RESUMO
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
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 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
We present here the complete genome sequence of Bradyrhizobium japonicum strain E109, one of the most used rhizobacteria for soybean inoculation in Argentina since the 1970s. The genome consists of a 9.22-Mbp single chromosome and contains several genes related to nitrogen fixation, phytohormone biosynthesis, and a rhizospheric lifestyle.
RESUMO
We present the complete genome sequence of Azospirillum brasilense Az39, isolated from wheat roots in the central region of Argentina and used as inoculant in extensive and intensive agriculture during the last four decades. The genome consists of 7.39 Mb, distributed in six replicons: one chromosome, three chromids, and two plasmids.
RESUMO
Twitching motility in Acinetobacter baylyi ADP1 is inhibited by moderate intensities of blue light in a temperature-dependent manner (maximally at 20 °C). We analysed the involvement of four predicted blue-light sensing using flavin (BLUF)-domain-containing proteins encoded in the genome of this strain in the twitching motility phenotype. All four genes were expressed both in light and in darkness. A phylogenetic tree showed that one BLUF domain, ACIAD2110, grouped separately from the other three (ACIAD1499, ACIAD2125 and ACIAD2129). Individual knockout mutants of the latter three, but not of ACIAD2110, fully abolished the light dependency of the twitching motility response. Quantitative analysis of transcript level of the three genes showed a decreased expression in the light, with dark/light ratios of 1.65±0.28, 1.79±0.21 and 2.69±0.39, for ACIAD2125, ACIAD2129 and ACIAD1499, respectively. Double and triple knockouts of ACIAD1499, ACIAD2125 and ACIAD2129 confirmed the same phenotype as the corresponding single knockouts. Complementation of all the single knockouts and the triple knockout mutants with any of the three BLUF-domain-encoding genes fully restored the inhibition of twitching motility by blue light that is observed in the wild-type strain. A. baylyi ADP1 therefore shows a high degree of redundancy in the genes that encode BLUF-containing photoreceptors. Moreover, all plasmid-complemented strains, expressing any of the BLUF proteins irrespective of the specific set of deleted photoreceptors, displayed increased light-dependent inhibition of twitching motility, as compared to the wild-type (P<0.001). We conclude that the three genes ACIAD1499, ACIAD2125 and ACIAD2129 are jointly required to inhibit twitching motility under moderate blue-light illumination.
Assuntos
Acinetobacter/citologia , Acinetobacter/efeitos da radiação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Acinetobacter/classificação , Acinetobacter/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Luz , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
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 abortusâ 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.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Bactérias/metabolismo , Brucella abortus/fisiologia , Regulação Bacteriana da Expressão Gênica , Oxigênio/farmacologia , Proteínas Quinases/metabolismo , Animais , Proteínas de Bactérias/genética , Brucella abortus/efeitos dos fármacos , Brucella abortus/genética , Brucella abortus/metabolismo , Brucelose/microbiologia , Desnitrificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Histidina Quinase , Camundongos , Oxirredução , Consumo de Oxigênio/fisiologia , Proteínas Quinases/genética , Transdução de SinaisRESUMO
Paramyxoviruses share the essential RNA polymerase complex components, namely, the polymerase (L), phosphoprotein (P), and nucleoprotein (N). Human respiratory syncytial virus (RSV) P is the smallest polypeptide among the family, sharing a coiled coil tetramerization domain, which disruption renders the virus inactive. We show that unfolding of P displays a first transition with low cooperativity but substantial loss of α-helix content and accessibility to hydrophobic sites, indicative of loose chain packing and fluctuating tertiary structure, typical of molten globules. The lack of unfolding baseline indicates a native state in conformational exchange and metastable at 20 °C. The second transition starts from a true intermediate state, with only the tetramerization domain remaining folded. The tetramerization domain undergoes a two-state dissociation/unfolding reaction (37.3 kcal mol(-1)). The M(2-1) transcription antiterminator, unique to RSV and Metapneumovirus, forms a nonglobular P:M(2-1) complex with a 1:1 stoichiometry and a K(D) of 8.1 nM determined by fluorescence anisotropy, far from the strikingly coincident dissociation range of P and M(2-1) tetramers (10(-28) M(3)). The M(2-1) binding region has been previously mapped to the N-terminal module of P, strongly suggesting the latter as the metastable molten globule domain. Folding, oligomerization, and assembly events between proteins and with RNA are coupled in the RNA polymerase complex. Quantitative assessment of the hierarchy of these interactions and their mechanisms contribute to the general understanding of RNA replication and transcription in Paramyxoviruses. In particular, the unique P-M(2-1) interface present in RSV provides a valuable antiviral target for this worldwide spread human pathogen.
