Biomimetic Materials to Characterize Bacteria-host Interactions

The overall goal of this procedure is to produce biomimetic materials consisting of pure recombinant adhesions, coupled to polystyrene beads, which can then be used to dissect the biochemical and functional interactions between individual bacterial adhesions and host cell receptors. This method can help answer key questions in the field of host pathogen interactions such as measuring the effects on host cell signaling and inhibition of pathogen mediated cytotoxicity. The main advantage to this technique is that purified adhesions, a AEAs are directionally and covalently coupled to polymer particles that are similar in size to bacteria and thus mimic the bacterial surface display.

Demonstrating the procedure with me will be TU A-C-E-D-A PhD student from our laboratory. Thal amine directional coupling will be demonstrated here. This protocol is suitable for coupling of cystine containing proteins to amine functionalized polymer beads using SUL succinyl for p meli phenyl butyrate, or SMPB As a cross-linking agent, prepare all necessary reagents just prior to use as described in the protocol text to begin the procedure for bead activation.

Mixed a bead suspension by gently inverting and then transfer the required amount of bead suspension into a sterile 1.5 milliliter tube. Containing one milliliter of sterile PBS pH 7.0 gently pipette up and down to wash the beads and pellet by centrifugation in a micro centrifuge. Huge carefully remove the snat with a pipette and a scar.

Reese suspend the bead pellet in one milliliter of fresh sterile PBS and repeat the washing step. Resuspend the bead pellet. In 0.8 milliliters of PBS add 200 microliters of freshly prepared 10 millimolar SFO SMPB.

To give a final concentration of two millimolar, incubate the bead suspension for one hour at 25 degrees Celsius on a rotating wheel while the beads are incubating. Prepare the protein for the following coupling step so it can be immediately added to the activated beads. Check the protein concentration and adjust it to the final concentration required for the coupling reaction.Six.

Micromolar of protein and PBS and a volume of one milliliter are usually used for protein reduction. Add a TEP stock solution to give a final concentration of five millimolars. Incubate the solution for 30 minutes At room temperature, retain a few microliters of the protein solution for determining the initial protein concentration and a calculation of coupling efficiency.

Start the protein coupling procedure by pelleting the activated beads and washing the pellet. Once in one milliliter of fresh, sterile PBS, re suspend the pellet in the prepared protein solution to give the desired protein concentration. Incubate the protein bead suspension for two hours at 25 degrees Celsius on a rotating wheel After two hours, deactivate the remaining activated groups on the beads by adding a Sistine stock solution to a final concentration of 50 millimolar and incubating to suspension for 30 minutes at 25 degrees Celsius on a rotating wheel.

Held the beads by centrifugation. Keep the snat for determining the protein concentration and calculation of coupling efficiency. Wash the bead pellet twice with one milliliter of PBS and Resus bend and one milliliter of fresh PBS to give the final product one day before the competition assay seed each well of a 24 well plate with one milliliter of hela cells at a concentration of 150, 000 cells per milliliter.

This will allow cells to reach approximately 80%co fluency prior to starting the experiment. Inoculate a five milliliter marine LB culture with a fresh colony of V para hemolytic and grow overnight at 30 degrees Celsius with shaking. Prepare sufficient bead coupled multivalent adhesion molecule as demonstrated earlier.

Allow for 100 microliters of 10 x bead stock per well or a final concentration of 500 nanomolar protein on the day of the competition assay. Measure the OD 600 of the bacterial culture. Prepare infection media by diluting bacterial cultures into colorless DMM without additives.

Prewarm to 37 degrees Celsius containing 10%volume to volume beat suspension. To give an MOI of 10, prepare one milliliter per well and 10 to 20%excess volume per sample. Remove the old medium from the wells and wash the cultured.

Heal the cells by adding to each well one milliliter of sterile PBS prewarm to 37 degrees Celsius. Remove the PBS and add one milliliter of infection medium per well. Add solutions containing control beads and bacteria as positive controls and adhesion beads and no bacteria as negative.Controls.

Add DMM containing 0.1%TRITTON X 100 as lysis controls. Set up each experimental condition including the controls triplicate. Incubate the plate in a tissue culture incubator at 37 degrees Celsius for the desired amount of time.

Cytotoxicity is assessed. Four hours post-infection. Remove 200 microliters from each well used in the 24 well plate and transfer to a new 96 well plate.

Spin the 96 well plate at 1, 500 G for five minutes and transfer 100 microliters from each well into a fresh 96 well plate. Add 100 microliters of the media used during the infection experiments to three fresh wells of the 96 well plate to be used as blanks. Carry out the lactate dehydrogenase release assay using an LDH Cytotoxicity detection kit and following the manufacturer’s instructions.

First, calculate the amount of reagent needed in increments of 25. For example, for 100 samples. Mix 11.25 milliliters of reagent A with 250 microliters of reagent B invert to avoid foaming do not vortex.

Next, put the mixture in a reservoir. Then add 100 microliters of the reagent mix to each sample and incubate the plate at room temperature at 10, 20 and 30 minutes. Read the absorbance at 490 nanometers on a plate reader.

Subsequently calculate percent cytotoxicity as described in the manuscript measurements of bacterial adhesion are made one hour after infection. Remove the media from the cell layer and thoroughly wash cell layer with sterile prewarm PBS to remove any unattached cells. Wash at least three to four times with one milliliter of PBS each time lyce to host cells by adding to each well one milliliter of a sterile 1%volume to volume Triton X 100 solution in PBS incubate the plate at 37 degrees Celsius for five minutes.

Pipette each sample up and down several times before transferring the contents of each well each to separate. 1.5 milliliter tubes. Prepare tenfold serial dilutions of the samples into sterile PBS plate, 100 microliters of each sample on marine LB agar and spread using a cell spreader optimize which dilution to plate depending on the bacterial strains and time point for this experimental setup.

A 10 to the fifth or 10 to the sixth fold dilution will give a suitable number of CFUs. Incubate the plates at 37 degrees Celsius overnight night on the following day, enumerate bacteria by colony counting. In competition assays, heela cells were infected with V para hemolytic strain.

Poor one indicated with positive or left uninfected in the presence of different competing entities. In vitro infection with poor one resulted in very high levels of cell lysis which was inhibited by MAM seven coupled beads, but not MA’AM one coupled or GST coupled beads. Enumeration of v paralytic attached to either untreated HELOC cells or cells incubated with MAM.

Seven MAM one or GST control beads revealed that MAM. Seven beads but not MAM one or GST Control beads. Outcompete v para hemolytic for attachment to host cell surface receptors.

Adhesions coupled to fluoro. Four Labeled beads can mimic bacterial adhesion to host cells and are powerful tools for cellular imaging. The tube on the right contains a suspension of fluorescent blue biomimetic beads.MAM.

Seven coupled fluorescent blue beads were used to characterize the process of MAM seven mediated attachment to epithelial cells. Attachment of MAM seven to host cells resulted in actin rearrangements and formation of stress fibers which were co visualized using Rumine Phin to stain for f actin. Following this procedure, other methods like affinity purification coupled with proteomics or western blot can be performed in order to answer additional questions such as which host cellular factors are involved in signaling processes downstream of bacterial attachment.