RESUMO
On June 13, 2014, two patients went to the Hennepin County Medical Center Emergency Department in Minneapolis, Minnesota, with symptoms suggestive of tetrodotoxin poisoning (i.e., oral paresthesias, weakness, and dyspnea) after consuming dried puffer fish (also known as globefish) purchased during a recent visit to New York City. The patients said two friends who consumed the same fish had similar, although less pronounced, symptoms and had not sought care. The Minnesota Department of Health conducted an investigation to determine the source of the product and samples were sent to the Food and Drug Administration (FDA) Center for Food Safety and Applied Nutrition for chemical and genetic analysis. Genetic analysis identified the product as puffer fish (Lagocephalus lunaris) and chemical analysis determined it was contaminated with high levels of tetrodotoxin. A traceback investigation was unable to determine the original source of the product. Tetrodotoxin is a deadly, potent poison; the minimum lethal dose in an adult human is estimated to be 2-3 mg. Tetrodotoxin is a heat-stable and acid-stable, nonprotein, alkaloid toxin found in many species of the fish family Tetraodontidae (puffer fish) as well as in certain gobies, amphibians, invertebrates, and the blue-ringed octopus. Tetrodotoxin exerts its effects by blocking voltage-activated sodium channels, terminating nerve conduction and muscle action potentials, leading to progressive paralysis and, in extreme cases, to death from respiratory failure. Because these fish were reportedly purchased in the United States, they pose a substantial U.S. public health hazard given the potency of the toxin and the high levels of toxin found in the fish.
Assuntos
Surtos de Doenças , Peixes Venenosos , Doenças Transmitidas por Alimentos/diagnóstico , Doenças Transmitidas por Alimentos/epidemiologia , Tetraodontiformes , Tetrodotoxina/intoxicação , Adulto , Animais , Serviço Hospitalar de Emergência , Feminino , Contaminação de Alimentos , Humanos , Masculino , Minnesota/epidemiologia , Cidade de Nova Iorque , Tetraodontiformes/genética , Tetrodotoxina/análiseRESUMO
α4ß1 integrin regulates cell migration via cytoplasmic interactions. Here, we report an association between the cytoplasmic tail of α4 integrin (α4 tail) and non-muscle myosin IIA (MIIA), demonstrated by co-immunoprecipitation of the MIIA heavy chain (HC) with anti-α4-integrin antibodies and pull-down of MIIA-HC with recombinant α4 tail from cell lysates. The association between the α4 tail and MIIA does not require paxillin binding or phosphorylation at Ser988 in the α4 tail. We found that substituting Glu982 in the α4 tail with alanine (E982A) disrupts the α4-MIIA association without interfering with the paxillin binding or Ser988 phosphorylation. By comparing stably transfected CHO cells, we show that the E982A mutation reduces the ability of α4ß1 integrin to mediate cell spreading and to promote front-back polarization. In addition, we show that E982A impairs shear-flow-induced migration of the α4-integrin-expressing CHO cells by reducing their migration speed and directional persistence. The E982A mutation also leads to defects in the organization of MIIA filament bundles. Furthermore, when cells are plated on fibronectin and simulated with shear flow, α4ß1 integrin forms filament-like patterns that co-align with MIIA filament bundles. These results provide a new mechanism for linking integrins to the actomyosin cytoskeleton and for regulating cell migration by integrins and non-muscle myosin II.
Assuntos
Movimento Celular/fisiologia , Integrina alfa4/metabolismo , Integrina alfa4beta1/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Camundongos , Paxilina/metabolismo , Ligação ProteicaRESUMO
The toxic plant protein ricin has gained notoriety due to wide availability and potential use as a bioterrorism agent, with particular concern for food supply contamination. We have developed a sensitive and selective mass spectrometry-based method to detect ricin in tap water, 2% milk, apple juice, and orange juice. Ricin added to beverage matrices was extracted using antibody-bound magnetic beads and digested with trypsin. Absolute quantification was performed using isotope dilution mass spectrometry with a linear ion trap operating in product-ion-monitoring mode. The method allows for identification of ricin A chain and B chain and for distinction of ricin from ricin agglutinin within a single analytical run. Ricin-bound beads were also tested for deadenylase activity by incubation with a synthetic ssDNA oligomer. Depurination of the substrate by ricin was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS). This method was used successfully to extract ricin from each beverage matrix. The activity of recovered ricin was assessed, and quantification was achieved, with a limit of detection of 10 fmol/mL (0.64 ng/mL).
