A Fijivirus Major Viroplasm Protein Shows RNA-Stimulated ATPase Activity by Adopting Pentameric and Hexameric Assemblies of Dimers.

Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. IMPORTANCE The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.

Crystal structures of peanut lectin in the presence of synthetic ß-N- and ß-S-galactosides disclose evidence for the recognition of different glycomimetic ligands.

Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation and tumor metastasis. Hence, structural studies of lectin-synthetic glycan complexes are essential for understanding lectin-recognition processes and for the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular-recognition process. Among them, peanut lectin (PNA) is highly relevant in the field of glycobiology because of its specificity for ß-galactosides, showing high affinity towards the Thomsen-Friedenreich antigen, a well known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of ß-galactoside and ß-lactoside ligands. Here, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable ß-N- and ß-S-galactosides. These complexes disclosed key molecular-binding interactions of the different sugars with PNA at the atomic level, revealing the roles of specific water molecules in protein-ligand recognition. Furthermore, binding-affinity studies by isothermal titration calorimetry showed dissociation-constant values in the micromolar range, as well as a positive multivalency effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides a qualitative structural rationale for the upcoming synthesis of optimized glycoclusters designed for the study of lectin-mediated biological processes. The understanding of the recognition of ß-N- and ß-S-galactosides by PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands that do not belong to the family of O-linked glycosides.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Three-Dimensional Structure of Full-Length NtrX, an Unusual Member of the NtrC Family of Response Regulators.

Bacteria sense and adapt to environmental changes using two-component systems. These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator (RR), which finally modulates the transcription of target genes. The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response. NtrX is a modular protein with REC, AAA+, and DNA-binding domains, an architecture that classifies it among the NtrC subfamily of RRs. However, it lacks the signature GAFTGA motif that is essential for activating transcription by the mechanism proposed for canonical members of this subfamily. In this article, we present the first crystal structure of full-length NtrX, which is also the first structure of a full-length NtrC-like RR with all the domains solved, showing that the protein is structurally similar to other members of the subfamily. We also report that NtrX binds nucleotides and the structures of the protein bound to ATP and ADP. Despite binding ATP, NtrX does not have ATPase activity and does not form oligomers in response to phosphorylation or nucleotide binding. We also identify a nucleotide sequence recognized by NtrX that allows it to bind to a promoter region that regulates its own transcription and to establish a negative feedback mechanism to modulate its expression. Overall, this article provides a detailed description of the NtrX RR and supports that it functions by a mechanism different to classical NtrC-like RRs.

Structural and functional characterization of a cold adapted TPM-domain with ATPase/ADPase activity.

The Pfam PF04536 TPM_phosphatase family is a broadly conserved family of domains found across prokaryotes, plants and invertebrates. Despite having a similar protein fold, members of this family have been implicated in diverse cellular processes and found in varied subcellular localizations. Very recently, the biochemical characterization of two evolutionary divergent TPM domains has shown that they are able to hydrolyze phosphate groups from different substrates. However, there are still incorrect functional annotations and uncertain relationships between the structure and function of this family of domains. BA41 is an uncharacterized single-pass transmembrane protein from the Antarctic psychrotolerant bacterium Bizionia argentinensis with a predicted compact extracytoplasmic TPM domain and a C-terminal cytoplasmic low complexity region. To shed light on the structural properties that enable TPM domains to adopt divergent roles, we here accomplish a comprehensive structural and functional characterization of the central TPM domain of BA41 (BA41-TPM). Contrary to its predicted function as a beta-propeller methanol dehydrogenase, light scattering and crystallographic studies showed that BA41-TPM behaves as a globular monomeric protein and adopts a conserved Rossmann fold, typically observed in other TPM domain structures. Although the crystal structure reveals the conservation of residues involved in substrate binding, no putative catalytic or intramolecular metal ions were detected. Most important, however, extensive biochemical studies demonstrated that BA41-TPM has hydrolase activity against ADP, ATP, and other di- and triphosphate nucleotides and shares properties of cold-adapted enzymes. The role of BA41 in extracellular ATP-mediated signaling pathways and its occurrence in environmental and pathogenic microorganisms is discussed.

