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Find video protocols related to scientific articles indexed in Pubmed.
Solution and crystal structure of BA42, a protein from the Antarctic bacterium Bizionia argentinensis comprised of a stand-alone TPM domain.
Proteins
PUBLISHED: 09-03-2014
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The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X-ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand-alone TPM domain. The structure reveals a new topological variant of the four ?-strands constituting the central ?-sheet of the ??? architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42. Proteins 2014; 82:3062-3078. © 2014 Wiley Periodicals, Inc.
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Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility.
Acta Crystallogr. D Biol. Crystallogr.
PUBLISHED: 01-28-2014
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Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C3 symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.
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Protection of mice against Shiga toxin 2 (Stx2)-associated damage by maternal immunization with a Brucella lumazine synthase-Stx2 B subunit chimera.
Infect. Immun.
PUBLISHED: 01-13-2014
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Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life.
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Identification and characterization of RibN, a novel family of riboflavin transporters from Rhizobium leguminosarum and other proteobacteria.
J. Bacteriol.
PUBLISHED: 08-09-2013
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Rhizobia are symbiotic bacteria able to invade and colonize the roots of legume plants, inducing the formation of nodules, where bacteria reduce atmospheric nitrogen (N2) to ammonia (NH3). Riboflavin availability influences the capacity of rhizobia to survive in the rhizosphere and to colonize roots. In this study, we identified the RL1692 gene of Rhizobium leguminosarum downstream of a flavin mononucleotide (FMN) riboswitch. RL1692 encodes a putative transmembrane permease with two EamA domains. The presence of an FMN riboswitch regulating a transmembrane protein is usually observed in riboflavin transporters, suggesting that RL1692 may be involved in riboflavin uptake. The product of RL1692, which we named RibN, is conserved in members of the alpha-, beta-, and gammaproteobacteria and shares no significant identity with any riboflavin transporter previously identified. In this work, we show that RibN is localized in the membrane cellular fraction and its expression is downregulated by riboflavin. By heterologous expression in a Brucella abortus mutant auxotrophic for riboflavin, we demonstrate that RibN possesses flavin transport activity. Similarly, we also demonstrate that RibN orthologues from Ochrobactrum anthropi and Vibrio cholerae (which lacks the FMN riboswitch) are able to transport riboflavin. An R. leguminosarum ribN null mutant exhibited lower nodule occupancy levels in pea plants during symbiosis assays. Thus, we propose that RibN and its homologues belong to a novel family of riboflavin transporters. This work provides the first experimental description of riboflavin transporters in Gram-negative bacteria.
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Immunization with a chimera consisting of the B subunit of Shiga toxin type 2 and brucella lumazine synthase confers total protection against Shiga toxins in mice.
J. Immunol.
PUBLISHED: 08-05-2013
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The striking feature of enterohemorrhagic Escherichia coli (EHEC) infections is the production of Shiga toxins (Stx) implicated in the development of the life-threatening hemolytic uremic syndrome. Despite the magnitude of the social impact of EHEC infections, no licensed vaccine or effective therapy is available for human use. One of the biggest challenges is to develop an effective and safe immunogen to ensure nontoxicity, as well as a strong input to the immune system to induce long-lasting, high-affinity Abs with anti-Stx-neutralizing capacity. The enzyme lumazine synthase from Brucella spp. (BLS) is a highly stable dimer of pentamers and a scaffold with enormous plasticity on which to display foreign Ags. Taking into account the advantages of BLS and the potential capacity of the B subunit of Stx2 to induce Abs that prevent Stx2 toxicity by blocking its entrance into the host cells, we engineered a new immunogen by inserting the B subunit of Stx2 at the amino termini of BLS. The resulting chimera demonstrated a strong capacity to induce a long-lasting humoral immune response in mice. The chimera induced Abs with high neutralizing capacity for Stx2 and its variants. Moreover, immunized mice were completely protected against i.v. Stx2 challenge, and weaned mice receiving an oral challenge with EHEC were completely protected by the transference of immune sera. We conclude that this novel immunogen represents a promising candidate for vaccine or Ab development with preventive or therapeutic ends, for use in hemolytic uremic syndrome-endemic areas or during future outbreaks caused by pathogenic strains of Stx-producing E. coli.
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The vaccine candidate BLSOmp31 protects mice against Brucella canis infection.
