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

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.

The overall goal of the following experiment is to study interference of bacterial virulence factors with intracellular signaling. This is achieved by establishing stable cell lines, expressing the protein of interest To study its activity on bulk cell level as a second step, an inducible expression vector system is created, which allows activating a host cell signaling cascade at a defined step. Next, it will be analyzed whether the protein of interest can interfere with the activation of the host cell signaling cascade in order to narrow down the point of activity of the protein of interest, the results show that the cozy yellow Burnett virulence factor KA B interferes with the apoptotic cascade downstream of backs and upstream of cascade three activation based on western blotting analysis.

This method can help answer key questions in the infectious biology field, such as identifying the key step in a signal transduction cascade at which a given bacterial effector protein interferes. This will not only help us better understand how pathogenic microorganisms manipulate their hosts, but also how these basic cellular processes function. Though this method can provide insight into bacterial strategies to prevent apoptotic cell death from occurring, it can also be applied to other signal transduction systems in the cell death field, such as ons or pyro ptosis, or also to signaling networks that are involved in cell proliferation, gene regulation or reactions by the immune system.

Demonstrating this procedure will be Stephanie Besler, a graduate student from the laboratory of Mann. Begin this procedure by seeding HEK 2 93 cells stably expressing GFP or GFP, say B in a 12. Well plate at a density of 100, 000 cells per well.

Next, prepare DNA and polyethylene mean transfection reagent separately in 75 microliters of opt medium, and incubate for five minutes at room temperature for complex formation. Incubate both mixtures together for 15 minutes at room temperature. After that, add the polyethylene DNA solution dropwise to the cells and incubate at 37 degrees Celsius in 5%carbon dioxide.

Five hours after transfection induce expression of the pro apoptotic proteins by adding one microgram per milliliter of doxycycline to the culture medium. Then incubate the cells for 18 hours at 37 degrees Celsius in 5%carbon dioxide under the light microscope. Inspect the cells for cell death induction prior to harvesting.

Examine the induced apoptotic cells, which are detectable by the presence of apoptotic bodies. Next, remove the supernatant and wash the adhered cells. Once with warm PBS resuspend the cells in 100 microliters of two x lamby sample buffer.

Then heat for five minutes at 95 degrees Celsius and for later use store at negative 20 degrees Celsius afterward. After that, load six microliters of a commercial protein ladder as a molecular weight marker. Then load approximately 40, 000 cells per sample on a 12%SDS page gel.

Run the gels in a gel electrophoresis system under a constant voltage of 180 volts for 45 to 60 minutes with one x laly running buffer. Afterward, BLO the proteins onto PVDF membranes at a constant voltage of 16 volts for 60 minutes. Using a trans blot SD semi-dry transfer cell, then block the membranes in 10 milliliters of MPT for one hour at room temperature.

Dilute seven microliters of anti cleaved PARP antibody in seven milliliters of MPT. Incubate the PVDF membrane with the antibody solution overnight at four degrees Celsius. Remove the antibody solution and perform three 10 minute washes with PBS 0.05%Tween 20, incubate the membranes with 1.4 microliters of horse radish peroxidase conjugated secondary antibodies diluted in seven milliliters of MPT for one hour at room temperature after the incubation.

Wash the membranes three times with PBS 0.05%Tween 20, detect horseradish peroxidase activity with a commercial kit according to the manufacturer's protocol. And apply standard equipment for film development to visualize the protein bands. Then remove bound antibodies by incubating the membranes with seven milliliters of western blot stripping buffer for 30 minutes at room temperature after three washes with PBS 0.05%Tween 20, probe the membranes with four microliters of anti actin antibody in four milliliters of MPT overnight at four degrees Celsius.

The next day, perform three 10 minute washing steps on the membranes with approximately 10 milliliters of PBS 0.05%Tween 20, then incubate the membranes with 1.4 microliters of horse radish peroxidase conjugated secondary antibodies diluted in seven milliliters of MPT After one hour of incubation at room temperature. Wash the membranes three times with PBS 0.05%Tween 20, detect horse radish peroxidase activity with a commercial kit and apply standard equipment for film development to visualize protein bans. In this experiment, whole cell lysates of HEK 2 93 GFP and HEK 2 93 GFP sabe cells were immuno blooded for GFP to show stable expression.

The representative flow cytometry analysis of HEK 2 93 GFP and HEK 2 93 GFP sabe cells show the intensity of the GFP expression. HEK 2 93 GFP and HEK 2 93 GFP sabe cells were treated with four micromolar sporin for six hours to induce intrinsic apoptosis. Apoptosis was analyzed by cleaved PARP immuno blot analysis in which PARP cleavage was found to decrease in HK 2 9 3 GFP SA B cells in comparison to HK 2 93 GFP cells.

CB protects from bax induced apoptosis, but not from caspase three induced apoptosis. While implementing this procedure, it is important to probe as many components of the signaling pathway as possible to help identify the interaction step. It is also good to test proteins that are activated in both their ground and activated states to determine if the chosen effector protein interferes with the pathway itself, or rather with the activation step following this procedure.

Other methods like knockdown, knockout yeast two hybrid pull down post-translational modification or proteolytic stability assays can be performed to answer additional questions such as, what are the exact molecular mechanisms these microbial effectors employ to manipulate normal host cell physiology? And how does this benefit pathogen survival and dissemination.