RESUMO
BACKGROUND: Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. METHODS: We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. RESULTS: Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. CONCLUSIONS: Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Linfoma/imunologia , Proteínas Recombinantes de Fusão/imunologia , Neoplasias da Bexiga Urinária/imunologia , Animais , Brucella/enzimologia , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Linfoma/patologia , Linfoma/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapiaRESUMO
Vaccination is as one of the most beneficial biopharmaceutical interventions against pathogens due to its ability to induce adaptive immunity through targeted activation of the immune system. Each vaccine needs a tailor-made set of tests in order to monitor its quality throughout the development and manufacturing. The analysis of the conformational state of protein nanoparticles is one of the key steps in vaccine quality control. The enzyme lumazine synthase from Brucella spp. (BLS) acts as a potent oral and systemic immunogen. BLS has been used as a carrier of foreign peptides, protein domains and whole proteins, serving as a versatile platform for vaccine engineering purposes. Here, we show the generation and characterization of four families of nanobodies (Nbs) which only recognize BLS in its native conformational state and that bind to its active site. The present results support the use of conformation-sensitive Nbs as molecular probes during the development and production of vaccines based on the BLS platform. Finally, we propose Nbs as useful molecular tools targeting other protein scaffolds with potential applications in nano-and biotechnology.
Assuntos
Complexos Multienzimáticos , Anticorpos de Domínio Único , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Brucella/enzimologia , Escherichia coli/genética , Células HEK293 , Humanos , Complexos Multienzimáticos/química , Complexos Multienzimáticos/fisiologia , Conformação Proteica , Dobramento de Proteína , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/fisiologia , Vacinas de Subunidades AntigênicasRESUMO
Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responses.
Assuntos
Brucella , Flagelina , Complexos Multienzimáticos , Proteínas Recombinantes de Fusão , Salmonella typhimurium , Animais , Brucella/enzimologia , Brucella/genética , Brucella/imunologia , Células CACO-2 , Feminino , Flagelina/biossíntese , Flagelina/genética , Flagelina/imunologia , Humanos , Imunidade Humoral , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Salmonella typhimurium/metabolismo , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/imunologiaRESUMO
Shiga toxin producing Escherichia coli (STEC) are bacterial pathogens involved in food-borne diseases. Shiga toxin (Stx) is the main virulence factor of STEC and is responsible for systemic complications including Hemolytic Uremic Syndrome (HUS). It has been previously demonstrated that Shiga toxin type 2 (Stx2) induces pregnancy loss in rats in early stage of pregnancy. The main purpose of this study was to determine if an active immunization prevents Stx2 mediated pregnancy loss and confers passive protective immunity to the offspring. For that purpose Sprague Dawley female rats were immunized with the chimera based on the enzyme lumazine synthase from Brucella spp. (BLS) and the B subunit of Shiga toxin 2 (Stx2B) named BLS-Stx2B. After immunization females were mated with males. At day 8 of gestation, dams were challenged intraperitoneally with a sublethal and abortifacient dose of Stx2. The immunization induced high anti-Stx2B-specific antibody titers in sera and most important, prevented pregnancy loss. Pups born and breastfeed by immunized dams had high anti-Stx2B-specific antibody titers in sera. Cross-fostering experiments indicated that passive protective immunity against Stx2 was transmitted through lactation. These results indicate that immunization of adult female rats with BLS-Stx2B prevents Stx2-induced pregnancy loss and confers anti Stx2 protective immunity to the offspring.
Assuntos
Aborto Espontâneo/prevenção & controle , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/imunologia , Imunidade Materno-Adquirida , Toxina Shiga II/imunologia , Aborto Espontâneo/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Brucella/enzimologia , Feminino , Doenças Transmitidas por Alimentos/prevenção & controle , Síndrome Hemolítico-Urêmica/prevenção & controle , Masculino , Complexos Multienzimáticos/imunologia , Gravidez , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/imunologia , Escherichia coli Shiga ToxigênicaRESUMO
In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK.
