RESUMO
Red/far-red light-sensing bacteriophytochrome photoreceptor (BphP) pathways play key roles in bacterial physiology and ecology. These bilin-binding proteins photoswitch between two states, Pr (red absorbing) and Pfr (far-red absorbing). The isomerization of the chromophore and the downstream structural changes result in the light signal transduction. The agricultural pathogen Xanthomonas campestris pv. campestris (Xcc) code for a single bathy-like type BphP (XccBphP), previously shown to negatively regulate several light-mediated biological processes involved in virulence. Here, we generated three different full-length variants with single amino acid changes within its GAF domain that affect the XccBphP photocycle favouring its Pr state: L193Q, L193N and D199A. While D199A recombinant protein locks XccBphP in a Pr-like state, L193Q and L193N exhibit a significant enrichment of the Pr form in thermal equilibrium. The X-ray crystal structures of the three variants were solved, resembling the wild-type protein in the Pr state. Finally, we studied the effects of altering the XccBphP photocycle on the exopolysaccharide xanthan production and stomatal aperture assays as readouts of its bacterial signalling pathway. Null-mutant complementation assays show that the photoactive Pr-favoured XccBphP variants L193Q and L193N tend to negatively regulate xanthan production in vivo. In addition, our results indicate that strains expressing these variants also promote stomatal apertures in challenged plant epidermal peels, compared to wild-type Xcc. The findings presented in this work provide new evidence on the Pr state of XccBphP as a negative regulator of the virulence-associated mechanisms by light in Xcc.
Assuntos
Arabidopsis/microbiologia , Pigmentos Biliares/metabolismo , Fitocromo/química , Fitocromo/genética , Doenças das Plantas/microbiologia , Virulência , Xanthomonas campestris/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Luz , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fitocromo/metabolismoRESUMO
Phytochromes are photosensitive proteins with a covalently bound open-chain chromophore that can switch between two principal states: red light absorbing Pr and far-red light absorbing Pfr. Our group has previously shown that the bacteriophytochrome from Xanthomonas campestris pv. campestris (XccBphP) is a bathy-like phytochrome that uses biliverdin IXα as a co-factor and is involved in bacterial virulence. To date, the XccBphP crystal structure could only be solved in the Pr state, while the structure of its Pfr state remains elusive. The aims of this work were to develop an efficient screening methodology for the rapid characterization and to identify XccBphP variants that favor the Pfr form. The screening approach developed here consists in analyzing the UV-Vis absorption behavior of clarified crude extracts containing recombinant phytochromes. This strategy has allowed us to quickly explore over a hundred XccBphP variants, characterize multiple variants and identify Pfr-favored candidates. The high-quality data obtained enabled not only a qualitative, but also a quantitative characterization of their photochemistry. This method could be easily adapted to other phytochromes or other photoreceptor families.
Assuntos
Fotoquímica/métodos , Fitocromo/química , Espectrofotometria Ultravioleta/métodos , Xanthomonas campestris/química , Cristalografia por Raios XRESUMO
BACKGROUND: Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. METHODS: We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. RESULTS: Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. CONCLUSIONS: Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Linfoma/imunologia , Proteínas Recombinantes de Fusão/imunologia , Neoplasias da Bexiga Urinária/imunologia , Animais , Brucella/enzimologia , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Linfoma/patologia , Linfoma/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapiaRESUMO
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
Many signaling pathways that control cellular development, cell-cycle progression and nutritional versatility have been studied in Caulobacter crescentus. For example, it was suggested that the response regulator NtrX is conditionally essential for this bacterium and that it might be necessary for responding to a signal produced in phosphate-replete minimal medium. However, such signal has not been identified yet and the role of NtrX in C. crescentus biology remains elusive. Here, using wild-type C. crescentus and a strain with a chromosomally myc-tagged ntrX gene, we demonstrate that high concentrations of phosphate (10 mM) regulate ntrX transcription and the abundance of the protein. We also show that the pH of the medium acts as a switch able to regulate the phosphorylation status of NtrX, promoting its phosphorylation under mildly acidic conditions and its dephosphorylation at neutral pH. Moreover, we demonstrate that the ntrX gene is required for survival in environments with low pH and under acidic stress. Finally, we prove that NtrX phosphorylation is also triggered by low pH in Brucella abortus, a pathogenic alphaproteobacterium. Overall, our work contributes to deepen the general knowledge of this system and provides tools to elucidate the NtrX regulon.