Assuntos
Bebidas/análise , Marcação por Isótopo/métodos , Ricina/análise , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Pesticides such as insecticides, fungicides, and herbicides can protect crops from insects, fungi, weeds, and other pests but must be applied following label instructions so that the pesticide residues in human and animal foods do not exceed maximum residue limits (MRLs, known in the US as pesticide tolerances). The US Food and Drug Administration (FDA) collects and tests foods for pesticide residues to enforce compliance with tolerances and publishes annual reports on pesticide testing results. In this study, results for over 56,000 human food samples collected and analysed under the FDA pesticide residue monitoring programme between fiscal years (FY) 2009 to 2017 were reviewed to identify trends not apparent in annual reports. The overwhelming majority of these samples, 98.0% of domestic and 90.9% of import human foods, were compliant with federal standards. Although herbicides may be more widely used, the 10 most frequently detected residues were insecticides and fungicides. On a yearly basis, the violation rate for imported samples is 3-5 times higher than the rate for domestic samples. The import violation rate increased over time, as did the number of residues detected. Targeted sampling of foods with higher commodity-specific violation rates appears to be a major contributor to the increased violation rate. Mismatches between US tolerances and international MRLs can lead to violations; this was especially marked for rice. Overall, the majority of violations are due to residues of pesticides not authorised for use in the US (lack of tolerances). While DDT continues to persist in the environment and was found in 2.2% of domestic samples and 0.6% of imported samples, 42.3% of DDT-positive samples were below the limit of quantitation. The trends and analyses identified in this paper may help FDA plan future sampling and continue to protect the food supply.
Assuntos
Análise de Alimentos , Contaminação de Alimentos/análise , Resíduos de Praguicidas/análise , Abastecimento de Alimentos , Humanos , Estados Unidos , United States Food and Drug AdministrationRESUMO
Acrylamide is a contaminant that can form in certain plant-based foods during high-temperature cooking. From 2011-2015, the Food and Drug Administration conducted extensive sampling and analyses of acrylamide in foods, as a follow-up to surveys from 2002-2006. We compared acrylamide occurrence data and exposure estimates based on 2011-2015 data with data and exposure estimates from 2002-2006. Acrylamide levels in selected food categories generally did not decrease significantly in 2011-2015 compared with 2002-2006. However, significant decreases in acrylamide concentrations were observed for potato chips and crackers, which may be related to the availability and use of mitigation techniques for reducing acrylamide in foods. Mean dietary intake for those 2 years and older based on 2011-2015 data was 0.36 µg/kg bw/day, comparable to the 0.44 µg/kg bw/day reported by FDA in 2006. French fries and potato products, breakfast cereal, cookies, potato chips, and crackers continue to be the greatest contributors to dietary intake of acrylamide. Infant snack foods were identified as an important contributor to acrylamide intake relative to infant jarred foods. The continued presence of acrylamide in food suggests that manufacturers and governments should continue to pursue efforts to reduce acrylamide in foods that are important contributors to acrylamide intake.