Assuntos
Brucella/enzimologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Cristalografia por Raios X , Análise Mutacional de DNA , Histidina Quinase , Simulação de Dinâmica Molecular , Fosforilação , Conformação Proteica , Proteínas Quinases/genética , Multimerização ProteicaRESUMO
Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Brucella/enzimologia , Brucella/imunologia , Imunomodulação , Complexos Multienzimáticos/metabolismo , Sequência de Aminoácidos , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Brucella/genética , DNA Complementar , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Proteínas RecombinantesRESUMO
Brucella Lumazine Synthase (BLS) is a highly immunogenic decameric protein which can accept the fusion of foreign proteins at its ten N-termini. These chimeras are very efficient to elicit systemic and oral immunity without adjuvants. BLS signaling via Toll-Like Receptor 4 (TLR4) regulates innate and adaptive immune responses, inducing dendritic cell maturation and CD8(+) T-cell cytotoxicity. In this work we study the effect induced by BLS in TLR4-expressing B16 melanoma. In order to evaluate the effectiveness of BLS as a preventive vaccine, C57BL/6J mice were immunized with BLS or BLS-OVA, and 35 days later were subcutaneously inoculated with B16-OVA melanoma. BLS or BLS-OVA induced a significant inhibition of tumor growth, and 50% of mice immunized with the highest dose of BLS did not develop visible tumors. This effect was not observed in TLR4-deficient mice. For treatment experiments, mice were injected with BLS or BLS-OVA 2 days after the inoculation of B16 cells. Both treatments induced significant and equal tumor growth delay and increased survival. Moreover, BLS and BLS-OVA stimulation were also effective in TLR4-deficient mice. In order to study whether BLS has a direct effect on tumor cells, B16 cells were preincubated with BLS, and after 48h, cells were inoculated. Tumors induced by BLS-stimulated cells had inhibited growth and survival was increased. In the BLS group, 40% of mice did not develop tumors. This effect was abolished by the addition of TLR4/MD2 blocking antibody to cells before BLS stimulation. Our work demonstrates that BLS immunization induces a preventive antitumor response that depends on mice TLR4. We also show that BLS generates a therapeutic effect in mice inoculated with B16 cells. Our results show that BLS acts directly in cultured tumor cells via TLR4, highly suggesting that BLS elicits its therapeutic effects acting on the TLR4 from B16 melanoma cells.
Assuntos
Brucella/enzimologia , Complexos Multienzimáticos/metabolismo , Receptor 4 Toll-Like/genética , Animais , Apoptose , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/mortalidade , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Ovalbumina/genética , Ovalbumina/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Taxa de Sobrevida , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/metabolismo , Transplante HomólogoRESUMO
One of the most remarkable characteristics of Brucella lumazine synthase (BLS) is its versatility to undergo reversible dissociation and reassociation as a polymeric scaffold. We have proposed a mechanism of dissociation and unfolding of BLS. Using static light scattering (SLS) analysis, we were able to demonstrate that the decameric assembly dissociates into two different conditions [pH 5 or 2M guanidinium chloride (GdnHCl) pH 7] forming stable folded pentamers. The transition from folded pentamers to unfolded monomers by GdnHCl denaturation is highly cooperative and can be measured by different spectroscopic techniques. In this work, we show the successful insertion of an intrinsic probe to study in more detail the equilibria described in previous publications. For that purpose, we performed single-point mutations of Phe residues 121 and 127, located at the pentamer-pentamer and monomer-monomer interface, respectively, to Trp residues. These mutations produced only a marginal perturbation of the BLS structure. We analyzed the unfolding and stability of the mutants through different techniques: far-and near-UV CD, SLS, dynamic light scattering, and fluorescence spectroscopy. The introduced intrinsic probe could be used to gain insights into the detailed folding and assembly mechanism of this protein.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação Puntual , Algoritmos , Proteínas de Bactérias/genética , Brucella/enzimologia , Guanidina , Concentração de Íons de Hidrogênio , Luz , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Análise Espectral , Termodinâmica , TriptofanoRESUMO
