RESUMO
The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Brucella abortus Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain.IMPORTANCE Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen Brucella abortus in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Brucella abortus/metabolismo , Cor , Histidina Quinase/química , Histidina Quinase/metabolismo , Luz , Proteínas de Bactérias/genética , Brucella abortus/genética , Brucella abortus/patogenicidade , Histidina Quinase/genética , Modelos Moleculares , Domínios Proteicos , Transdução de SinaisRESUMO
Brucella spp. are pathogenic intracellular Gram-negative bacteria adapted to life within cells of several mammals, including humans. These bacteria are the causative agent of brucellosis, one of the zoonotic infections with the highest incidence in the world and for which a human vaccine is still unavailable. Current therapeutic treatments against brucellosis are based on the combination of two or more antibiotics for prolonged periods, which may lead to antibiotic resistance in the population. Riboflavin (vitamin B2) is biosynthesized by microorganisms and plants but mammals, including humans, must obtain it from dietary sources. Owing to the absence of the riboflavin biosynthetic enzymes in animals, this pathway is nowadays regarded as a rich resource of targets for the development of new antimicrobial agents. In this work, we describe a high-throughput screening approach to identify inhibitors of the enzymatic activity of riboflavin synthase, the last enzyme in this pathway. We also provide evidence for their subsequent validation as potential drug candidates in an in vitro brucellosis infection model. From an initial set of 44 000 highly diverse low molecular weight compounds with drug-like properties, we were able to identify ten molecules with 50% inhibitory concentrations in the low micromolar range. Further Brucella culture and intramacrophagic replication experiments showed that the most effective bactericidal compounds share a 2-Phenylamidazo[2,1-b][1,3]benzothiazole chemical scaffold. Altogether, these findings set up the basis for the subsequent lead optimization process and represent a promising advancement in the pursuit of novel and effective antimicrobial compounds against brucellosis.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Brucella abortus/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Riboflavina Sintase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Linhagem Celular , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala/métodos , Camundongos , Ligação Proteica , Riboflavina Sintase/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The histidine kinase (HK) domain belonging to the light-oxygen-voltage histidine kinase (LOV-HK) from Brucella abortus is a member of the HWE family, for which no structural information is available, and has low sequence identity (20%) to the closest HK present in the PDB. The `off-edge' S-SAD method in macromolecular X-ray crystallography was used to solve the structure of the HK domain from LOV-HK at low resolution from crystals in a low-symmetry space group (P21) and with four copies in the asymmetric unit (â¼108â kDa). Data were collected both from multiple crystals (diffraction limit varying from 2.90 to 3.25â Å) and from multiple orientations of the same crystal, using the κ-geometry goniostat on SOLEIL beamline PROXIMA 1, to obtain `true redundancy'. Data from three different crystals were combined for structure determination. An optimized HK construct bearing a shorter cloning artifact yielded crystals that diffracted X-rays to 2.51â Å resolution and that were used for final refinement of the model. Moreover, a thorough a posteriori analysis using several different combinations of data sets allowed us to investigate the impact of the data-collection strategy on the success of the structure determination.
Assuntos
Brucella abortus/enzimologia , Proteínas Quinases/química , Brucella abortus/química , Cristalização , Cristalografia por Raios X/métodos , Histidina Quinase , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Brucella abortus is an important pathogenic bacterium that has to overcome oxygen deficiency in order to achieve a successful infection. Previously, we proved that a two-component system formed by the histidine kinase NtrY and the response regulator NtrX is essential to achieve an adaptive response to low oxygen tension conditions. Even though the relevance of this signaling pathway has already been demonstrated in other microorganisms, its molecular activation mechanism has not yet been described in detail. In this article, we report the first crystal structures from different conformations of the NtrX receiver domain from B. abortus, and we propose a sequence of events to explain the structural rearrangements along the activation process. The analysis of the structures obtained in the presence of the phosphoryl group analog beryllofluoride led us to postulate that changes in the interface formed by the α4 helix and the ß5 strand are important for the activation, producing a reorientation of the α5 helix. Also, a biochemical characterization of the NtrX receiver domain enzymatic activities was performed, describing its autophosphorylation and autodephosphorylation kinetics. Finally, the role of H85, an important residue, was addressed by site-directed mutagenesis. Overall, these results provide significant structural basis for understanding the response regulator activation in this bacterial two-component system.