Assuntos
Acrilamida/análise , Exposição Dietética/análise , Análise de Alimentos , Contaminação de Alimentos/análise , Alimentos Infantis/análise , Humanos , Lactente , Estados UnidosRESUMO
Fish within the family Tetraodontidae are potential sources of both endogenous tetrodotoxins (TTXs) and dietary derived saxitoxins (STXs). Ingestion of fish tissues containing these toxins by other vertebrates can lead to severe illness and death. The Caribbean sharpnose puffer (Canthigaster rostrata) is a widespread tetraodontid species within the western Atlantic. Mass settlement of juveniles into foraging habitats have been associated with large-scale puffer fish mortality events. In 2013, 2014, and 2017, puffer mortality events on the southern Caribbean coast of Costa Rica were also associated with strandings of green turtles (Chelonia mydas) found to have fed on C. rostrata. Stranded sea turtles were found dead without apparent cause or alive with severe neurological signs that resolved during short periods of captivity. Puffer fish and turtle organ samples were analyzed for both TTXs and STXs. Concentrations of TTXs were extremely low in the fish (0.5-0.7 µg/g) and undetectable in turtle stomach contents. However, concentrations of STXs in whole fish (16.6-47.5 µg STX-eq/g) exceeded the 0.8 µg STX-eq/g human seafood safety threshold for STXs by orders of magnitude. Saxitoxins were also detected in samples of stomach contents (ingested fish), brain, lung, kidney, and serum from three affected turtles. Study results indicate that saxitoxicosis resulting from opportunistic foraging on C. rostrata during fish mortality events may be a significant factor in episodic stranding of green sea turtles in this region.
RESUMO
Sesqui- and oxy-mustards pose a significant threat to military forces and civilians because they are potent vesicants. We have developed an isotope-dilution high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry method utilizing negative ion multiple reaction monitoring for the analysis of sesqui-mustard metabolites bis(2-hydroxyethylthio)alkanes (n = 1-5) and oxy-mustard metabolite bis(2-hydroxyethylthioethyl)ether in human urine. Relative standard deviations were < 10% and the reportable limits of detection were 1 ng/mL in 0.5 mL of urine. We applied this method to 100 samples collected from individuals with no known exposure to sesqui- or oxy-mustards, and no urines showed detectable levels of any of the analytes, suggesting that these metabolites may be used for monitoring exposure to sesqui- and oxy-mustards.
Assuntos
Monitoramento Ambiental/métodos , Gás de Mostarda/análise , Espectrometria de Massas em Tandem/métodos , Biomarcadores/urina , Cromatografia Líquida/métodos , Exposição Ambiental/análise , Humanos , Hidróxidos/química , Silicatos de Magnésio/química , Estrutura Molecular , Gás de Mostarda/análogos & derivados , Gás de Mostarda/metabolismo , Compostos de Potássio/química , Reprodutibilidade dos Testes , Extração em Fase Sólida , Titânio/químicaRESUMO
Preparations of palytoxin (PLTX, derived from Japanese Palythoa tuberculosa) and the congeners 42-OH-PLTX (from Hawaiian P. toxica) and ovatoxin-a (isolated from a Japanese strain of Ostreopsis ovata), as well as a 50:50 mixture of PLTX and 42-OH-PLTX derived from Hawaiian P. tuberculosa were characterized as to their concentration, composition, in-vitro potency and interaction with an anti-PLTX monoclonal antibody (mAb), after which they were evaluated for lethality and tissue histopathology after intraperitoneal (IP) and aerosol administration to rats. Once each preparation was characterized as to its toxin composition by LC-HRMS and normalized to a total PLTX/OVTX concentration using HPLC-UV, all four preparations showed similar potency towards mouse erythrocytes in the erythrocyte hemolysis assay and interactions with the anti-PLTX mAb. The IP LD50 values derived from these experiments (0.92, 1.93, 1.81 and 3.26⯵g/kg, for the 50:50 mix, 42-OH-PLTX, PLTX, and ovatoxin-a, respectively) were consistent with published values, although some differences from the published literature were seen. The aerosol LD50 values (0.063, 0.045, 0.041, and 0.031⯵g/kg for the 50:50 mix, 42-OH PLTX, PLTX, and ovatoxin-a, respectively) confirmed the exquisite potency of PLTX suggested by the literature. The tissue histopathology of the different toxin preparations by IP and aerosol administration were similar, albeit with some differences. Most commonly affected tissues were the lungs, liver, heart, salivary glands, and adrenal glands. Despite some differences, these results suggest commonalities in potency and mechanism of action among these PLTX congeners.
