In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Our work unveils how VCP or p97 acts as a cytosolic defender against multiple different pathogens. This unlocks the potential of VCP or p97 for therapeutic applications and bio-engineering strategies. In addition to routine biochemistry, super resolution microscopy, cryo EM, cryo ET, are utilized to figure out how these host effectors organize on pathogen to destroy them.

To begin, grow the streptococcus pneumonia are six strain serotype two overnight at 37 degrees Celsius with 5%carbon dioxide in Todd Hewitt yeast medium. Inoculate one part of the overnight culture into 10 parts of fresh Todd Hewitt yeast medium, and incubate at 37 degrees Celsius with 5%carbon dioxide until the optical density at 600 nanometers reaches 0.4. Then mix 900 microliters of the streptococcus pneumonia culture with 600 microliters of 50%glycerol and store the glycerol stock at minus 80 degrees Celsius.

Then thaw a fresh stock of streptococcus pneumonia from minus 80 degrees Celsius and completely inoculate it into five milliliters of Todd Hewitt yeast medium. Take approximately 10 to the power of seven streptococcus pneumonia cells. To wash the cells thoroughly, re-suspend the bacteria in the reaction buffer and centrifuge at 21, 000 G for five minutes at room temperature.

Re-suspend the pellet with a 549 mammalian cell lysate. Then add purified ubiquitin and one millimolar adenosine triphosphate and incubate the mixture for one to two hours at 27 degrees Celsius. Then wash the bacteria once with reaction buffer containing one molar urea, followed by two additional washes with only the reaction buffer.

Fix the bacteria with 1%paraformaldehyde. After fixation, incubate the cells with PBS, containing one milligram per milliliter glycine for 15 minutes. Then wash twice with PBS.

Prepare a reaction mixture by combining ubiquitin with other required components in the reaction buffer. Adjust the total volume of the reaction mixture to 100 to 150 microliters. Re-suspend the bacteria in the ubiquitin enzyme containing reaction mixture, and incubate the suspension for two hours at 27 degrees Celsius.

After washing the bacteria three times with reaction buffer, containing urea, fix them with 1%paraformaldehyde. After fixation, incubate the cells with PBS, containing one milligram per milliliter glycine for 15 minutes. Then incubate the sample in 3%BSA for one hour at 27 degrees Celsius, and wash the sample twice with PBS.

Re-suspend the ubiquitinated bacteria in a primary antibody solution, prepared at higher concentrations than typically used in immunofluorescence assays. Then incubate for one hour at 27 degrees Celsius. After washing the bacteria twice with PBS, re-suspend them in the corresponding secondary antibody conjugated with Alexa Fluor 488 and Alexa Fluor 555.

Now drop cast approximately 10 to 20 microliters of the labeled bacterial sample onto a glass slide and mix in five to 10 microliters of mounting medium with or without DAPI. Place a cover slip over the sample and use tissue paper to blot any excess liquid. Combine six micromolar VCP p97 with one micromolar NPLOC4 and one micromolar UFD-1.

Adjust the volume of the reaction mixture to approximately 90 microliters, using reaction buffer, and incubate on ice for two hours. Next, add one to two millimolar adenosine triphosphate to the reaction mixture containing p97 UFD-1, NPLOC4. Then combine this with 10 to the power of seven ubiquitinated streptococcus pneumonia cells, and incubate for two hours at 37 degrees Celsius.

Immediately after treatment, take a 10 microliter aliquot from the reaction mixture and perform a serial dilution. Spot 10 to 20 microliters of each dilution onto brain heart infusion auger plates. Assess colony forming units between the zero hour and two hour time points.

Calculate bacterial viability percentage by dividing the two hour CFU count by the zero hour CFU count and multiplying by 100. Significant ubiquitination of streptococcus pneumoniae was observed when treated with either mammalian cell lysate or a reconstituted E-1, E-2, E-3 enzyme system. Ubiquitination signal was absent in the control and markedly diminished when ubiquitinated streptococcus pneumoniae was exposed to OTUB1, confirming K48 linked poly ubiquitin modification.

A line profile analysis showed full overlap between ubiquitin and bacterial signals confirming their spatial co-localization. Three-dimensional reconstruction confirmed the spatial overlap of ubiquitin and streptococcus pneumoniae signals. Wild type p97 when combined with its co-factors, UFD1 and NPLOC4, reduced bacterial viability to 10 to 20%after two hours, regardless of whether ubiquitination was performed with cell lysate or with purified enzyme system.

No reduction in bacterial viability was observed when ubiquitination was omitted, when p97 was heat inactivated, or when using the catalytically inactive p97, E57 8Q mutant, confirming that both ubiquitination and active p97 are essential for bacterial killing. Our study opens the door about how host E3 ubiquitin ligases modify different pathogens and investigate the effector mechanisms to clear the infectious threats. We next aim to explore how p97 or VCP is regulated, and farther engineer it to increase its antibacterial activity.