Vaccine
PUBLISHED: 04-19-2013
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Canine brucellosis represents a major reproductive problem worldwide and it is considered a zoonotic disease. New approaches are therefore urgently needed to develop an effective and safe immunization strategy against Brucella canis. In the present study, BALB/c mice were subcutaneously immunized with the recombinant chimera rBLSOmp31 formulated in different adjuvants. The different strategies induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. Besides, spleen cells from rBLSOmp31-immunized mice produced gamma interferon and IL-4, suggesting the induction of a mixed Th1-Th2. Vaccination with rBLSOmp31-IFA formulation provided the best protection levels comparable with that given by control vaccines. None of the immunization strategies induced serological interference in diagnosis. Hitherto, this is the first report that a recombinant vaccine confers protection against B. canis in mice.
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Immune response and serum bactericidal activity against Brucella ovis elicited using a short immunization schedule with the polymeric antigen BLSOmp31 in rams.
Vet. Immunol. Immunopathol.
PUBLISHED: 04-04-2013
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Brucella ovis is the etiologic agent of ovine brucellosis. The control measures for this disease are periodical diagnosis by serological tests and/or bacteriological culture and culling of positive animals. Vaccination with Brucella melitensis Rev 1 is recommended when prevalence is high. This attenuated strain vaccine gives protection against B. ovis but it has important disadvantages associated with the development of antibodies interfering with serodiagnosis, virulence for humans and the prohibition of its use in countries considered free of B. melitensis. Consequently, there is a need for new safe and effective brucellosis vaccines to be developed. We have previously reported that the polymeric subcellular vaccine BLSOmp31 confers protection against experimental challenge with B. ovis when rams are immunized three times. In the present work we evaluated and characterized, along 56 weeks after the first immunization of adult rams, the evolution of the immune response elicited by BLSOmp31 using a short immunization schedule.
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The art of blocking ADP-ribosyltransferases (ARTs): nanobodies as experimental and therapeutic tools to block mammalian and toxin ARTs.
FEBS J.
PUBLISHED: 03-07-2013
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In 1901, the first Nobel Prize in Physiology or Medicine was awarded to Emil von Behring for his ground-breaking discovery of serum therapy: serum from horses vaccinated with toxin-containing culture medium of Corynebacterium diphtheriae contained life-saving antitoxins. The molecular nature of the ADP-ribosylating toxin and the neutralizing antibodies were unraveled only 50 years later. Today, von Behrings antibody therapy is being refined with a new generation of recombinant antibodies and antibody fragments. Nanobodies, which are single-domain antibodies derived from the peculiar heavy-chain antibodies of llamas and other camelids, are emerging as a promising new class of highly specific enzyme inhibitors. In this review, we illustrate the potential of nanobodies as tools to block extracellular and intracellular ADP-ribosyltransferases (ARTs), using the toxin-related membrane-bound mammalian ecto-enzyme ARTC2 and the actin-ADP-ribosylating Salmonella virulence plasmid factor B toxin of Salmonella enterica as examples.
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The two-component systems PrrBA and NtrYX co-ordinately regulate the adaptation of Brucella abortus to an oxygen-limited environment.
Mol. Microbiol.
PUBLISHED: 02-09-2013
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Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. The success of Brucella as pathogen relies in its ability to adapt to the harsh environmental conditions found in mammalian hosts. One of its main adaptations is the induction of the expression of different genes involved in respiration at low oxygen tension. In this report we describe a regulatory network involved in this adaptation. We show that Brucella abortus?PrrBA is a functional two-component signal transduction system that responds to the redox status and acts as a global regulator controlling the expression of the regulatory proteins NtrY, FnrN and NnrA, which are involved in the adaptation to survive at low oxygen tension. We also show that the two-component systems PrrBA and NtrYX co-ordinately regulate the expression of denitrification and high-affinity cytochrome oxidase genes. Strikingly, a double mutant strain in the prrB and ntrY genes is severely impaired in growth and virulence, while the ntrY and prrB single mutant strains are similar to wild-type B.?abortus. The proposed regulatory network may contribute to understand the mechanisms used by Brucella for a successful adaptation to its replicative niche inside mammalian cells.
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Heterologous prime-boost oral immunization with GK-1 peptide from Taenia crassiceps cysticerci induces protective immunity.
Clin. Vaccine Immunol.
PUBLISHED: 05-18-2011
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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 Peyers 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.
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YqiC of Salmonella enterica serovar Typhimurium is a membrane fusogenic protein required for mice colonization.
BMC Microbiol.