Assuntos
Acrilamidas/química , Acrilamidas/toxicidade , Acrilamidas/administração & dosagem , Acrilamidas/metabolismo , Aerossóis , Animais , Venenos de Cnidários , Dinoflagellida/metabolismo , Relação Dose-Resposta a Droga , Feminino , Injeções Intraperitoneais , Toxinas Marinhas/administração & dosagem , Toxinas Marinhas/química , Toxinas Marinhas/toxicidade , Estrutura Molecular , Ratos , Ratos Endogâmicos F344RESUMO
PURPOSE: Pragmatic language difficulties have been documented as part of the FMR1 premutation phenotype, yet the interplay between these features in mothers and the language outcomes of their children with fragile X syndrome is unknown. This study aimed to determine whether pragmatic language difficulties in mothers with the FMR1 premutation are related to the language development of their children. METHOD: Twenty-seven mothers with the FMR1 premutation and their adolescent/young adult sons with fragile X syndrome participated. Maternal pragmatic language violations were rated from conversational samples using the Pragmatic Rating Scale (Landa et al., 1992). Children completed standardized assessments of vocabulary, syntax, and reading. RESULTS: Maternal pragmatic language difficulties were significantly associated with poorer child receptive vocabulary and expressive syntax skills, with medium effect sizes. CONCLUSIONS: This work contributes to knowledge of the FMR1 premutation phenotype and its consequences at the family level, with the goal of identifying modifiable aspects of the child's language-learning environment that may promote the selection of treatments targeting the specific needs of families affected by fragile X. Findings contribute to our understanding of the multifaceted environment in which children with fragile X syndrome learn language and highlight the importance of family-centered intervention practices for this group.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/psicologia , Idioma , Mães/psicologia , Adolescente , Adulto , Feminino , Técnicas de Genotipagem , Humanos , Inteligência , Testes de Inteligência , Testes de Linguagem , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fenótipo , Análise de Regressão , Classe Social , Adulto JovemRESUMO
The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity.
Assuntos
Lectinas de Plantas/química , Ricina/química , Ricinus communis/química , Ricinus communis/enzimologia , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases/metabolismo , Imunoensaio , Extratos Vegetais/química , Sementes/química , Espectrometria de Massas em TandemRESUMO
Global and targeted mass spectrometry-based proteomic approaches were developed to discover, evaluate, and apply gluten peptide markers to detect low parts per million (ppm) wheat contamination of oats. Prolamins were extracted from wheat, barley, rye, and oat flours and then reduced, alkylated, and digested with chymotrypsin. The resulting peptides were subjected to LC-MS/MS analysis and database matching. No peptide markers common to wheat, barley, and rye were identified that could be used for global gluten detection. However, many grain-specific peptide markers were identified, and a set of these markers was selected for gluten detection and grain differentiation. Wheat flour was spiked into gluten-free oat flour at concentrations of 1-100,000 ppm and analyzed to determine the lowest concentration at which the wheat "contaminant" could be confidently detected in the mixture. The same 2D ion trap instrument that was used for the global proteomics approach was used for the targeted proteomics approach, providing a seamless transition from target discovery to application. A powerful, targeted MS/MS method enabled detection of two wheat peptide markers at the 10 ppm wheat flour-in-oat flour concentration. Because gluten comprises approximately 10% of wheat flour protein, the reported wheat gluten-specific peptides can enable detection of approximately 1 ppm of wheat gluten in oats.
Assuntos
Contaminação de Alimentos/análise , Glutens/química , Peptídeos/química , Espectrometria de Massas em Tandem/métodos , Triticum/química , Análise Discriminante , Grão Comestível/química , Especificidade da EspécieRESUMO
In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin.