Brucella spp. lumazine synthase (BLS) is a highly immunogenic decameric protein. It has been previously evaluated as a carrier to increase the immunogenicity of peptides fused to its N-termini. VP8 is a sialic acid binding domain of rotavirus external capsid protein VP4, which is involved in virus adhesion to host cells. In this work, the C486 bovine rotavirus (BRV) VP8 core protein (VP8d) was fused to the structure of BLS with the aim to produce an enhancement of the immune response against BRV VP8 and to evaluate the possible use of this antigen for vaccine development. The feasibility of using BLS as an antigen delivery system of polypeptides larger in size than those previously tested was also evaluated. Groups of female mice were immunized with BLS-VP8d fusion protein, VP8d or an equimolar mixture of purified VP8d and BLS (BLS+VP8d). Dams immunized with BLS-VP8 induced 97.5-100% protection against homologous challenge with C486 BRV; while pups born to dams immunized either with VP8d or BLS+VP8d presented a significant lower level of protection. The neutralizing antibody pattern was also significantly different among these experimental groups, and in concordance with challenge experiment. Overall, these results demonstrate that the BLS-VP8d chimeric protein is properly folded and stable, and that the BLS scaffold is a potent antigen delivery system that enhances the antibody response against BRV and elicits complete homotypic passive protection in a suckling mouse model.
Assuntos
Brucella/enzimologia , Sistemas de Liberação de Medicamentos , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus/imunologia , Rotavirus/química , Vacinas Sintéticas/imunologia , Animais , Animais Recém-Nascidos , Animais Lactentes/imunologia , Anticorpos Antivirais/sangue , Brucella/imunologia , Bovinos , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Rotavirus/imunologia , Vacinas contra Rotavirus/administração & dosagem , Células Th1/imunologia , Vacinas Sintéticas/administração & dosagemRESUMO
6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase; LS) catalyzes the penultimate step in the biosynthesis of riboflavin in plants and microorganisms. This protein is known to exhibit different quaternary assemblies between species, existing as free pentamers, decamers (dimers of pentamers) and icosahedrally arranged dodecamers of pentamers. A phylogenetic analysis on eubacterial, fungal and plant LSs allowed us to classify them into two categories: Type I LSs (pentameric or icosahedral) and Type II LSs (decameric). The Rhizobiales represent an order of alpha-proteobacteria that includes, among others, the genera Mesorhizobium, Agrobacterium and Brucella. Here, we present structural and kinetic studies on several LSs from Rhizobiales. Interestingly, Mesorhizobium and Brucella encode both a Type-I LS and a Type-II LS called RibH1 and RibH2, respectively. We show that Type II LSs appear to be almost inactive, whereas Type I LSs present a highly variable catalytic activity according to the genus. Additionally, we have solved four RibH1/RibH2 crystallographic structures from the genera Mesorhizobium and Brucella. The relationship between the active-site architecture and catalytic properties in these isoenzymes is discussed, and a model that describes the enzymatic behavior is proposed. Furthermore, sequence alignment studies allowed us to extend our results to the genus Agrobacterium. Our results suggest that the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress.
