Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Genetic code expansion, or GCE, overcomes limitations of other labeling strategies. It enables us to site specifically labeled proteins, and we have bright organic fluorophores that enable their visualization, so we can avoid strategies that make, say, fluorescent protein fusions, which can disrupt protein function. This method can be applied to any other system.

For example, we've used it to label Zika CO protein, and that enables us to provide and produce labeled Zika virus that we can then observe infecting host cells. To begin, transfer 100 microliters of primary culture into five milliliters of LB medium containing 35 micrograms per milliliter chloramphenicol and 100 micrograms per milliliter ampicillin. Incubate at 37 degrees Celsius with shaking at 250 RPM until the optical density at 600 nanometers reaches 0.6.

Replace the LB medium with the modified N-minimal medium supplemented with one millimolar TCO, a non-canonical amino acid. And grow the bacteria at 34 degrees Celsius for 30 minutes. Add 0.2%arabinose, 25 milligrams per milliliter of chloramphenicol, and 100 milligrams per milliliter of ampicillin, and grow the cells for another six hours, shaking at 250 RPM.

After six hours, wash the bacteria four times over an interval of 30 minutes with fresh MgM media without non-canonical amino acids or, ncAA. Centrifuge the bacteria, re-suspend in PBS buffer, and incubate for one hour at four degrees Celsius in the dark to remove excess ncAAs. After one hour, centrifuge the bacteria at 3, 000 G for 15 minutes at four degrees Celsius, and store them for further use.

For a control experiment, repeat the same experiment by expressing ncAA bearing proteins in the absence of ncAA. Re-suspend salmonella cells expressing SseJ incorporated with TCO in the absence or presence of TCO in PBS. Adjust the optical density at 600 nanometers to four in PBS, and incubate the cells with 20 micromolar Janelia Fluor 646 tetrazene or 20 micromolar BDPFL tetrazine at 37 degrees Celsius in the dark, and shake for one to two hours at 250 RPM.

Pellet the cells and wash three to four times with PBS containing 5%DMSO and 0.2%Pluronic F-127. Re-suspend the pellet in PBS containing 5%DMSO. After incubating overnight at four degrees Celsius in the dark, wash twice again with PBS.

Image the cells immediately using a confocal microscope, or fix them with 1.5%paraformaldehyde in PBS for 30 to 45 minutes at room temperature in the dark. Culture and maintain HeLa cells at 37 degrees Celsius, 5%carbon dioxide, and 95%humidity in high glucose DMEM supplemented with 10%FBS in addition to penicillin streptomycin. Culture bacteria one day before infection and inoculate a single colony of wild-type salmonella harboring pEVOL plasmid containing orthogonal tRNA synthetase pair and pWSK29 plasmid containing SseJ gene in five millimeters of antibiotic containing standard LB broth overnight at 37 degrees Celsius with shaking at 250 RPM.

Repair a diluted cell stock at 100, 000 cells per milliliter and seed 50, 000 HeLA cells per well in 500 microliters of DMEM containing 10%FBS growth medium in an eight well chamber slide. Keep the chamber slide in an incubator for 24 hours at 37 degrees Celsius, 5%carbon dioxide, and 95%humidity. For the bacterial infection, subculture wild-type salmonella harboring pEVOL plasmid and pWSK29 plasmid containing SseJ gene by diluting 100 microliters of overnight bacterial culture into three milliliters of LB medium containing 35 micrograms per milliliter chloramphenicol and 100 micrograms per milliliter ampicillin.

Incubate at 37 degrees Celsius with shaking at 250 RPM for five to seven hours. To initiate the HeLa cell infection, after taking out the HeLa cells from the incubator, wash the cells with prewarmed DPBS, and add 500 microliters of fresh DMEM containing 10%FBS to each well. Place the chamber slide back in the carbon dioxide incubator until the infection begins.

After five to seven hours of incubation, dilute the salmonella culture so that the optical density at 600 nanometers is 0.2 in one milliliter of DMEM growth medium, and add the requisite amounts of the salmonella inoculum to each well of the chamber slide, so that the multiplicity of infection is 100. After incubating the infected cells in a carbon dioxide incubator for 30 minutes, wash the cells three times with prewarmed DPBS to remove extracellular salmonella. Set this time point to zero hours post-infection, and add 500 microliters of complete medium containing 100 micrograms per milliliter of gentamicin for one hour.