PUBLISHED: 01-24-2011
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Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen which can colonize a variety of hosts, including human, causing syndromes that vary from gastroenteritis and diarrhea to systemic disease.
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Single-domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP-ribosylating toxin of Salmonella typhimurium.
FASEB J.
PUBLISHED: 10-12-2010
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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.
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An atypical riboflavin pathway is essential for Brucella abortus virulence.
PLoS ONE
PUBLISHED: 02-04-2010
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Brucellosis is a worldwide zoonosis that affects livestock and humans and is caused by closely related Brucella spp., which are adapted to intracellular life within cells of a large variety of mammals. Brucella can be considered a furtive pathogen that infects professional and non-professional phagocytes. In these cells Brucella survives in a replicative niche, which is characterized for having a very low oxygen tension and being deprived from nutrients such as amino acids and vitamins. Among these vitamins, we have focused on riboflavin (vitamin B2). Flavin metabolism has been barely implicated in bacterial virulence. We have recently described that Brucella and other Rhizobiales bear an atypical riboflavin metabolic pathway. In the present work we analyze the role of the flavin metabolism on Brucella virulence. Mutants on the two lumazine synthases (LS) isoenzymes RibH1 and RibH2 and a double RibH mutant were generated. These mutants and different complemented strains were tested for viability and virulence in cells and in mice. In this fashion we have established that at least one LS must be present for B. abortus survival and that RibH2 and not RibH1 is essential for intracellular survival due to its LS activity in vivo. In summary, we show that riboflavin biosynthesis is essential for Brucella survival inside cells or in mice. These results highlight the potential use of flavin biosynthetic pathway enzymes as targets for the chemotherapy of brucellosis.
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The polymeric antigen BLSOmp31 confers protection against Brucella ovis infection in rams.
Vaccine
PUBLISHED: 07-15-2009
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We have engineered the polymeric vaccine BLSOmp31 by decorating the highly immunogenic and decameric Brucella lumazine synthase with an exposed loop of the Brucella outer membrane protein Omp31. In the present study, we have immunized different groups of rams with the recombinant chimera rBLSOmp31 in two different adjuvants (Incomplete Freund Adjuvant-IFA and QUIL A) and with the plasmid pCIBLSOmp31 administered either by i.m. injection alone or by using electroporation. In addition, we have used a heterologous prime-boost strategy consisting of repeated pCIBLSOmp31 electroporation priming followed by a single protein boost. Both, chimera rBLSOmp31 in IFA and the prime-boost strategy induced the highest IgG specific antibodies with bacteriolytic activity. While electroporation-enhanced humoral immune responses as compared to pCIBLSOmp31 injection alone, the highest levels of specific IFN-gamma and protection against bacterial challenge were achieved with prime-boost (76%) and chimera rBLSOmp31 in IFA (63%). Taken together these results strongly support the usefulness of the chimera BLSOmp31 as a vaccine against Brucella ovis in ovine brucellosis.
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Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.
Med. Microbiol. Immunol.
PUBLISHED: 05-15-2009
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Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.
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Brucella spp. lumazine synthase as a bovine rotavirus antigen delivery system.
Vaccine
PUBLISHED: 03-12-2009
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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.
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Tandem repeats of the extracellular domain of Matrix 2 influenza protein exposed in Brucella lumazine synthase decameric carrier molecule induce protection in mice.
Vaccine
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The antigenic variation of influenza virus represents a major prevention problem. However, the ectodomain of the protein Matrix 2 (M2e) is nearly invariant in all human influenza A strains and has been considered as a promising candidate for a broadly protective vaccine because antibodies to M2e are protective in animal models. In this work we evaluated the possible use of Brucella abortus lumazine synthase protein (BLS), a highly immunogenic decameric protein, as a carrier of the M2e peptide. Chimeric proteins generated by the fusion of one or four in tandem copies of M2e to BLS were efficiently expressed in Escherichia coli and assembled in decameric subunits similarly to the wild type BLS enzyme, as demonstrated by the comparative circular dichroism spectra and size exclusion chromatography and static light scattering analysis. The M2e peptides were stably exposed at the ten N-terminal ends of each BLS molecule. Immunization of mice with purified chimeras carrying only one M2e (BLS-M2e) copy elicited a significant humoral immune response with the addition of different adjuvants. The fusion of four in tandem copies of the M2e peptide (BLS-4M2e) resulted in similar levels of humoral immune response but in the absence of adjuvant. Survival of mice challenged with live influenza virus was 100% after vaccination with BLS-4M2e adjuvanted with Iscomatrix(®) (P<0.001) and 80% when adjuvanted with alum (P<0.01), while the chimera alone protected 60% of the animals (P<0.05). The approach described in this study is intended as a contribution to the generation of universal influenza immunogens, through a simple production and purification process and using safe carriers that might eventually avoid the use of strong adjuvants.