Assuntos
Proteínas de Bactérias/fisiologia , Caulobacter crescentus/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Brucella abortus/genética , Brucella abortus/metabolismo , Caulobacter crescentus/genética , Deleção de Genes , Concentração de Íons de Hidrogênio , Fosfatos/química , Fosforilação , Regiões Promotoras Genéticas , Proteólise , Regulon , Transdução de Sinais , Estresse Fisiológico , TemperaturaRESUMO
Phytochromes constitute a major superfamily of light-sensing proteins that are reversibly photoconverted between a red-absorbing (Pr) and a far-red-absorbing (Pfr) state. Bacteriophytochromes (BphPs) are found among photosynthetic and non-photosynthetic bacteria, including pathogens. To date, several BphPs have been biophysically characterized. However, it is still not fully understood how structural changes are propagated from the photosensory module to the output module during the signal transduction event. Most phytochromes share a common architecture consisting of an N-terminal photosensor that includes the PAS2-GAF-PHY domain triad and a C-terminal variable output module. Here we present the crystal structure of the full-length BphP from the plant pathogen Xanthomonas campestris pv. campestris (XccBphP) bearing its photosensor and its complete output module, a PAS9 domain. In the crystals, the protein was found to be in the Pr state, whereas diffraction data together with resonance Raman spectroscopic and theoretical results indicate a ZZZssa and a ZZEssa chromophore configuration corresponding to a mixture of Pr and Meta-R state, the precursor of Pfr. The XccBphP quaternary assembly reveals a head-to-head dimer in which the output module contributes to the helical dimer interface. The photosensor, which is shown to be a bathy-like BphP, is influenced in its dark reactions by the output module. Our structural analyses suggest that the photoconversion between the Pr and Pfr states in the full-length XccBphP may involve changes in the relative positioning of the output module. This work contributes to understand the light-induced structural changes propagated from the photosensor to the output modules in phytochrome signaling.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fitocromo/química , Fitocromo/metabolismo , Transdução de Sinais , Xanthomonas campestris/química , Cristalografia por Raios X , Luz , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Análise Espectral Raman , Difração de Raios XRESUMO
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
We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii.
Assuntos
Antígenos/metabolismo , Proteínas de Bactérias/farmacologia , Brucella/química , Imunidade nas Mucosas , Inibidores de Proteases/farmacologia , Vacinas/imunologia , Administração Oral , Sequência de Aminoácidos , Animais , Feminino , Camundongos , Camundongos Endogâmicos , Dados de Sequência MolecularRESUMO
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
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
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.
Assuntos
Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Brucella abortus/metabolismo , Riboflavina/biossíntese , Animais , Proteínas de Bactérias/genética , Western Blotting , Brucella abortus/genética , Brucella abortus/patogenicidade , Brucelose/microbiologia , Linhagem Celular , Feminino , Teste de Complementação Genética , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutação , VirulênciaRESUMO
Food intended for celiac patients' consumption must be analyzed for the presence of toxic prolamins using high detectability tests. Though 60% ethanol is the most commonly used solvent for prolamins extraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increase protein recovery. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In the present work, a new method for gliadins quantification is shown. The method is based on the selection of llama single domain antibody fragments able to operate under denaturing conditions. Six out of 28 VHH-phages obtained retained their binding capacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additional cysteines that could be responsible for the higher stability. One of the clones (named VHH26) was fully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA using VHH26 was able to detect gliadins in samples shown as negatives by conventional ELISA. Therefore, this new strategy appears as an excellent platform for quantitative determination of proteins or any other immunogenic compound, in the presence of denaturing agents, when specific recognition units with high stability are required.
Assuntos
Anticorpos/química , Doença Celíaca/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Gliadina/sangue , Sequência de Aminoácidos , Animais , Anticorpos/genética , Anticorpos/imunologia , Modelos Animais de Doenças , Gliadina/administração & dosagem , Gliadina/química , Humanos , Masculino , Dados de Sequência Molecular , Ligação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The penultimate step in the biosynthesis of riboflavin (vitamin B2) involves the condensation of 3,4-dihydroxy-2-butanone 4-phosphate with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase). Pathogenic Brucella species adapted to an intracellular lifestyle have two genes involved in riboflavin synthesis, ribH1 and ribH2, which are located on different chromosomes. The ribH2 gene was shown previously to specify a lumazine synthase (type II lumazine synthase) with an unusual decameric structure and a very high Km for 3,4-dihydroxy-2-butanone 4-phosphate. Moreover, the protein was found to be an immunodominant Brucella antigen and was able to generate strong humoral as well as cellular immunity against Brucella abortus in mice. We have now cloned and expressed the ribH1 gene, which is located inside a small riboflavin operon, together with two other putative riboflavin biosynthesis genes and the nusB gene, specifying an antitermination factor. The RibH1 protein (type I lumazine synthase) is a homopentamer catalyzing the formation of 6,7-dimethyl-8-ribityllumazine at a rate of 18 nmol mg(-1) min(-1). Sequence comparison of lumazine synthases from archaea, bacteria, plants, and fungi suggests a family of proteins comprising archaeal lumazine and riboflavin synthases, type I lumazine synthases, and the eubacterial type II lumazine synthases.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Complexos Multienzimáticos/metabolismo , Riboflavina Sintase/metabolismo , Riboflavina/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Fases de Leitura Aberta , Filogenia , Riboflavina Sintase/química , Riboflavina Sintase/genética , Alinhamento de SequênciaRESUMO
We have previously demonstrated that immunization of normal mice with a defined protein:DNA complex comprising the C-terminal DNA-binding domain of the human papillomavirus E2 protein and its cognate site 35 oligonucleotide, results in high antibody titers against both E2 and its target DNA sequence. Here we show that repeated immunization with the isolated form of the E2 domain also elicits anti-DNA antibodies, but in this case, no preferential binding for a given sequence was observed, indicating that these antibodies have broad specificity for DNA. Taken together our results indicate that this viral protein can induce two classes of anti-DNA antibody responses: one directed against endogenous DNA and other in which anti-site 35-specific antibodies are produced. In both cases, the character of the resulting anti-DNA response seems to be directed by the DNA molecule that the protein binds in vivo. Evaluation of the fine specificity of the antibodies induced by the free and bound states of this single foreign DNA-binding protein would contribute to the understanding of the processes involved in the acquisition of particular DNA specificities by anti-DNA antibodies.