Brucella spp. Lumazine Synthase Induces a TLR4-Mediated Protective Response against B16 Melanoma in Mice.

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.

Expression of the Multimeric and Highly Immunogenic Brucella spp. Lumazine Synthase Fused to Bovine Rotavirus VP8d as a Scaffold for Antigen Production in Tobacco Chloroplasts.

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein which can accommodate foreign polypeptides or protein domains fused to its N-termini, markedly increasing their immunogenicity. The inner core domain (VP8d) of VP8 spike protein from bovine rotavirus is responsible for viral adhesion to sialic acid residues and infection. It also displays neutralizing epitopes, making it a good candidate for vaccination. In this work, the BLS scaffold was assessed for the first time in plants for recombinant vaccine development by N-terminally fusing BLS to VP8d and expressing the resulting fusion (BLSVP8d) in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern, northern and western blot. BLSVP8d was highly expressed, representing 40% of total soluble protein (4.85 mg/g fresh tissue). BLSVP8d remained soluble and stable during all stages of plant development and even in lyophilized leaves stored at room temperature. Soluble protein extracts from fresh and lyophilized leaves were able to induce specific neutralizing IgY antibodies in a laying hen model. This work presents BLS as an interesting platform for highly immunogenic injectable, or even oral, subunit vaccines. Lyophilization of transplastomic leaves expressing stable antigenic fusions to BLS would further reduce costs and simplify downstream processing, purification and storage, allowing for more practical vaccines.

A polymeric protein induces specific cytotoxicity in a TLR4 dependent manner in the absence of adjuvants.

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA(257-264) and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA(257-264)in vivo. BLS-OVA(257-264) immunization induced the proliferation of OVA(257-264)-specific CD8+ lymphocytes and also increased the percentage of OVA(257-264)-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA(257-264)-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA(257-264) induced the generation of CD8+ effector cells. BLS-OVA(257-264) was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA(257-264) without adjuvants induced a strong OVA(257-264)-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor. These data show that TLR4 signaling is necessary for the induction of a cytotoxic response but not for antigen cross presentation.

Polymeric Display of Proteins through High Affinity Leucine Zipper Peptide Adaptors.

The polymeric display of proteins is a method that could be used to increase the immunogenicity of antigens and to enhance the interaction strength of binding domains for their target ligands through an avidity effect. However, the coupling of proteins to oligomeric scaffolds is challenging. The chemical conjugation and recombinant fusion techniques have limitations that prevent their general use. In this work we describe a simple and effective method for coupling proteins to the decameric structure of Brucella abortus Lumazine Synthase based on the use of a pair of high affinity heterodimeric coiled coil peptides complementary fused to the scaffold and the target protein. Results obtained with a series of proteins demonstrate the capability of this approach to generate polyvalent particles. Furthermore, we show that the method is able to increase the immunogenicity of antigens and produce polyfunctional particles with promising biomedical and nanotechnological applications.

Heterologous prime-boost oral immunization with GK-1 peptide from Taenia crassiceps cysticerci induces protective immunity.

Oral immunization is a goal in vaccine development, particularly for pathogens that enter the host through the mucosal system. This study was designed to explore the immunogenic properties of the Taenia crassiceps protective peptide GK-1 administered orally. Mice were orally immunized with the synthetic GK-1 peptide in its linear form with or without the Brucella lumazine synthase (BLS) protein adjuvant or as a chimera recombinantly bound to BLS (BLS-GK-1). Mice were boosted twice with GK-1 only at 15-day intervals. A significant rate of protection of 64.7% was achieved in GK-1-immunized mice, and that rate significantly increased to 91.8 and 96% when mice were primed with GK-1 coadministered with BLS as an adjuvant and BLS as a carrier, respectively. Specific antibodies and T cell activation and proliferation accompanied the protection induced, revealing the potent immunogenicity of GK-1. Through immunohistochemical studies, GK-1 was detected in T and B cell zones of the Peyer's patches (PP) and mesenteric lymph nodes. In the latter, abundant proliferating cells were detected by 5'-bromo-2'-deoxyuridine incorporation. No proliferation was detected in PP. Altogether, these results portray the potent immunogenic properties of GK-1 administered orally and reinforce the usefulness of BLS as an adjuvant and adequate vaccine delivery system for oral vaccines.

