A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

This method can reveal spatial patterns of bacterial gene expression and strategies for physiological adaptation to host tissue micro-environments that are lost when averaged across the entire population in tissues. With this technique potential heterogeneities can be detected that must be considered when identifying new bacterial pathways and proteins to be targeted by antivirulence or antibacterial compounds. Begin by streaking S.aureus strains of interest on blood agar plates from a frozen glycerol stock and incubating the plates at 37 degrees Celsius for 16 to 24 hours to confirm their hemolytic phenotypes based on their ability to lyse red blood cells and to form clear transparent zones.

Inoculate single colony isolates of each strain in four milliliters of tryptic soy broth, or TSB, in sterile glass tubes and rotate the tubes at 37 degrees Celsius for 16 to 24 hours in a tube roller at an approximately 70 degree angle and 70 rotations per minute. The next morning measure the optical density at 600 nanometers, or OD 600, of the cultures on a spectrophotometer and dilute the cells to an OD 600 of 0.05 in 25 milliliters of sterile TSB at a five to one flask to volume ratio in 125 milliliter Delong flasks. Incubate the cultures in a 37 degree Celsius water bath at 280 rotations per minute and harvest the cells during the exponential phase.

Pellet the cells by centrifugation and wash the cells two times in an equal volume of sterile PBS. Then resuspend the cells in fresh PBS to a one times 10 to the eighth colony forming units per milliliter concentration for their subsequent injection into a recipient animal. At the appropriate experimental end point, harvest the kidney, heart, liver, lungs, and spleen into 15 milliliter polypropylene tubes containing 10%buffered formalin for a 24 to 48 hour incubation in the dark at room temperature with gentle shaking or rotation.

At the end of the fixation, embed the organs in clear tissue freezing medium for storage at minus 80 degrees Celsius. Use a cryostat to acquire 10 micrometer thick tissue sections and dry the sections on individual pre-cleaned charged glass slides for 20 minutes in the dark before applying hard mounting medium supplemented with DAPI. Then apply cover slips and cure the slides at room temperature overnight before transferring the samples to long term storage at four degrees Celsius.

To identify lesions within the samples, place a slide on the stage of a laser scanning confocal microscope and use an appropriate objective for visualizing individual cells to acquire images of the lesions. To measure the fluorescence intensities within the confocal images, open an image in ImageJ and adjust the brightness and contrast to properly visualize the fluorescence signal of the lesion. Define the region of interest by using the polygon selections tool and add the ROI to the ROI manager under the Edit tab in ImageJ.

Select Edit, then Selection, and finally Add to Manager. Next, rename the ROI and label the respective image. To define the centroid, open the Analyze tab and select Area, Mean Gray Value, Centroid, and Limit to Threshold.

Extract the centroid fluorescence intensity value or measure the mean fluorescence intensity in a given lesion per unit area. This can be achieved by using the thresholding function to set the lower and upper fluorescence limits as necessary. Export the data to an Excel file and prepare a column indicating the mean fluorescence intensity per unit area for the periphery and core.

To calculate the mean fluorescence intensities per unit area in the periphery and core, manually define the upper and lower limits of thresholding. Staphylococcus aureus thermonuclease GFP fusion fluorescence is on average nearly ninefold higher in cells carrying the fusion gene than in cells not carrying the reporter fusion. Similarly, tandem dimeric Tomato fusion fluorescence is about sixfold higher than the null reporter control.

The pattern of reporter activities can be confirmed by flow cytometry with tissue homogenates. Although the fold differences in fluorescence are typically lower. Variations in the fluorescence measurements can be due to heterogeneous expression of the reporters.

For example, in these representative samples it was observed that some lesions expressed either one or both of the reporters within approximately 100 micrometers of distance in the same tissue sample. Examining the reporter activity to a single cell resolution in staphylococcal abcess communities reveals a spatial regulation of staphylococcus aureus thermonuclease expression in abscesses circumscribed with strong DAPI staining, likely associated with the formation and release of neutrophil extracellular traps. For example, significantly higher staphylococcus aureus thermonuclease GFP fusion fluorescence is observed in the interior core of the staphylococcal abcess community compared to localized to the periphery.

In the same abcess, the pattern for the tandem dimeric Tomato fusion fluorescence appears to be inverted. However, the pattern in this experiment was not statistically significant. Obtaining intact cryo-embedded dissections is vital for the fluorescence image analysis and should be done carefully.

Sorting of the bacterial cells to capture sub-populations expressing the fusion to varying extents, coupled with RNA-Seq analysis could illuminate genome wide changes in the transcriptome. Take care when working with formalin as it is a carcinogen and may cause skin irritation, allergic reactions, or eye damage upon direct exposure. Generating mutant derivatives of strains carrying fluorescent reporters of interest can help reveal the genetic basis for the observed spatial regulatory patterns.