Assuntos
Proteínas de Bactérias/ultraestrutura , Brucella abortus/enzimologia , Proteínas Quinases/ultraestrutura , Brucella abortus/metabolismo , Hipóxia Celular/fisiologia , Cristalografia por Raios X , Histidina Quinase , Oxigênio/metabolismo , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms.
Assuntos
Brucella abortus/genética , Genes Bacterianos , Óperon , Proteínas Quinases/metabolismo , Estresse Fisiológico , Brucella abortus/enzimologia , Histidina Quinase , RNA Bacteriano/isolamento & purificação , Técnicas do Sistema de Duplo-HíbridoRESUMO
This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD) using the Toll-like receptor 2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPPcysMPEG) as an adjuvant. Intranasal coadministration of BPPcysMPEG with a plasmid carrying the SOD-encoding gene (pcDNA-SOD) into BALB/c mice elicited antigen-specific humoral and cellular immune responses. Humoral responses were characterized by the stimulation of IgG2a and IgG1 and by the presence of SOD-specific secretory IgA in nasal and bronchoalveolar lavage fluids. Furthermore, T-cell proliferative responses and increased production of gamma interferon were also observed upon splenocyte restimulation with recombinant SOD. Cytotoxic responses were also stimulated, as demonstrated by the lysis of RB51-SOD-infected J774.A1 macrophages by cells recovered from immunized mice. The pcDNA-SOD/BPPcysMPEG formulation induced improved protection against challenge with the virulent strain B. abortus 2308 in BALB/c mice over that provided by pcDNA-SOD, suggesting the potential of this vaccination strategy against Brucella infection.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacina contra Brucelose/imunologia , Brucella abortus/enzimologia , Brucelose/prevenção & controle , Polietilenoglicóis/administração & dosagem , Superóxido Dismutase/imunologia , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/farmacologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Vacina contra Brucelose/administração & dosagem , Vacina contra Brucelose/genética , Brucella abortus/genética , Brucelose/imunologia , Proliferação de Células , Testes Imunológicos de Citotoxicidade , Modelos Animais de Doenças , Feminino , Imunoglobulina A Secretora/análise , Imunoglobulina G/sangue , Interferon gama/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , Polietilenoglicóis/farmacologia , Baço/imunologia , Superóxido Dismutase/genética , Linfócitos T/imunologia , Receptor 2 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
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.
Assuntos
Brucella abortus/enzimologia , Riboflavina Sintase/química , Riboflavina Sintase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Riboflavina/química , Riboflavina Sintase/genética , Homologia de Sequência de AminoácidosRESUMO
Brucellosis, a disease caused by the gram-negative bacterium Brucella spp., is a widespread zoonosis that inflicts important animal and human health problems, especially in developing countries. One of the hallmarks of Brucella infection is its capacity to establish a chronic infection, characteristic that depends on a wide repertoire of virulence factors among which are immunomodulatory proteins such as PrpA (encoding the proline racemase protein A or hydroxyproline-2-epimerase), involved in the establishment of the chronic phase of the infectious process that we have previously identified and characterized. We report here that, in vivo, Brucella abortus prpA is responsible for an increment in the B-cell number and in the specific antibody response and that these antibodies promote cell infection. We additionally found that Brucella alters the cytokine levels of IFN-γ, IL-10, TGFß1 and TNFα during the acute phase of the infectious process in a prpA dependent manner.
Assuntos
Isomerases de Aminoácido/imunologia , Proteínas de Bactérias/imunologia , Brucella abortus/enzimologia , Brucelose/imunologia , Brucelose/microbiologia , Isomerases de Aminoácido/genética , Animais , Anticorpos Antibacterianos/imunologia , Linfócitos B/imunologia , Proteínas de Bactérias/genética , Brucella abortus/genética , Brucella abortus/imunologia , Brucelose/genética , Feminino , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
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.
Assuntos
Brucella abortus/genética , Nitrogênio/metabolismo , Oxigênio/metabolismo , Proteínas Quinases/metabolismo , Adaptação Fisiológica , Animais , Brucella abortus/enzimologia , Linhagem Celular , Desnitrificação , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Histidina Quinase , Camundongos , Óperon , Oxirredução , Fosforilação , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Transdução de SinaisRESUMO
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.