Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

Summary

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Abstract

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

Introduction

The virulence of many Gram-negative bacterial pathogens depends on specialized secretion systems to hijack eukaryotic host cells. Bacteria use these secretion systems to inject bacterial virulence proteins (effectors) into the host cell to modulate a variety of cellular and biochemical activities. The study of effector proteins has not only provided remarkable insight into fundamental aspects of host/pathogen interactions but also into the basic biology of eukaryotic cells¹. Modulation of host cell apoptosis has been shown to be an important virulence mechanism for many intracellular pathogens, and a number of effector proteins modulating apoptosis have bee....

Protocol

1. Generation of Stable Cell Lines Expressing the Protein of Interest

1. Prepare media by adding heat-inactivated FCS and 1% Penicillin/Streptomycin to commercially available Dulbecco´s Modified Eagle Medium (DMEM) supplemented with GlutaMAX-I, Pyruvate, and 4.5 g/L D-Glucose.
2. Cultivate HEK293 cells in media at 37 °C in 5% CO₂. Sub-cultivate cells every third day. Remove the media and resuspend the cells in 15 ml fresh media. Pipette 1 ml into a new 75 cm² cell culture flask and add 14 ml media.
3. Analyze the cell number by using a hemocytometer.
4. Seed HEK293 cells in a 6-well plat....

Results

First, HEK293 cell lines stably expressing the protein of interest (CaeB) as a GFP-fusion protein were established. As a control, HEK293 cell lines stably expressing GFP were also generated. Expression of GFP and GFP-CaeB was verified by immunoblot analysis. The representative immunoblot (Figure 4A) demonstrates stable and clearly detectable expression of GFP and GFP-CaeB. However, this assay cannot determine whether all cells express GFP or GFP-CaeB. Therefore, the stably transfected HEK293 cell lines w.......

Discussion

Many pathogenic bacteria harbor secretion systems to secrete or translocate bacterial effector proteins into the host cell. These effector proteins have the capacity to modulate processes and pathways in the host cell, allowing the bacteria to survive and replicate within their respective intracellular niche. Understanding the biochemical activities and the molecular mechanisms of the effector proteins will help towards a better understanding of pathogenicity and may help to develop new therapeutic tools to combat diseas.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	life technologies	31966-021
FCS	Biochrom	S0115
Pen/Strep	life technologies	15140-122
OptiMEM	life technologies	51985
X-tremeGENE 9	Roche	6365752001
Geneticin	Roth	CP11.3
Polyethylenimine	Polyscienes	23966
Doxycycline	Sigma Aldrich	D9891
Mini-PROTEAN Tetra Cell	Bio-Rad	165-8000EDU
Trans-Blot SD Semi-Dry Transfer Cell	Bio-Rad	170-3940
PageRuler Prestained Protein Ladder	Thermo Scientific	26616
PVDF membrane	Millipore	IPVH00010
anti-GFP	life technologies	A6455
anti-cleaved PARP	BD Bioscience	611038
anti-actin	Sigma Aldrich	A2066
Mouse IgG (H+L)-HRPO	Dianova	111-035-062
Rabbit IgG (H+L)-HRPO	Dianova	111-035-045
ECL Western Blotting Substrate	Thermo Scientific	32106
Restore Plus Western Blot Stripping Buffer	Thermo Scientific	46428