RESUMO
Listeriolysin O (LLO) has been proposed as a potential carrier or adjuvant molecule in the vaccination field. However, the cytotoxic and pro-apoptotic effects of LLO are the major limitations for this purpose. Here, we have performed a preclinical safety evaluation and characterized a new potential adjuvant application for a non-cytolytic LLO mutant (dtLLO) to enhance and modulate the immune response against the envelope (E) protein from dengue virus. In addition, we have studied the adjuvant effects of dtLLO on human immune cells and the role of membrane cholesterol for the binding and proinflammatory property of the toxoid. Our in-vivo results in the murine model confirmed that dtLLO is a safer molecule than wild-type LLO (wtLLO), with a significantly increased survival rate for mice challenged with dtLLO compared with mice challenged with wtLLO (P < 0·001). Histopathological analysis showed non-toxic effects in key target organs such as brain, heart, liver, spleen, kidney and lung after challenge with dtLLO. In vitro, dtLLO retained the capacity of binding to plasma membrane cholesterol on the surface of murine and human immune cells. Immunization of 6-8-week-old female BALB/c mice with a combination of dtLLO mixed with E protein elicited a robust specific humoral response with isotype diversification of immunoglobulin (Ig)G antibodies (IgG1 and IgG2a). Finally, we demonstrated that cholesterol and lipid raft integrity are required to induce a proinflammatory response by human cells. Taken together, these findings support a potential use of the dtLLO mutant as a safe and effective adjuvant molecule in vaccination.
Assuntos
Adjuvantes Imunológicos , Antígenos Virais/imunologia , Toxinas Bacterianas/imunologia , Vírus da Dengue/imunologia , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/imunologia , Imunidade Humoral , Proteínas Mutantes/imunologia , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Toxinas Bacterianas/genética , Colesterol/metabolismo , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Dengue/imunologia , Dengue/patologia , Dengue/prevenção & controle , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Hemólise/imunologia , Imunização , Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipídeos de Membrana/metabolismo , Camundongos , Proteínas Mutantes/genética , Ligação Proteica/imunologiaRESUMO
The basic replicon of Rhizobium etli CE3, like other members of the repABC plasmid family, is constituted by the repABC operon. RepC is essential for replication, and RepA and RepB play a role in plasmid segregation. It has been shown that deletion derivatives lacking the repAB genes have an increased copy number, indicating that these genes participate in the control of plasmid copy number. RepA is also a trans-incompatibility factor. To understand the regulation of the repABC operon, in this paper: (i) the transcription start site of the repABC operon was determined; (ii) the promoter region was identified by site-directed mutagenesis of the putative -35 and -10 hexameric elements; and (iii) RepA was recognized as a negative regulator of the transcription of the repABC operon.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases , Proteínas de Ligação a DNA , Óperon/genética , Proteínas/metabolismo , Rhizobium/genética , Transativadores , Transcrição Gênica , Proteínas de Bactérias/genética , Sequência de Bases , Pegada de DNA , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rhizobium/fisiologia , Alinhamento de SequênciaRESUMO
The symbiotic plasmid of Rhizobium etli CE3 belongs to the RepABC family of plasmid replicons. This family is characterized by the presence of three conserved genes, repA, repB, and repC, encoded by the same DNA strand. A long intergenic sequence (igs) between repB and repC is also conserved in all members of the plasmid family. In this paper we demonstrate that (i) the repABC genes are organized in an operon; (ii) the RepC product is essential for replication; (iii) RepA and RepB products participate in plasmid segregation and in the regulation of plasmid copy number; (iv) there are two cis-acting incompatibility regions, one located in the igs (incalpha) and the other downstream of repC (incbeta) (the former is essential for replication); and (v) RepA is a trans-acting incompatibility factor. We suggest that incalpha is a cis-acting site required for plasmid partitioning and that the origin of replication lies within incbeta.
