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Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum
Ureaplasma diversum is a bacterial pathogen that infects cattle and can cause severe inflammation of the genital and reproductive systems. Lipid-associated membrane proteins (LAMPs), including GUDIV-103, are the main virulence factors in this bacterium. In this study, we heterologously expressed rec...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9147684/ https://www.ncbi.nlm.nih.gov/pubmed/35630474 http://dx.doi.org/10.3390/microorganisms10051032 |
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author | Santos-Junior, Manoel Neres Neves, Wanderson Souza Santos, Ronaldo Silva Almeida, Palloma Porto Fernandes, Janaina Marinho Guimarães, Bruna Carolina de Brito Barbosa, Maysa Santos da Silva, Lucas Santana Coelho Gomes, Camila Pacheco Sampaio, Beatriz Almeida Rezende, Izadora de Souza Correia, Thiago Macedo Lopes Neres, Nayara Silva de Macedo Campos, Guilherme Barreto Bastos, Bruno Lopes Timenetsky, Jorge Marques, Lucas Miranda |
author_facet | Santos-Junior, Manoel Neres Neves, Wanderson Souza Santos, Ronaldo Silva Almeida, Palloma Porto Fernandes, Janaina Marinho Guimarães, Bruna Carolina de Brito Barbosa, Maysa Santos da Silva, Lucas Santana Coelho Gomes, Camila Pacheco Sampaio, Beatriz Almeida Rezende, Izadora de Souza Correia, Thiago Macedo Lopes Neres, Nayara Silva de Macedo Campos, Guilherme Barreto Bastos, Bruno Lopes Timenetsky, Jorge Marques, Lucas Miranda |
author_sort | Santos-Junior, Manoel Neres |
collection | PubMed |
description | Ureaplasma diversum is a bacterial pathogen that infects cattle and can cause severe inflammation of the genital and reproductive systems. Lipid-associated membrane proteins (LAMPs), including GUDIV-103, are the main virulence factors in this bacterium. In this study, we heterologously expressed recombinant GUDIV-103 (rGUDIV-103) in Escherichia coli, purified it, and evaluated its immunological reactivity and immunomodulatory effects in bovine peripheral blood mononuclear cells (PBMCs). Samples from rabbits inoculated with purified rGUDIV-103 were analysed using indirect enzyme-linked immunosorbent assay and dot blotting to confirm polyclonal antibody production and assess kinetics, respectively. The expression of this lipoprotein in field isolates was confirmed via Western blotting with anti-rGUDIV-103 serum and hydrophobic or hydrophilic proteins from 42 U. diversum strains. Moreover, the antibodies produced against the U. diversum ATCC 49783 strain recognised rGUDIV-103. The mitogenic potential of rGUDIV-103 was evaluated using a lymphoproliferation assay in 5(6)-carboxyfluorescein diacetate succinimidyl ester–labelled bovine PBMCs, where it induced lymphocyte proliferation. Quantitative polymerase chain reaction analysis revealed that the expression of interleukin-1β, toll-like receptor (TLR)-α, TLR2, TLR4, inducible nitric oxide synthase, and caspase-3–encoding genes increased more in rGUDIV-103–treated PBMCs than in untreated cells (p < 0.05). Treating PBMCs with rGUDIV-103 increased nitric oxide and hydrogen peroxide levels. The antigenic and immunogenic properties of rGUDIV-103 suggested its suitability for immunobiological application. |
format | Online Article Text |
id | pubmed-9147684 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-91476842022-05-29 Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum Santos-Junior, Manoel Neres Neves, Wanderson Souza Santos, Ronaldo Silva Almeida, Palloma Porto Fernandes, Janaina Marinho Guimarães, Bruna Carolina de Brito Barbosa, Maysa Santos da Silva, Lucas Santana Coelho Gomes, Camila Pacheco Sampaio, Beatriz Almeida Rezende, Izadora de Souza Correia, Thiago Macedo Lopes Neres, Nayara Silva de Macedo Campos, Guilherme Barreto Bastos, Bruno Lopes Timenetsky, Jorge Marques, Lucas Miranda Microorganisms Article Ureaplasma diversum is a bacterial pathogen that infects cattle and can cause severe inflammation of the genital and reproductive systems. Lipid-associated membrane proteins (LAMPs), including GUDIV-103, are the main virulence factors in this bacterium. In this study, we heterologously expressed recombinant GUDIV-103 (rGUDIV-103) in Escherichia coli, purified it, and evaluated its immunological reactivity and immunomodulatory effects in bovine peripheral blood mononuclear cells (PBMCs). Samples from rabbits inoculated with purified rGUDIV-103 were analysed using indirect enzyme-linked immunosorbent assay and dot blotting to confirm polyclonal antibody production and assess kinetics, respectively. The expression of this lipoprotein in field isolates was confirmed via Western blotting with anti-rGUDIV-103 serum and hydrophobic or hydrophilic proteins from 42 U. diversum strains. Moreover, the antibodies produced against the U. diversum ATCC 49783 strain recognised rGUDIV-103. The mitogenic potential of rGUDIV-103 was evaluated using a lymphoproliferation assay in 5(6)-carboxyfluorescein diacetate succinimidyl ester–labelled bovine PBMCs, where it induced lymphocyte proliferation. Quantitative polymerase chain reaction analysis revealed that the expression of interleukin-1β, toll-like receptor (TLR)-α, TLR2, TLR4, inducible nitric oxide synthase, and caspase-3–encoding genes increased more in rGUDIV-103–treated PBMCs than in untreated cells (p < 0.05). Treating PBMCs with rGUDIV-103 increased nitric oxide and hydrogen peroxide levels. The antigenic and immunogenic properties of rGUDIV-103 suggested its suitability for immunobiological application. MDPI 2022-05-16 /pmc/articles/PMC9147684/ /pubmed/35630474 http://dx.doi.org/10.3390/microorganisms10051032 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Santos-Junior, Manoel Neres Neves, Wanderson Souza Santos, Ronaldo Silva Almeida, Palloma Porto Fernandes, Janaina Marinho Guimarães, Bruna Carolina de Brito Barbosa, Maysa Santos da Silva, Lucas Santana Coelho Gomes, Camila Pacheco Sampaio, Beatriz Almeida Rezende, Izadora de Souza Correia, Thiago Macedo Lopes Neres, Nayara Silva de Macedo Campos, Guilherme Barreto Bastos, Bruno Lopes Timenetsky, Jorge Marques, Lucas Miranda Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum |
title | Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum |
title_full | Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum |
title_fullStr | Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum |
title_full_unstemmed | Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum |
title_short | Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum |
title_sort | heterologous expression, purification, and immunomodulatory effects of recombinant lipoprotein gudiv-103 isolated from ureaplasma diversum |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9147684/ https://www.ncbi.nlm.nih.gov/pubmed/35630474 http://dx.doi.org/10.3390/microorganisms10051032 |
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