Assuntos
Substâncias para a Guerra Química/análise , Extratos Vegetais/química , Proteínas de Plantas/isolamento & purificação , Ricina/análise , Ricinus communis/química , Ricinus communis/genética , Primers do DNA , Sondas de DNA , Humanos , Espectrometria de Massas , Extratos Vegetais/genética , Lectinas de Plantas/genética , Reação em Cadeia da Polimerase , Proteômica , Saúde Pública , Ricina/genéticaRESUMO
Francisella tularensis and related intracellular pathogens synthesize lipid A molecules that differ from their Escherichia coli counterparts. Although a functional orthologue of lpxK, the gene encoding the lipid A 4'-kinase, is present in Francisella, no 4'-phosphate moiety is attached to Francisella lipid A. We now demonstrate that a membrane-bound phosphatase present in Francisella novicida U112 selectively removes the 4'-phosphate residue from tetra- and pentaacylated lipid A molecules. A clone that expresses the F. novicida 4'-phosphatase was identified by assaying lysates of E. coli colonies, harboring members of an F. novicida genomic DNA library, for 4'-phosphatase activity. Sequencing of a 2.5-kb F. novicida DNA insert from an active clone located the structural gene for the 4'-phosphatase, designated lpxF. It encodes a protein of 222 amino acid residues with six predicted membrane-spanning segments. Rhizobium leguminosarum and Rhizobium etli contain functional lpxF orthologues, consistent with their lipid A structures. When F. novicida LpxF is expressed in an E. coli LpxM mutant, a strain that synthesizes pentaacylated lipid A, over 90% of the lipid A molecules are dephosphorylated at the 4'-position. Expression of LpxF in wild-type E. coli has no effect, because wild-type hexaacylated lipid A is not a substrate. However, newly synthesized lipid A is not dephosphorylated in LpxM mutants by LpxF when the MsbA flippase is inactivated, indicating that LpxF faces the outer surface of the inner membrane. The availability of the lpxF gene will facilitate re-engineering lipid A structures in diverse bacteria.
Assuntos
Proteínas de Bactérias/genética , Francisella/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas de Bactérias/análise , Sequência de Bases , Membrana Celular , Clonagem Molecular , Escherichia coli , Expressão Gênica , Lipídeo A/metabolismo , Dados de Sequência Molecular , Periplasma , Monoéster Fosfórico Hidrolases/análise , Rhizobium etli/genética , Rhizobium leguminosarum/genética , Análise de Sequência de DNARESUMO
Francisella tularensis subsp. novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15% of the total phospholipids, as judged by 32Pi labeling. Although lipopolysaccharide is detectable in F. tularensis subsp. novicida U112, less than 5% of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry, and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2' and a secondary palmitoyl residue at position 2'. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4'- and 3'-positions of A1 and A2 are not derivatized, and 3-deoxy-d-manno-octulosonic acid (Kdo) is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an alpha-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An alpha-linked glucose moiety is attached to the 6'-position of A2. The lipid A released by mild acid hydrolysis of F. tularensis subsp. novicida lipopolysaccharide consists solely of component A1. F. tularensis subsp. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. tularensis subsp. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism.
Assuntos
Francisella tularensis/química , Francisella tularensis/metabolismo , Lipídeo A/biossíntese , Lipídeo A/química , Álcalis/química , Configuração de Carboidratos , Galactosamina/química , Galactosamina/metabolismo , Hidrólise , Íons/química , Lipídeo A/isolamento & purificação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Fosfolipídeos/química , Fosfolipídeos/metabolismoRESUMO
Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxEHP-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxEHP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 microg/ml). However, disruption of lpxEHP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 microg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.