Single-domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP-ribosylating toxin of Salmonella typhimurium.

ADP-ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial toxins. Antibodies induced by immunization with inactivated ADP-ribosylating toxins provide efficient protection in case of some secreted toxins, e.g., diphtheria and pertussis toxins. However, other ADP-ribosylating toxins, such as Salmonella SpvB toxin, are secreted directly from the Salmonella-containing vacuole into the cytosol of target cells via the SPI-2 encoded bacterial type III secretion system, and thus are inaccessible to conventional antibodies. Small-molecule ADP-ribosylation inhibitors are fraught with potential side effects caused by inhibition of endogenous ADP-ribosyltransferases. Here, we report the development of a single-domain antibody from an immunized llama that blocks the capacity of SpvB to ADP-ribosylate actin at a molar ratio of 1:1. The single-domain antibody, when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric C2IN-C/SpvB toxin. Transfected cells were also protected against cytoskeletal alterations induced by wild-type SpvB-expressing strains of Salmonella. This proof of principle paves the way for developing new antidotes against intracellular toxins.

Brucella spp. lumazine synthase as a bovine rotavirus antigen delivery system.

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.

Renewed hope for a vaccine against the intestinal adult Taenia solium.

Review of experimental and observational evidence about various cestode infections of mammalian hosts revives hope for the development of an effective vaccine against adult intestinal tapeworms, the central protagonists in their transmission dynamics. As for Taenia solium, there are abundant immunological data regarding cysticercosis in humans and pigs, but information about human taeniasis is scarce. A single publication reporting protection against T. solium taeniasis by experimental primo infection and by vaccination of an experimental foster host, the immunocompetent female hamster, kindles the hope of a vaccine against the tapeworm to be used in humans, its only natural definitive host.

Blue-light-activated histidine kinases: two-component sensors in bacteria.

Histidine kinases, used for environmental sensing by bacterial two-component systems, are involved in regulation of bacterial gene expression, chemotaxis, phototaxis, and virulence. Flavin-containing domains function as light-sensory modules in plant and algal phototropins and in fungal blue-light receptors. We have discovered that the prokaryotes Brucella melitensis, Brucella abortus, Erythrobacter litoralis, and Pseudomonas syringae contain light-activated histidine kinases that bind a flavin chromophore and undergo photochemistry indicative of cysteinyl-flavin adduct formation. Infection of macrophages by B. abortus was stimulated by light in the wild type but was limited in photochemically inactive and null mutants, indicating that the flavin-containing histidine kinase functions as a photoreceptor regulating B. abortus virulence.

Improvement of the synthetic tri-peptide vaccine (S3Pvac) against porcine Taenia solium cysticercosis in search of a more effective, inexpensive and manageable vaccine.

Vaccination of pigs may curtail Taenia solium transmission by reducing the number of cysticerci, the precursors of adult intestinal tapeworms in humans. Several antigen preparations induce protection against porcine cysticercosis in experimental settings but only one subunit vaccine (S3Pvac) has been tested and proved effective in the field against naturally acquired disease. Besides improving of the vaccine's effectiveness, significant reductions in production costs and in the logistics of its administration are necessary for the feasibility of nationwide control programs. This review highlights the development of several versions of S3Pvac aimed to increase effectiveness, reduce costs and increase feasibility by novel delivery systems and alternative routes of administration.

Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2.