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A polymeric protein induces specific cytotoxicity in a TLR4 dependent manner in the absence of adjuvants.
PLoS ONE
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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-7R?, 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.
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Light regulates attachment, exopolysaccharide production, and nodulation in Rhizobium leguminosarum through a LOV-histidine kinase photoreceptor.
Proc. Natl. Acad. Sci. U.S.A.
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Rhizobium leguminosarum is a soil bacterium that infects root hairs and induces the formation of nitrogen-fixing nodules on leguminous plants. Light, oxygen, and voltage (LOV)-domain proteins are blue-light receptors found in higher plants and many algae, fungi, and bacteria. The genome of R. leguminosarum bv. viciae 3841, a pea-nodulating endosymbiont, encodes a sensor histidine kinase containing a LOV domain at the N-terminal end (R-LOV-HK). R-LOV-HK has a typical LOV domain absorption spectrum with broad bands in the blue and UV-A regions and shows a truncated photocycle. Here we show that the R-LOV-HK protein regulates attachment to an abiotic surface and production of flagellar proteins and exopolysaccharide in response to light. Also, illumination of bacterial cultures before inoculation of pea roots increases the number of nodules per plant and the number of intranodular bacteroids. The effects of light on nodulation are dependent on a functional lov gene. The results presented in this work suggest that light, sensed by R-LOV-HK, is an important environmental factor that controls adaptive responses and the symbiotic efficiency of R. leguminosarum.
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The NtrY/X two-component system of Brucella spp. acts as a redox sensor and regulates the expression of nitrogen respiration enzymes.
Mol. Microbiol.
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Brucella spp. are facultative intracellular bacteria pathogenic for many mammalian species including humans, causing a disease called brucellosis. Learning how Brucella adapts to its intracellular niche is crucial for understanding its pathogenesis mechanism, allowing for the development of new and more effective vaccines and treatments against brucellosis. Brucella pathogenesis resides mostly in its ability to adapt to the harsh environmental conditions encountered during host infection such as the oxygen depletion. The mechanism by which Brucella senses the oxygen tension and triggers its environmental adaptation is unknown. In this work we show that the Brucella abortus NtrY/NtrX two-component system is involved in oxygen sensing through a haem group contained in a Per-ARNT-SIM (PAS) domain of the NtrY histidine kinase. The NtrY haem iron can be reduced to the ferrous form and is rapidly oxidized to the ferric form in presence of oxygen. Importantly, we show that the oxidation state of the haem iron modulates the autokinase activity, being the anoxygenic reduced ferrous form the signalling state of NtrY. Also, we show that ntrY gene expression increases under low oxygen tension and that NtrY transfers its signal to its cognate response regulator NtrX, regulating in this way the expression of nitrogen respiration enzymes. Based on these findings, we postulate that NtrY acts as a redox sensor in Brucella spp.
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The ?-scaffold of the LOV domain of the Brucella light-activated histidine kinase is a key element for signal transduction.
J. Mol. Biol.
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Light-oxygen-voltage (LOV) domains are blue-light-activated signaling modules present in a wide range of sensory proteins. Among them, the histidine kinases are the largest group in prokaryotes (LOV-HK). Light modulates the virulence of the pathogenic bacteria Brucella abortus through LOV-HK. One of the striking characteristic of Brucella LOV-HK is the fact that the protein remains activated upon light sensing, without recovering the basal state in the darkness. In contrast, the light state of the isolated LOV domain slowly returns to the dark state. To gain insight into the light activation mechanism, we have characterized by X-ray crystallography and solution NMR spectroscopy the structure of the LOV domain of LOV-HK in the dark state and explored its light-induced conformational changes. The LOV domain adopts the ?/? PAS (PER-ARNT-SIM) domain fold and binds the FMN cofactor within a conserved pocket. The domain dimerizes through the hydrophobic ?-scaffold in an antiparallel way. Our results point to the ?-scaffold as a key element in the light activation, validating a conserved structural basis for light-to-signal propagation in LOV proteins.
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Polymeric Display of Proteins through High Affinity Leucine Zipper Peptide Adaptors.
Biomacromolecules
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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.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.