After incubation, again, wash the cells three times with DPBS as shown earlier and add 500 microliters of the complete medium supplemented with 0.2%arabinose, 10 micrograms per milliliter of gentamicin to each well of the chamber slide. In a control experiment, perform similar infections of HeLa cells without TCO. After incubating the chamber slide for 10 hours in the carbon dioxide incubator, replace the complete medium with fresh complete medium without TCO, and wash the HeLa cells four times over an interval of 30 minutes each with prewarmed DPBS.

Then wash the cells with fresh complete medium without TCO. After 12 hours post-infection, aspirate off the medium from the cells and wash the cell with prewarmed DPBS twice or three times. In one group of the wells, add 500 microliters of the dye solution mixture one, and in another group of the wells of the same chamber slide, add 500 microliters of the dye solution mixture two.

Place the chamber slides back into the carbon dioxide incubator for one and 1/2 to two hours. After 13.5 to 14 hours post-infection, rinse the HeLa cells twice with prewarmed DPBS, and add 500 microliters of fresh DMEM supplemented with FBS. After incubating for 30 minutes, wash the cells again with prewarmed DPBS as shown earlier, and then wash with fresh DMEM four times over an interval of 30 minutes.

At 16 hours post-infection, fix the HeLa cells with paraformaldehyde by adding 200 microliters of 4%paraformaldehyde to each well and incubating for 10 minutes at room temperature in the dark. Aspirate off the paraformaldehyde. Rinse three times with PBS, and store the cells in PBS at four degrees Celsius in the dark.

Bring the cells to the microscope, and place the imaging chamber on the microscope stage in the sample holder. Then change the medium in the well with 0.4 milliliters of the freshly made GLOX BME imaging buffer. Decrease the laser power to around one milliwatt to identify a HeLa cell of interest and adjust the focal plane and laser beam angle while illuminating the sample with low 647 nanometer laser densities.

Adjust the HILO illumination angle. Capture a reference to fraction limited image of the target structure, and switch the fluorophores to the dark state by turning the laser to its maximum power. Set the pre-amplifier gain to three and activate the frame transfer.

Then set EM gain to 200 for higher sensitivity in dSTORM measurements. Adjust the laser strength to a suitable level where blinking events are separated in space and time. Set the exposure time to 30 milliseconds and begin the acquisition.

Acquire 10, 000 to 30, 000 frames. Repeat the similar acquisition at different ROIs. For the imagery reconstruction of dSTORM, open image J and import the raw data.

Open the Thunderstorm plugin, and configure the camera setting corresponding to the device. Go to run analysis and set the appropriate settings, such as image filtering, localization methods, and subpixel localization of molecules. Click Okay to start the image reconstruction and begin the post-processing end result analysis.

Salmonella expressing SseJ incorporated with TCO are treated with Janelia Fluor 646 tetrazine and imaged with Janelia Fluor 646 fluorescence in the absence or presence of TCO. The fluorescence of SseJ is detected only when TCO is present. HeLa cells are infected with salmonella harboring psseJ-HA for 16 hours, fixed and immunostained with anti HA antibody, LPS, and DAPI.

As expected, SseJ dependent salmonella induced filaments, or SIFs, are formed in the infected cells. In the absence of TCO, the amber codon is not suppressed, and SseJ dependent SIFs are absent in infected HeLa cells. SseJ dependent SIFs are observed in the presence of TCO, which clearly indicates that SseJ was rescued by the expression of SseJF10TCOHA using genetic code expansion, or GCE.

Labeling specificity of secreted SseJF10TCOHA in HeLa cells through SPIEDAC click reactions using two alternate dye mixture one and dye mixture two are shown here. Click dye Janelia Fluor 646 TZ was further used to observe whether there was any background labeling in HeLa cells infected with wild-type Salmanella carrying SseJ HA in the presence of TCO and P vault plasmid. SseJ was released into the cytoplasm of the host cell with no intracellular or non-specific fluorescent signals demonstrating that the fluorescent signal was specific to SseJ.

Super resolution imaging of GCE labeled SseJ in HeLa cells is presented in this figure. A brightfield image of a HeLa cell under observation is shown here. The diffraction limited widefield image of secreted SseJ labeled with TCO and Janelia Fluor 646 and the corresponding dSTORM image of the SseJ decorated SIFs are presented here.

The insert shows a magnified view of a super resolved SseJ in the boxed region. NCAA stock solutions were prepared in NaOH. Do not forget to neutralize it before or after addition to the media.

After labeling protein of interest, wash the cell extensively, so that background signal is minimal. Following our GCE procedure, now, we can label any virulence factor in bacteria and follow its activity using our imaging techniques.