Assuntos
Brucella/enzimologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Rhizobium/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Brucella/genética , Catálise , Cristalografia por Raios X , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Estrutura Quaternária de Proteína , Pteridinas/metabolismo , Rhizobium/genética , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The enzyme lumazine synthase (LS) catalyzes the penultimate step of riboflavin biosynthesis in plants, fungi and bacteria. The quaternary structure of the polypeptide differs between species, existing as pentamers or as icosahedrally arranged dodecamers of pentamers with 60 subunits. The pathogen Brucella spp. expresses two proteins that exhibit LS activity, RibH1 and RibH2. The latter enzyme belongs to a novel third category of quaternary arrangement for LS, that of a decameric structure assembled as a head-to-head oriented dimer of pentamers. In contrast, the RibH1 enzyme is assembled as a pentamer, as noted for several other LS enzymes. RibH1 appears to be the functional LS in Brucella spp., whereas RibH2, an enzyme of lower catalytic activity, is a virulence factor presumably acting in response to oxidative stress. The latter observation prompted us to further investigate the structural and catalytic properties of RibH2 in order to clarify the biological function of this enzyme. Here, we present a detailed analysis of two new crystallographic forms of RibH2 that explain the low catalytic activity of this enzyme in comparison with RibH1 and other LSs. Additionally, we analyze the effect of pH on the structure of this enzyme, and the binding of riboflavin and 6,7-dimethyl-8-ribityllumazine to its active site.
Assuntos
Brucella/enzimologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Sítios de Ligação , Brucella/genética , Catálise , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Histidina/genética , Histidina/metabolismo , Ligantes , Modelos Moleculares , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/genética , Fosfatos/metabolismo , Estrutura Quaternária de Proteína , Pteridinas/metabolismo , Riboflavina/biossíntese , Riboflavina/metabolismo , Especificidade por SubstratoRESUMO
The multiple display of protein domains on polymeric scaffolds is an emerging technology for many applications. BLS is a highly immunogenic protein that has an oligomeric structure formed by a 17.2 kDa subunit arranged as a dimer of pentamers. Here we describe the production as well as the structural, functional, and immunological properties of a 9 kDa double-stranded RNA-binding domain (RBD3) fused to the structure of BLS. We demonstrate that the BLS and RBD3 modules are stably and independently folded in the structure of the chimera and form a decameric structure of 255 kDa as the native BLS oligomers. The polymeric display of RBD3 in the structure of BLS increases the dsRNA binding strength of this domain both in vitro and in vivo, and also enhances its immunogenicity to the point that it breaks the tolerance of mice to the RBD3 self-antigen. Our results underscore the BLS display strategy as a powerful tool for biotechnological and therapeutic applications.
Assuntos
Complexos Multienzimáticos/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sítios de Ligação , Brucella/enzimologia , Dicroísmo Circular , Clonagem Molecular , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Desnaturação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , TermodinâmicaRESUMO
Protein assemblies with a high degree of repetitiveness and organization are known to induce strong immune responses. For that reason they have been postulated for the design of subunit vaccines by means of protein engineering. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably owing to its homodecameric arrangement and remarkable thermodynamic stability. Structural analysis has shown that it is possible to insert foreign peptides at the ten amino terminus of BLS without disrupting its general folding. These peptides would be displayed to the immune system in a highly symmetric three-dimensional array. In the present work, BLS has been used as a protein carrier of foreign peptides. We have established a modular system to produce chimeric proteins decorated with ten copies of a desired peptide as long as 27 residues and have shown that their folding and stability is similar to that of the wild-type protein. The knowledge about the mechanisms of dissociation and unfolding of BLS allowed the engineering of polyvalent chimeras displaying different predefined peptides on the same molecular scaffold. Moreover, the reassembly of mixtures of chimeras at different steps of the unfolding process was used to control the stoichiometry and spatial arrangement for the simultaneous display of different peptides on BLS. This strategy would be useful for vaccine development and other biomedical applications.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biblioteca de Peptídeos , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Biopolímeros/química , Biopolímeros/metabolismo , Brucella/enzimologia , Dicroísmo Circular , Expressão Gênica , Vetores Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18-kDa monomers, each extensively contacting neighboring monomers. The icosahedrical structure consists of 60 LS monomers arranged as 12 pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella sp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedric enzymes. This unusual divergence prompted us to further investigate its quaternary arrangement. In the present work, we demonstrate by means of solution light scattering and x-ray structural analyses that BLS assembles as a very stable dimer of pentamers, representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 degrees C compared with pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins.
