in 1 ml of PBS (repeat the washing step 3 more times).
5. Read the bacterial optical density at 600 nm and estimate the number of bacteria (OD=1 is equivalent to 1E9 bacteria/ml).
6. Prepare the adequate L. monocytogenes dilution in DMEM supplemented with 1% FBS: using highly invasive strains like EGDe.PrfA*, we suggest the use of 5E4 bacteria in 30 μl of medium per well (the multiplicity of infection is estimated as 25 for 2,000 cells).
7. Remove the cell culture medium in each well (80 μl) and replace it by adding 30 μl of the L. monocytogenes-containing medium.
8. Centrifuge the plate at 200 rcf for 5 min at room temperature to synchronize the infection process.
9. Incubate the plate for 1 hr in a humidified 5% CO₂-containing atmosphere at 37 °C on a prewarmed aluminum block.
10. Remove the L. monocytogenes-containing medium from each well and add 30 μl of prewarmed DMEM supplemented with 10% FBS and 40 μg/ml gentamicin to kill extracellular L. monocytogenes.
11. Incubate for 4 hr in a 5% CO₂-containing atmosphere at 37 °C on a prewarmed metal block.
12. Prepare a solution of PBS supplemented with 8% formaldehyde (this solution should be prepared fresh so that the formaldehyde monomers, rather than the paraformaldehyde polymers, are used).
13. Without discarding the cell culture medium, add 30 μl of PBS supplemented with 8% formaldehyde (final concentration: 4%) and incubate for 15 min at room temperature.
14. Remove the fixative and wash the cells three times with 80 μl of PBS per well (keep the cells in a final volume of 80 μl of PBS per well).
15. Prepare a 1:250 dilution of the rabbit anti-InlC serum in PBS supplemented with 0.2% saponin.
16. Add 10 μl of the primary antibody solution to each well after removing the PBS and incubate for 30 min at room temperature.
17. Discard the primary antibody solution and wash four times with 40 μl of PBS per well.
18. Dilute the secondary Alexa Fluor 546-coupled anti-rabbit antibody (1:250), the DAPI solution (1:1,500) and phalloidin-Dy647 (1:150) in PBS supplemented with 0.2% saponin.
19. Add 10 μl of this secondary staining solution to each well and incubate for 30 min at room temperature.
20. Discard the secondary staining solution and wash four times with 40 μl of PBS per well (leave a final volume of 40 μl in each well and seal the plate).
21. The plate can be imaged immediately or can be stored at 4 °C protected from light (cover with aluminum foil) for subsequent analysis.

4. Image Acquisition and Analysis

1. Acquire images in three different channels (350 nm, 546 nm and 647 nm) using a 10X objective mounted on an automated microscope (acquire preferentially 9 images per well).
2. The InlC signal can be measured using image analysis software like CellProfiler that allows automated segmentation of nuclei and cell bodies using the DAPI and the phalloidin staining, respectively.

Wyniki

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...

Dyskusje

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...

Materiały

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/