Interplay between Outer Membrane Vesicles and Host Defense Peptides : Bacterial shields versus Host swords

Abstract

The scope of this thesis is the interplay between Host Defense Peptides (HDPs) and Outer Membrane Vesicles (OMVs). HDPs have both antimicrobial and immunomodulatory properties. OMVs contain multiple antigens in their native environment and are therefore promising for vaccine development. It was also thought that OMVs could protect bacteria against killing by HDPs, but additionally it was investigated whether OMV release could be a response of bacteria upon HDPs in the environment. This increase in OMV yield could also be used for vaccine production, if OMV properties are not significantly altered. HDPs are known to have membrane-active properties, but the role of LPS-binding in killing of Gram-negative bacteria is not yet evident. Therefore, LPS-binding was correlated with HDP antibacterial effectivity, to distinguish between LPS acting as anchor and facilitating HDP functions or LPS acting as a sink and inhibiting HDP functions. Furthermore, immunomodulatory capabilities of HDPs were investigated, since this neutralization has not yet been described for OMV-induced activation. If HDPs could balance OMV-induced immune responses, the combination could be promising for vaccine development.

In chapter 3, it was shown that OMVs can indeed act as a bacterial defense against HDPs. Not only were OMV quantities increased after stimulation with sub-lethal concentrations of HDPs, but also could addition of isolated OMVs to bacterial cultures protect against HDP killing. However, this was not true for all HDPs tested. The differences in the mechanisms of action of HDPs were further elucidated in chapter 4. CATH-2 and PMAP-36 were shown to be membrane active, but their effectivity did not correlate with LPS-binding. PMAP-23 showed an interaction with LPS and probably acts via a carpet model, but antibacterial effectivity was not correlated with LPS affinity. PR-39 was shown to be purely intracellularly active and to not affect bacterial membranes at all but did show binding to LPS. Stronger LPS-binding even correlated with enhanced bacterial killing for PR-39. Not only HDPs were shown to affect bacterial membranes, but also heat was shown to affect bacteria and enhance OMV release. In chapter 5, PMAP-36 was studied in more detail with respect to OMV induction and also to the immunomodulation of OMVs. Induction with PMAP-36 resulted in the presence of the peptide in isolated OMVs but this presence did not affect immunomodulation. When PMAP-36 was added to spontaneously formed OMVs (sOMVs) after isolation, it did show a neutralizing effect. Therefore, a large array of HDPs was investigated for their immunomodulatory capabilities in combination with OMVs (chapter 6). Four out of the eight HDPs tested showed immunomodulatory effects, being LL-37, CATH-2, PMAP-36 and K9CATH. They were further investigated for their specific TLR neutralizing capabilities. This revealed that OMVs were not only able to stimulate TLR4 and TLR2, but also TLR5 and TLR9. TLR neutralization was HDP specific, but TLR5 was consistently not neutralized by any peptide tested. Concluding, OMVs indeed play a role in defense against HDPs and HDPs are able to modulate OMV-induced immune responses. However, LPS-binding did not seem to correlate with HDP antibacterial activity.