This article describes a method to quantify the polarity and dynamics of secretion components in Legionella pneumophila, a respiratory pathogen. The approach involves fluorescence imaging and statistical analysis of intensity gradients to assess component recruitment at bacterial poles.
Begin with Legionella pneumophila, a respiratory pathogen expressing a fluorescently tagged secretion system component required for host infection.
To quantify the polarity of these components, acquire fluorescence images, and adjust contrast to distinguish individual cells.
Place rectangular regions from the cell pole toward the cytoplasm to measure fluorescence gradients.
Mask these regions to extract mean intensity and variance, then calculate polarity scores as variance-to-mean intensity ratios.
High polarity scores indicate targeted recruitment of components at the bacterial poles.
For dynamic measurements, capture two time-lapse images and adjust the contrast to visualize the cells clearly.
Place square regions at the cell center to measure cytosolic fluorescence.
Mask entire cells to correct for photobleaching and define background regions between cells for noise subtraction.
Export all intensities and calculate the change in fluorescence over time.
Increased intensity indicates clustering, while decreased intensity reflects the dynamic redistribution of secretion components within the bacteria.
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.
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 successive 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.
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