Outer Membrane Vesicles From Brucella melitensis Modulate Immune Response and Induce Cytoskeleton Rearrangement in Peripheral Blood Mononuclear Cells.

Eric Daniel Avila-Calderón,Leopoldo Flores-Romo,Ahidé López-Merino,Araceli Contreras-Rodríguez,Olín Medina-Chávez,José Manuel Hernández-Hernández,Ma Guadalupe Aguilera-Arreola,Luis Donis-Maturano,Zulema Gomez-Lunar,Sharon Witonsky,Enrico A Ruiz,Beatriz Arellano-Reynoso

doi:10.3389/fmicb.2020.556795

Abstract

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived rough-mutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O-polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified in OMVs from B. melitensis16M, and 43 in OMVs from B. melitensis VTRM1. Proteome comparison showed that 22 orthologous proteins were common in vesicles from both strains, and their core proteome contained Omp31, Omp25, GroL, and Omp16. After a subsequent detergent and enzyme treatment, OMVs from B. melitensis VTRM1 exhibited higher sensitive compared to OMVs from the B. melitensis 16M strain. Neither OMVs induced IL-17, proliferation, apoptosis or DNA damage. Nonetheless, OMVs from the smooth and rough strains induced overproduction of TNFα and IL-6, as well as actin and tubulin rearrangements in the cytoskeleton. Moreover, OMVs from both strains inhibited PD-L1 expression in T-cells. These data revealed significant differences in OMVs derived from the rough and smooth Brucella strains, among which, the presence or absence of complete LPS appeared to be crucial to protect proteins contained within vesicles and to drive the immune response.

Highlights

Brucella is a Gram-negative pathogen that can cause brucellosis, a worldwide zoonotic disease still prevalent in many countries
Outer membrane vesicles released from the whole cell of B. melitensis 16M and VTRM1 and purified vesicles were observed as showed in the micrographs (Figure 1)
Outer membrane vesicles from B. melitensis were first observed by Gamazo and Moriyon (1987) using samples from the smooth, B. melitensis 16M and the rough, B. melitensis 115 strains, both of which had been cultured in tryptic soy broth

Summary

Introduction

Brucella is a Gram-negative pathogen that can cause brucellosis, a worldwide zoonotic disease still prevalent in many countries. Consumption of unpasteurized dairy-products made using milk from infected animals is the main infection cause for humans. Similar to other Gram-negative bacteria, B. melitensis releases OMVs (Lynch and Alegado, 2017). These vesicles are nanostructures consisting of a bi-lipid membrane and a spherical shape, ranging from 25 to 250 nm in size. Production of OMVs begins with membrane bulging and ends with membrane fusion, followed by the release of vesicles into the external space (Toyofuku et al, 2019). Many studies have contributed in determining the composition of bacterial vesicles, which include phospholipids, outer membrane proteins (OMPs), lipopolysaccharides (LPS), periplasmic/cytoplasmic proteins, as well as nucleic acids (Pathirana and Kaparakis-Liaskos, 2016; Bitto et al, 2017)

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