Proteomic Analysis of Membrane Blebs of Brucella abortus 2308 and RB51 and Their Evaluation as an Acellular Vaccine.

Minerva Araiza-Villanueva,Juana Calderón-Amador,Eric Daniel Avila-Calderón,Ma Guadalupe Aguilera-Arreola,Hamzeh Al Qublan,Nammalwar Sriranganathan,Francisco Suárez-Güemes,Zulema Gómez-Lunar,María Del Socorro Ruiz-Palma,Enrico A Ruiz,Leopoldo Flores-Romo,Sharon Witonsky,Araceli Contreras-Rodríguez

doi:10.3389/fmicb.2019.02714

Abstract

Membrane blebs are released from Gram-negative bacteria, however, little is known about Brucella blebs. This work pursued two objectives, the first was to determine and identify the proteins in the membrane blebs by proteomics and in silico analysis. The second aim was to evaluate the use of membrane blebs of Brucella abortus 2308 and B. abortus RB51 as an acellular vaccine in vivo and in vitro. To achieve these aims, membrane blebs from B. abortus 2308 and RB51 were obtained and then analyzed by liquid chromatography coupled to mass spectrometry. Brucella membrane blebs were used as a “vaccine” to induce an immune response in BALB/c mice, using the strain B. abortus RB51 as a positive vaccine control. After subsequent challenge with B. abortus 2308, CFUs in spleens were determined; and immunoglobulins IgG1 and IgG2a were measured in murine serum by ELISA. Also, activation and costimulatory molecules induced by membrane blebs were analyzed in splenocytes by flow cytometry. Two hundred and twenty eight proteins were identified in 2308 membrane blebs and 171 in RB51 blebs, some of them are well-known Brucella immunogens such as SodC, Omp2b, Omp2a, Omp10, Omp16, and Omp19. Mice immunized with membrane blebs from rough or smooth B. abortus induced similar protective immune responses as well as the vaccine B. abortus RB51 after the challenge with virulent strain B. abortus 2308 (P < 0.05). The levels of IgG2a in mice vaccinated with 2308 membrane blebs were higher than those vaccinated with RB51 membrane blebs or B. abortus RB51 post-boosting. Moreover, mice immunized with 2308 blebs increased the percentage of activated B cells (CD19+CD69+) in vitro. Therefore, membrane blebs are potential candidates for the development of an acellular vaccine against brucellosis, especially those derived from the rough strains so that serological diagnostic is not affected.

Highlights

The membrane blebs have been described in Gram-negative bacteria for more than 50 years (Manning and Kuehn, 2013)
Immunization with membrane blebs from both strains did not affect the expression of CD86 molecules since Mean Fluorescence Intensity (MFI) values did not change significantly compared with mice injected with saline
The MHC-II MFI values decreased drastically in mice immunized with RB51 membrane blebs while CD11c expression decreased in mice immunized with membrane blebs from both Brucella strains, so these results were merely based on activated cells but not by up- or downregulation of molecules analyzed (Supplementary Figure S3A) (Two-way ANOVA with Bonferroni post-test, confidence interval of 95%)

Summary

Introduction

The membrane blebs have been described in Gram-negative bacteria for more than 50 years (Manning and Kuehn, 2013). These membrane blebs are released from the outer membrane of the cell to the external milieu, showing spherical shapes, and ranging in sizes from 20 to 250 nm. Marion et al (2019), showed pulmonary inflammation and neutrophil recruitment as well as cytokine production in mice inoculated with Acinetobacter baumannii membrane blebs intranasally. Membrane blebs from Aeromonas hydrophila induced B and T cell activation and the production of TNFα, IL-1β, and stimulated peripheral blood mononuclear cells producing IL-8 (Avila-Calderón et al, 2018)

Methods

Results

Conclusion