Browsing by Author "Ruiz, Enrico A."

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    Outer Membrane Vesicles From Brucella melitensis Modulate Immune Response and Induce Cytoskeleton Rearrangement in Peripheral Blood Mononuclear Cells
    Avila-Calderon, Eric Daniel; Medina-Chavez, Olin; Flores-Romo, Leopoldo; Hernandez-Hernandez, Jose Manuel; Donis-Maturano, Luis; Lopez-Merino, Ahidé; Arellano-Reynoso, Beatriz; Aguilera-Arreola, Ma. Guadalupe; Ruiz, Enrico A.; Gomez-Lunar, Zulema; Witonsky, Sharon G.; Contreras-Rodriguez, Araceli (2020-10-19)
    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 TNF alpha 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.
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    Outer Membrane Vesicles of Gram-Negative Bacteria: An Outlook on Biogenesis
    Avila-Calderon, Eric Daniel; Ruiz-Palma, Maria del Socorro; Aguilera-Arreola, Ma. Guadalupe; Velazquez-Guadarrama, Norma; Ruiz, Enrico A.; Gomez-Lunar, Zulema; Witonsky, Sharon G.; Contreras-Rodriguez, Araceli (2021-03-04)
    Outer membrane vesicles (OMVs) from Gram-negative bacteria were first described more than 50 years ago. However, the molecular mechanisms involved in biogenesis began to be studied only in the last few decades. Presently, the biogenesis and molecular mechanisms for their release are not completely known. This review covers the most recent information on cellular components involved in OMV biogenesis, such as lipoproteins and outer membrane proteins, lipopolysaccharide, phospholipids, quorum-sensing molecules, and flagella.
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    Proteomic Analysis of Membrane Blebs of Brucella abortus 2308 and RB51 and Their Evaluation as an Acellular Vaccine
    Araiza-Villanueva, Minerva; Avila-Calderon, Eric Daniel; Flores-Romo, Leopoldo; Calderon-Amador, Juana; Sriranganathan, Nammalwar; Al Qublan, Hamzeh; Witonsky, Sharon G.; Aguilera-Arreola, Ma. Guadalupe; Ruiz-Palma, Maria del Socorro; Ruiz, Enrico A.; Suarez-Gueemes, Francisco; Gomez-Lunar, Zulema; Contreras-Rodriguez, Araceli (2019-11-29)
    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.