Assuntos
Brucella/enzimologia , Complexos Multienzimáticos/química , Estrutura Quaternária de Proteína , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Luz , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Desnaturação Proteica , Dobramento de Proteína , Espalhamento de Radiação , Espectrometria de Fluorescência , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The immunogenicity and protective efficacy of recombinant lumazine synthase from Brucella spp. (rBLS) administered with different adjuvants was evaluated in mice. Mice were immunized with rBLS in the absence or the presence of aluminum hydroxide gel (BLS-Al), monophosphoryl lipid A (BLS-MPA), or incomplete Freund's adjuvant (BLS-IFA). rBLS per se induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. All the adjuvants increased this response; the BLS-IFA formulation was the most effective at inducing BLS-specific IgG antibodies. In addition, after in vitro stimulation with rBLS, spleen cells from BLS-IFA-, BLS-Al-, or BLS-MPA-immunized mice proliferated and produced interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-10, and IL-4, suggesting the induction of a mixed Th1-Th2 response. Immunization with rBLS protected mice against challenge with B. abortus 544. The levels of protection in the spleen were similar for all adjuvants, but only BLS-Al and BLS-IFA were effective in the liver. Our results indicate that BLS might be a useful candidate for the development of subunit vaccines against brucellosis, since it elicits antigen-specific cellular responses, with production of IFN-gamma and protection, independently of the adjuvant formulation used.
Assuntos
Brucella/enzimologia , Brucella/imunologia , Brucelose/prevenção & controle , Complexos Multienzimáticos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Vacina contra Brucelose/administração & dosagem , Brucelose/imunologia , Feminino , Técnicas In Vitro , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/administração & dosagem , Células Th1/imunologia , Células Th2/imunologia , Vacinas Sintéticas/administração & dosagemRESUMO
Multilocus enzyme electrophoresis (MLEE) of 99 Brucella isolates, including the type strains from all recognized species, revealed a very limited genetic diversity and supports the proposal of a monospecific genus. In MLEE-derived dendrograms, Brucella abortus and a marine Brucella sp. grouped into a single electrophoretic type related to Brucella neotomae and Brucella ovis. Brucella suis and Brucella canis formed another cluster linked to Brucella melitensis and related to Rhizobium tropici. The Brucella strains tested that were representatives of the six electrophoretic types had the same rRNA gene restriction fragment length polymorphism patterns and identical ribotypes. All 99 isolates had similar chromosome profiles as revealed by the Eckhardt procedure.
Assuntos
Brucella/classificação , Brucelose/microbiologia , Laticínios/microbiologia , Variação Genética , Animais , Brucella/enzimologia , Brucella/genética , Brucella/isolamento & purificação , Bovinos , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Ribossômico/análise , DNA Ribossômico/genética , Cães , Eletroforese em Gel de Amido/métodos , Enzimas/análise , Humanos , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , RibotipagemRESUMO
Previous studies have shown that the detection of antibodies to an 18-kDa cytoplasmic protein of Brucella spp. is useful for the diagnosis of human and animal brucellosis. This protein has now been expressed in recombinant form in Escherichia coli. The recombinant protein is soluble only under reducing conditions, but alkylation with iodoacetamide renders it soluble in non-reducing media. As shown by gel exclusion chromatography, this soluble form arranges in pentamers of 90 kDa. The reactivity of human and animal sera against the recombinant protein was similar to that found with the native protein present in brucella cytoplasmic fraction, suggesting that the recombinant protein is correctly folded. The protein has low but significant homology (30%) with lumazine synthases involved in bacterial riboflavin biosynthesis, which also arrange as pentamers. Biological tests on the crude extract of the recombinant bacteria and on the purified recombinant protein showed that the biological activity of the Brucella spp. 18-kDa protein is that of lumazine synthase. Preliminary crystallographic analysis showed that the Brucella spp. lumazine synthase arranges in icosahedric capsids similar to those formed by the lumazine synthases of other bacteria. The high immunogenicity of this protein, potentially useful for the design of acellular vaccines, could be explained by this polymeric arrangement.