Assuntos
Biopolímeros/metabolismo , Fosfoproteínas/metabolismo , Desnaturação Proteica , Vírus Sincicial Respiratório Humano/metabolismo , Biopolímeros/química , Cromatografia em Gel , Dicroísmo Circular , Fosfoproteínas/química , Conformação Proteica , Dobramento de Proteína , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Aderência Bacteriana/fisiologia , Luz , Fotorreceptores Microbianos/metabolismo , Pisum sativum/microbiologia , Nodulação/fisiologia , Rhizobium leguminosarum/fisiologia , Simbiose , Sequência de Aminoácidos , Aderência Bacteriana/efeitos da radiação , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Western Blotting , Flagelos/metabolismo , Violeta Genciana , Histidina Quinase , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Nodulação/efeitos da radiação , Polissacarídeos Bacterianos/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína/genética , Reação em Cadeia da Polimerase em Tempo Real , Rhizobium leguminosarum/efeitos da radiação , Rhizobium leguminosarum/ultraestrutura , Alinhamento de Sequência , Análise de Sequência de DNA , Estatísticas não ParamétricasRESUMO
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
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 α/ß PAS (PER-ARNT-SIM) domain fold and binds the FMN cofactor within a conserved pocket. The domain dimerizes through the hydrophobic ß-scaffold in an antiparallel way. Our results point to the ß-scaffold as a key element in the light activation, validating a conserved structural basis for light-to-signal propagation in LOV proteins.
Assuntos
Brucella/química , Luz , Proteínas Quinases/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Brucella/patogenicidade , Cristalografia por Raios X , Mononucleotídeo de Flavina/metabolismo , Histidina Quinase , Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteínas Quinases/química , Estrutura Terciária de ProteínaRESUMO
The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.
Assuntos
Anticorpos/farmacologia , Variação Antigênica , Camelídeos Americanos/imunologia , Neuraminidase/imunologia , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Variação Antigênica/imunologia , Domínio Catalítico/imunologia , Inibidores Enzimáticos/imunologia , Inibidores Enzimáticos/farmacologia , Mapeamento de Epitopos , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Neuraminidase/metabolismo , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/metabolismoRESUMO
Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to beta-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T.rangeli sialidase (TrSA) and T.cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studying the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA(5mut)) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA(5mut) revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.
Assuntos
Mutação/genética , Ácido N-Acetilneuramínico/análogos & derivados , Neuraminidase/genética , Neuraminidase/metabolismo , Trypanosoma/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Glicoproteínas , Glicosilação , Hidrólise , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/farmacologia , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Trypanosoma/genéticaRESUMO
Chagas' disease, caused by Trypanosoma cruzi, affects about 18 million people in Latin America, and no effective treatment is available to date. To acquire sialic acid from the host glycoconjugates, T. cruzi expresses an unusual surface sialidase with trans-sialidase activity (TcTS) that transfers the sugar to parasite mucins. Surface sialic acid was shown to have relevant functions in protection of the parasite against the lysis by complement and in mammalian host cell invasion. The recently determined 3D structure of TcTS allowed a detailed analysis of its catalytic site and showed the presence of a lactose-binding site where the beta-linked galactose accepting the sialic acid is placed. In this article, the acceptor substrate specificity of lactose derivatives was studied by high pH anion-exchange chromatography with pulse amperometric detection. The lactose open chain derivatives lactitol and lactobionic acid, as well as other derivatives, were found to be good acceptors of sialic acid. Lactitol, which was the best of the ones tested, effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Furthermore, lactitol inhibited parasite mucins re-sialylation when incubated with live trypanosomes and TcTS. Lactitol also diminished the T. cruzi infection in cultured Vero cells by 20-27%. These results indicate that compounds directed to the lactose binding site might be good inhibitors of TcTS.
Assuntos
Inibidores Enzimáticos/química , Glicoproteínas/química , Neuraminidase/química , Álcoois Açúcares/química , Trypanosoma cruzi/enzimologia , Animais , Sítios de Ligação/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Chlorocebus aethiops , Glicoproteínas/metabolismo , Neuraminidase/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ácidos Siálicos/química , Especificidade por Substrato/efeitos dos fármacos , Álcoois Açúcares/metabolismo , Álcoois Açúcares/farmacologia , Trypanosoma cruzi/patogenicidade , Células Vero , Virulência/efeitos dos fármacosRESUMO
Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans-sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.