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

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.

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

“…Previous testing of OMVs purified from B. melitensis and B. abortus as an acellular vaccine in mice, resulted in a level of protection comparable to that obtained from live Brucella vaccines (Avila-Calderón et al, 2012;Araiza-Villanueva et al, 2019). Such results demonstrated that OMVs are not infectious unlike whole Brucella cells, and they elicited a cellmediated protective immune response to kill the intracellular pathogen (Avila-Calderón et al, 2012;Araiza-Villanueva et al, 2019).…”

“…Differences found in the number of proteins detected in OMVs from B. melitensis 16M could be attributed to the use of different equipment. In 2019, a recent study by Araiza-Villanueva et al (2019) reported 228 proteins identified in OMVs from B. abortus 2308 and 171 in membrane vesicles from the B. abortus rough strain RB51.…”

“…Boigegrain et al (2003) had previously identified Omp25 and Omp31 in OMVs from B. suis 1330. In 2019, a recent study by Araiza-Villanueva et al (2019) identified 171 proteins found in vesicles from the B. abortus RB51 rough-vaccine strain, while vesicles from the B. abortus 2308 smooth strain, yielded a total of 228 proteins. In the present work, a less number of proteins were identified in OMVs from B. melitensis VTRM1 rough mutant, in comparison to proteins found in OMVs from the B. melitensis 16M smooth reference strain.…”

“…OMVs purified from Aeromonas hydrophila induced the expression of the surface activation marker CD69 in B and T lymphocytes in vitro (Avila-Calderón et al, 2018). On the other hand, OMVs have been used as an in vivo vaccine, inducing protection in mice after exposing them with the virulent strain as observed in OMVs from Acinetobacter baumannii, Helicobacter pylori, Escherichia coli, and Brucella (Avila-Calderón et al, 2012;Araiza-Villanueva et al, 2019;Liu et al, 2019;Wang et al, 2019;Pulido et al, 2020).…”

“…Interestingly, when comparing OMVs from the B. melitensis and B. abortus rough strains against smooth strains from the same species, important differences were observed. For instance the absence of the O-polysaccharide side chain of the LPS in rough strains was shown to improve the exposition of protein epitopes in OMVs; thus, increasing immune protection in vivo (Avila-Calderón et al, 2012;Araiza-Villanueva et al, 2019). The LPS has been related to biogenesis of OMVs as well as to the selective mechanism of protein sorting (Bonnington and Kuehn, 2014;Murphy et al, 2014;Cahill et al, 2015).…”