We have obtained a monoclonal antibody (ED15) against the C-terminal DNA-binding domain of the high-risk human papillomavirus strain-16 E2 protein that strongly interferes with its DNA-binding activity. We here characterize the recognition mechanism of this antibody and find that the ED15-E2 interaction has a strong electrostatic component, which correlates with the high proportion of acidic residues found in the antibody combining site. Further circular dichroism experiments in the presence of phosphate show that, in addition to electrostatic screening of key potential interactions, ionic strength affects the conformation of the epitope. In addition, the interaction is strongly modulated by pH, which correlates with the local flexibility of the epitope rather than the presence of pH sensitive residues at the interface. Noticeably, this finding is well correlated with the strong entropic component of the interaction. Site directed mutagenesis indicates that the ED15 epitope involves at least part of the DNA-binding helix of E2, explaining the mAb inhibitory activity. At physiological salt concentrations, the equilibrium dissociation constant of the E2-ED15 interaction is 10(-7) M and the association rate is 10(4) M-1 s-1, at least 1 order of magnitude slower than those generally reported in the most extensively described "nonflexible" antibody-protein interactions, indicating the presence of a slow conformational rearrangement on the antigen as the rate-limiting step. The crucial role of antigen flexibility in antibody-protein recognition is discussed.

Brucella spp. lumazine synthase: a novel adjuvant and antigen delivery system to effectively induce oral immunity.

Brucella lumazine synthase (BLS) has been previously used with success as a delivery system for systemic immunization against murine cysticercosis. We herein determined the usefulness of BLS as a new antigen-delivery system and mucosal-adjuvant using KETc1, one of the peptides of the anti-cysticercosis vaccine. A protection of up to 98% was induced when KETc1 was used as a chimera fused to BLS. Used as adjuvant of KETc1, BLS also induced a high level of protection (79%), which did not significantly differ from that induced by the cholera toxin (74%). KETc1 and BLS administered separately also reduced the parasite load. KETc1 administered orally as a chimera, and to a lesser extent with BLS as adjuvant, elicited IgG and IgA specific antibodies, which were detectable both in fecal extracts and in sera, and increased B and CD4 activated cells. BLS-KETc1 also increased the levels of transcription of TNF-alpha, IL-2 and IFNgamma in Peyer's patches, and in spleen, only increased TNF-alpha was observed. Overall, these results showed that BLS can be used as both an antigen-carrier and as an adjuvant in the design of new oral subunit vaccines.

Exploring protein interfaces with a general photochemical reagent.

Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent-accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH(2)). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. (3)H-DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein-protein complexes. For this purpose we chose the complex formed between hen egg white lysozyme (HEWL) and the monoclonal antibody IgG(1) D1.3. :CH(2) labeling of free HEWL or complexed with IgG(1) D1.3 yields 2.76 and 2.32 mmol CH(2) per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH(2). Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H(15)GLDNYR(21), G(117)TDVQAWIR(125), andG(22)YSLGNWVCAAK(33). Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.

A polymeric bacterial protein activates dendritic cells via TLR4.

The enzyme lumazine synthase from Brucella spp. (BLS) is a highly immunogenic protein that folds as a stable dimer of pentamers. It is possible to insert foreign peptides and proteins at the 10 N terminus of BLS without disrupting its general folding, and these chimeras are very efficient to elicit systemic and oral immunity without adjuvants. In this study, we show that BLS stimulates bone marrow dendritic cells from mice in vitro to up-regulate the levels of costimulatory molecules (CD40, CD80, and CD86) and major histocompatibility class II Ag. Furthermore, the mRNA levels of several chemokines are increased, and proinflammatory cytokine secretion is induced upon exposure to BLS. In vivo, BLS increases the number of dendritic cells and their expression of CD62L in the draining lymph node. All of the observed effects are dependent on TLR4, and clearly independent of LPS contamination. The described characteristics of BLS make this protein an excellent candidate for vaccine development.

Crystallographic studies on decameric Brucella spp. Lumazine synthase: a novel quaternary arrangement evolved for a new function?

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.