High dissolved oxygen tension triggers outer membrane vesicle formation by Neisseria meningitidis

Background: Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria and can be used as vaccines. Often, detergents are used to promote release of OMVs and to remove the toxic lipopolysaccharides. Lipopolysaccharides can be detoxified by genetic modification such that vesicles spontaneously produced by bacteria can be directly used as vaccines. The use of spontaneous OMVs has the advantage that no separate extraction step is required in the purification process. However, the productivity of spontaneous OMVs by bacteria at optimal growth conditions is low. One of many methods for increasing OMV formation is to reduce the linkage of the outer membrane to the peptidoglycan layer by knocking out the rmpM gene. A previous study showed that for Neisseria meningitidis this resulted in release of more OMVs. Furthermore, cysteine depletion was found to trigger OMV release and at the same time cause reduced growth and oxidative stress responses. Here we study the effect of growth rate and oxidative stress on OMV release. Results: First, we identified using chemostat and accelerostat cultures of N. meningitidis that increasing the growth rate from 0.03 to 0.18 h-1 has a limited effect on OMV productivity. Thus, we hypothesized that oxidative stress is the trigger for OMV release and that oxidative stress can be introduced directly by increasing the dissolved oxygen tension of bacterial cultures. Slowly increasing oxygen concentrations in a N. meningitidis changestat showed that an increase from 30 to 150% air saturation improved OMV productivity four-fold. Batch cultures controlled at 100% air saturation increased OMV productivity three-fold over batch cultures controlled at 30% air saturation. Conclusion: Increased dissolved oxygen tension induces the release of outer membrane vesicles in N. meningitidis cultures. Since oxygen concentration is a well-controlled process parameter of bacterial cultures, this trigger can be applied as a convenient process parameter to induce OMV release in bacterial cultures. Improved productivity of OMVs not only improves the production costs of OMVs as vaccines, it also facilitates the use of OMVs as adjuvants, enzyme carriers, or cell-specific drug delivery vehicles.