Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

The overall goal of this study is to purify Outer Membrane Vesicles or OMVs from Legionella pneumophila in a standardized way and to analyze their influence on bacterial replication in human and murine macrophages. This method can help to answer key questions in the field of outer membrane vesicle research. For example, the standardized procedure for OMV preparation may help to study their impact on host-pathogen interaction.

The main advantage of this technique is that it will provide a standardized protocol to study the impact of Legionella pneumophila OMVs on human or murine macrophages and this will improve our understanding of Legionella pneumophila pathogenicity. We first had the idea for this method when we thought about the possible pro-inflammatory potential of OMVs and how this might influence the course of infection as OMVs are released by Legionella both inside the host cell and extracellularly. The procedure will be demonstrated by Kerstin Hoffmann, a technician from my laboratory and Analina Young, a post doctoral researcher.

To begin, while working under sterile conditions, spread L.pneumophila strain core B on BCYE agar plates and incubate at 37 degrees Celsius for three days. Use the L.pneumophila from a pre-culture plate to inoculate 10 milliliters of YEB media at an OD600 of 0.3 and incubate the bacteria at 37 degrees Celsius on a rotating shaker for six hours. Verify the purity of the liquid culture by spreading 100 microliters of the suspension on a blood agar plate.

Incubate the blood agar plate overnight at 37 degrees Celsius. Add the remaining liquid culture to 90 milliliters of fresh YEB medium and incubate the culture on a rotating shaker until it reaches at OD600 of 3.0 to 3.5. Centrifuge the liquid culture at 4, 000 g for 20 minutes to pellet the bacteria.

Then transfer the supernatant to fresh centrifuge tubes. Discard the bacterial pellets and centrifuge the samples again before repeating the centrifugation step once more. Next, filter sterilize the remaining supernatant twice.

Then transfer the bacteria-free supernatant to ultracentrifuge tubes and spin the samples at 100, 000 g in four degrees Celsius for three hours. Decant the supernatant and discard it. Then use sterile PBS to resuspend the OMV pellets and pool them.

Repeat the ultracentrifugation step to remove contaminating proteins and free LPS. Now, discard the supernatant and dry the ultracentrifuge tube with a sterile cotton swab. Resuspend the OMV pellet with 500 microliters of sterile PBS.

Then streak 20 microliters each onto blood agar and BCYE agar plates to exclude bacterial contamination of the prepared vesicle. Incubate the blood agar plate overnight and the BCYE agar plate for three days. Use the BCYE assay according to the manufacturer's instructions to quantify the amount of protein for the obtained OMV preparation.

After differentiating THP-1 cells into macrophages according to the text protocol, use 500 microliters of fresh medium to replace the differentiation medium and incubate the cells for another 24 hours at 37 degrees Celsius. To treat THP-1 or murine BMDM cells with OMVs, thaw previously prepared OMVs and add the appropriate amount to the cells. Incubate the macrophages with OMVs at 37 degrees Celsius for at least 20 hours to infect pre-treated or untreated THP-1 cells with wild-type L.pneumophila strain core B or murine BMDM cells with a flagella-lacking mutant of this strain without changing the medium from the cells.

Add bacteria at an MOI of 0.5 which amounts to one times 10 to the fifth L.pneumophila per well. Incubate the cultures for 24 and 48 hours respectively. To lyse the cells, add saponin at a final concentration of 0.1%and incubate the cells for five minutes.

Then resuspend the lysed macrophages by pipetting and transfer the suspension to a reaction vessel. Use sterile PBS to prepare serial dilutions of the L.pneumophila suspension. Then streak 50 microliters of the required dilutions on BCYE agar plates and incubate the plates for three days.

Visually count the formed colonies by eye and carry out calculations according to the text protocol. In this experiment, examining the pro-inflammatory potential of L.pneumophila OMVs, PMA-differentiated THP-1 cells respond with a time and dose-dependent increase in IL-8 and IL-6 secretion. Here, the influence of different toll-like receptors on L.pneumophila OMV recognition was analyzed in murine BMDMs with different genetic backgrounds using a CXCL-1 ELISA assay.

Murine BMDMs from wild-type mice secreted CXCL-1 after OMV stimulation while murine BMDMs from TLR2/4 double knockout secreted significantly less. To study the impact of L.pneumophila OMVs on bacterial replication in THP-1 macrophages, cells were pre-incubated with OMVs for 20 hours and then additionally infected with L.pneumophila. The pre-stimulation of THP-1 derived macrophages first reduces the bacterial replication dose dependently after 24 hours of infection while it leads to a doubling in CFU count at the later time point.

As shown in this experiment, the pre-stimulation of murine BMDMs with OMVs result in a ten-fold increase in CFU count in murine BMDM from wild-type animals while TLR2 knockout and TRIF/MyD88 macrophages do not show this increase in L.pneumophila replication after OMV pre-incubation. While attempting this procedure, it's important to remember to work under sterile conditions and to check for bacterial contamination of the purified OMVs. Otherwise, the downstream analysis doesn't give conclusive results.

Following this procedure, other methods like nanoparticle tracking analysis can be performed in order to answer additional questions like which Legionella mutant is producing more or less OMVs or to investigate the impact of different stress conditions on the secretion of OMVs. After its development, this technique paved the way for researchers in the field of OMV research to explore the impact of OMVs on host-pathogen interactions in vitro and then in vivo. After watching this video, you should have a good understanding of how to purify OMVs and how to start analyzing their effect on the subsequent infection of macrophages.

This protocol can then be applied for different gram-negative bacteria and their respective host cell after adapting incubation and growth times. Don't forget that working with Legionella pneumophila can be extremely infectious for humans so work should always be carried out under a sterile bench while handling Legionella and performing this procedure.