PMAP-36 reduces the innate immune response induced by Bordetella bronchiseptica-derived outer membrane vesicles

Melanie D Balhuizen,Henk P Haagsman,Eline F De Jonge,Chris H.A Van De Lest,Jan Tommassen,Edwin J.A Veldhuizen,Chantal M Versluis,Jos F Brouwers,Roel M Van Harten

doi:10.1016/j.crmicr.2020.100010

Melanie D Balhuizen, Henk P Haagsman + Show 7 more

Open Access

https://doi.org/10.1016/j.crmicr.2020.100010

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Abstract

Host defense peptides (HDPs), such as cathelicidins, are small, cationic, amphipathic peptides and represent an important part of the innate immune system. Most cathelicidins, including the porcine PMAP-36, are membrane active and disrupt the bacterial membrane. For example, a chicken cathelicidin, CATH-2, has been previously shown to disrupt both Escherichia coli membranes and to release, at sub-lethal concentrations, outer membrane vesicles (OMVs). Since OMVs are considered promising vaccine candidates, we sought to investigate the effect of sub-bactericidal concentrations of PMAP-36 on both OMV release by a porcine strain of Bordetella bronchiseptica and on the modulation of immune responses to OMVs. PMAP-36 treatment of bacteria resulted in a slight increase in OMV release. The characteristics of PMAP-36-induced OMVs were compared with those of spontaneously released OMVs and OMVs induced by heat treatment. The stability of both PMAP-36- and heat-induced OMVs was decreased compared to spontaneous OMVs, as shown by dynamic light scattering. Furthermore, treatment of bacteria with PMAP-36 or heat resulted in an increase in negatively charged phospholipids in the resulting OMVs. A large increase in lysophospholipid content was observed in heat-induced OMVs, which was at least partially due to the activity of the outer-membrane phospholipase A (OMPLA). Although PMAP-36 was detected in OMVs isolated from PMAP-36-treated bacteria, the immune response of porcine bone-marrow-derived macrophages to these OMVs was similar as those against spontaneous or heat-induced OMVs. Therefore, the effect of PMAP-36 addition after OMV isolation was investigated. This did decrease cytokine expression of OMV-stimulated macrophages. These results indicate that PMAP-36 is a promising molecule to attenuate undesirable immune responses, for instance in vaccines.

Highlights

Outer membrane vesicles (OMVs) are spherical particles, 20-300 nm in size, that are naturally produced by all Gram-negative bacteria (Schwechheimer and Kuehn, 2015, Deatherage et al, 2009)
We investigated the effects of sublethal concentrations of a very potent porcine Host defense peptides (HDPs), PMAP-36, on outer membrane vesicles (OMVs) release in a porcine, pathogen B. bronchiseptica, with the goal to increase release of OMVs that are suited for vaccine usage
OMV release was studied by analyzing the protein content of isolated OMV fractions using SDSPAGE, which revealed an increase in protein concentration with increasing PMAP-36 concentrations (Fig. 1a)

Summary

Introduction

Outer membrane vesicles (OMVs) are spherical particles, 20-300 nm in size, that are naturally produced by all Gram-negative bacteria (Schwechheimer and Kuehn, 2015, Deatherage et al, 2009). OMVs represent the outer membrane (OM) of the Gram-negative bacterium and comprise a large number and wide variety of surface-exposed antigens. This makes OMVs promising in vaccine development as has already been shown for Neisseria meningitidis and Bordetella pertussis One class of HDPs, cathelicidins, includes the porcine PMAP-36 This HDP is α-helical with a hinge region at the C-terminus containing a cysteine residue which allows the peptide to dimerize. Several models for the interaction between HDPs and bacterial membranes have been proposed, all leading to membrane permeabilization and cell death (Zhang and Gallo, 2019)

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