Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

This protocol is useful for characterizing the assembling functions of bacterial secretion complexes and can lead to a fundamental understanding of the molecular mechanisms that are required for host pathogen interactions. This technique integrates complementary approaches that preserve the native structure of the sample and allows and see to visualization of proteins complexes in intact bacterial cells. It increases our understanding of the structural function relationships of bacterial secretion systems.

This method can be adapted to study other types of secretion systems or other cellular complexes in a wide variety of bacterial species. Start by making agarose pads for live cell imaging. Prepare about 30 mL of 1%low melt agarose solution in water and microwave it in a glass flask for about 90 seconds swirling it occasionally until the agarose is completely dissolved.

Place two 22 by 22 by 0.15mm glass slides on the edge of a 25 by 75 by 1.1mm glass slide one on top of the other. Then, stack two more of the smaller slides on the other edge. Pipette about 1 mL of the molten agarose into the center slide between the two upper glass slides and place another large slide on top of the molten agarose making sure to avoid formation of air bubbles.

Cool the slides at four degrees celsius for 15 minutes then use a scalpel or razor blade to gently cut the pad into small squares. Fix a double-sided adhesive frame on a 25 by 75 by 1.1mm glass slide and place several pads on the slide. Dissolve a heavy patch of Legionella Pneumophila in 1 mL of double-distilled water.

Then vortex and pipette two to three microliters of the dilution onto the pads. Gently place a 58 by 24 by 0.15 mm cover slip over the adhesive frame. Select cells for imaging using bright field lighting.

Then, in the capture window of the microscope's software, Adjust the ND to 180 and the binning to two by two. Use the 488 nanometer channel to expose the sample for 500 to 1000 milliseconds and validate the specificity of the fluorescence by imaging untagged Legionella Pneumophila with the same parameters. To quantify polarity, adjust the image contrast so the bacteria are clearly visible.

Then zoom in on the region of interest and use the region tool to place a 0.25 by 1.3 micrometer rectangle starting at the pole and extending into the cytoplasm making sure that the rectangle remains within the bacterial borders. Mark at least 200 bacteria and create masks by using the region to mask button in the analyze menu. Click on mask statistics and choose object under mask scope.

Then mark mean intensity and variance under features and intensity. Export the data as a text file and use the spreadsheet application to calculate the polarity scores of each bacterium as the ratio between the variance and the mean intensity. For high throughput applications use face contrast microscopy to acquire dual channel images.

Make sure to choose fields of view where the bacteria are fully separated. Next, adjust the face channel contrast of the image to a level where the bacteria are clearly visible. Open the dual channel image and launch the create segment mask window.

Adjust the appropriate threshold, then remove the small objects by clicking the define objects button and adjusting the minimum size to 100 pixels. Under refine mask, select remove edges objects and separate masks of bacteria that are adjacent to each other. To quantify the dynamics of the fusion proteins, open the image capture window and mark time lapse.

Enter two in the number of time points box. Acquire two successful images of Legionella Pneumophila expressing the fluorescent protein of interest. Adjust the image contrast until the cells are clearly visible.

Then use the region tool to place a 0.25 by 0.25 micrometer square in the middle of at least 400 cells. Next, use the region to mask button in the analyze menu to create a mask of the squares of interest. Create a new empty mask and use the pixel tool to mark the entire cell area of at least 25 random cells.

Create another mask and use the large brush tool to mark areas between the cells. Finally, select mask statistics and make sure object is selected under mask scope for mask one. Under features and intensity, choose mean intensity.

Export the data and repeat the process for mask 2. For mask 3, select the entire mask and export the mean intensity. To prepare the electron cryotomography sample, grow a heavy patch of Legionella Pneumophila on CYE Agrastreptomisum plates for 48 hours at 37 degrees Celsius.

Resuspend the cells in water to an OD 600 of about 0.7. Then add five microliters of colloidal gold particles to 20 microliters of the cell suspension. Glow discharge a holey carbon grid and pipette five microliters of the cell mixture on it.

Let it stand for one minute. Blot the grid with filter paper and freeze it in liquid ethane using a gravity-driven plunger apparatus. After inserting superfolder GFP into the Legionella Pneumophila chromosome, live cell fluorescent microscopy was performed to compare the fluorescent signal with that of the parental superfolder GFP negative strain.

In addition, bright field microscopy was used to examine the proportion of cells with a specific fluorescent signal. Only a fraction of the cells that produced DotB superfolder GFP displayed polar localization of the fusion protein. When characterizing the distribution of DotB fluorescent signal, it was found that the intensity of DotB superfolder GFP at the poles was about two times higher than in the cytosol.

To investigate if DotB superfolder GFP polar localisation was dependent on a fully assembled type four secretion system, polarity scores of DotB superfolder GFP were determined for individual cells in a wild type, and in a type four secretion system mutant. Indeed, polar positioning of DotB superfolder GFP disappeared when the Dot/ICM system was deleted. Dynamic patterns indicated that the DotB ATPase was present in a dynamic cytosolic population and was recruited to the polar localized Dot/ICM type four secretion system at a late stage assembly reaction.

High throughput cryo-ET was used to visualize the intact type four secretion system apparatus in a Legionella Pneumophila strain expressing DotB superfolder GFP. Reconstruction of the cell revealed multiple Dot/ICM machines embedded in the cell envelope. The overall positioning of DotB and superfolder GFP in relation to the intact type four secretion system machine was also determined.

Three-dimensional surface renderings indicated that superfolder GFP is positioned below the DotB hexamer, which assembles as a disc at the base of the cytoplasmic ATPase complex. When attempting this technique, make sure to assess the stability of the fusion proteins and the functionality of the tag secretion system before proceeding to image acquisition. Modeling and fitting can be effective ways to analyze the dynamic patterns and structural densities as well as for evaluation of conformational changes or localisation of subunits into the structure.

This approach can help in understanding how different type four secret system components function, how they interact within the greater complex, and what functions different subassemblies perform.