Assuntos
Helicobacter pylori/enzimologia , Monoéster Fosfórico Hidrolases/fisiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Farmacorresistência Bacteriana , Etanolaminas , Helicobacter pylori/efeitos dos fármacos , Lipídeo A/química , Lipídeo A/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Polimixinas/farmacologiaRESUMO
The Salmonella and related bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental stimuli. Some lipid A modifications are required for virulence and resistance to cationic antimicrobial peptides. We now demonstrate that membranes of Salmonella typhimurium contain a novel hydrolase that removes the 3'-acyloxyacyl residue of lipid A in the presence of 5 mM Ca2+. We have identified the gene encoding the S. typhimurium lipid A 3'-O-deacylase, designated lpxR, by screening an ordered S. typhimurium genomic DNA library, harbored in Escherichia coli K-12, for expression of Ca2+-dependent 3'-O-deacylase activity in membranes. LpxR is synthesized with an N-terminal type I signal peptide and is localized to the outer membrane. Mass spectrometry was used to confirm the position of lipid A deacylation in vitro and the release of the intact 3'-acyloxyacyl group. Heterologous expression of lpxR in the E. coli K-12 W3110, which lacks lpxR, resulted in production of significant amounts of 3'-O-deacylated lipid A in growing cultures. Orthologues of LpxR are present in the genomes of E. coli O157:H7, Yersinia enterocolitica, Helicobacter pylori, and Vibrio cholerae. The function of LpxR is unknown, but it could play a role in pathogenesis because it might modulate the cytokine response of an infected animal.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Lipídeo A/metabolismo , Proteínas de Membrana/metabolismo , Salmonella typhimurium/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/genética , Biblioteca Gênica , Espectrometria de Massas , Proteínas de Membrana/genética , Dados de Sequência Molecular , Sinais Direcionadores de ProteínasRESUMO
At mammalian body temperature, the plague bacillus Yersinia pestis synthesizes lipopolysaccharide (LPS)-lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity. To address the effect of weak TLR4 stimulation on virulence, we modified Y. pestis to produce a potent TLR4-stimulating LPS. Modified Y. pestis was completely avirulent after subcutaneous infection even at high challenge doses. Resistance to disease required TLR4, the adaptor protein MyD88 and coreceptor MD-2 and was considerably enhanced by CD14 and the adaptor Mal. Both innate and adaptive responses were required for sterilizing immunity against the modified strain, and convalescent mice were protected from both subcutaneous and respiratory challenge with wild-type Y. pestis. Despite the presence of other established immune evasion mechanisms, the modified Y. pestis was unable to cause systemic disease, demonstrating that the ability to evade the LPS-induced inflammatory response is critical for Y. pestis virulence. Evading TLR4 activation by lipid A alteration may contribute to the virulence of various Gram-negative bacteria.
Assuntos
Lipídeo A/imunologia , Vacina contra a Peste/imunologia , Peste/prevenção & controle , Receptor 4 Toll-Like/agonistas , Fatores de Virulência/imunologia , Yersinia pestis/imunologia , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Células Cultivadas , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Lipídeo A/biossíntese , Lipídeo A/farmacologia , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos , Receptor 4 Toll-Like/antagonistas & inibidores , Vacinação , Virulência/imunologia , Fatores de Virulência/genética , Fatores de Virulência/farmacologia , Yersinia pestis/patogenicidadeRESUMO
The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.
Assuntos
Helicobacter pylori/enzimologia , Hidrolases/metabolismo , Lipopolissacarídeos/metabolismo , Açúcares Ácidos/metabolismo , Helicobacter pylori/genética , Hidrolases/genética , Lipídeo A/metabolismo , Lipopolissacarídeos/química , Fosfatos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Açúcares Ácidos/química , Temperatura , Transferases/genética , Transferases/metabolismoRESUMO
The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.
Assuntos
Proteínas de Drosophila/química , Proteínas Nucleares/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ubiquitina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Glicina/química , Guanidina/química , Íons , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Coelhos , Tripsina/química , Tripsina/farmacologiaRESUMO
Distinct from other spirochetes, cells of Leptospira interrogans contain orthologues of all the Escherichia coli lpx genes required for lipid A biosynthesis, but they synthesize a modified form of lipopolysaccharide that supposedly activates toll-like receptor 2 (TLR2) instead of TLR4. The recent determination of the L. interrogans lipid A structure revealed an unprecedented O-methylation of its 1-phosphate group (Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25420-25429). The enzymatic activity responsible for selective 1-phosphate methylation has not been previously explored. A membrane enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the 1-phosphate moiety of E. coli Kdo2-[4'-(32)P]lipid A has now been discovered. The gene encoding this enzyme was identified based on the hypothesis that methylation of a phosphate group is chemically analogous to methylation of a carboxylate moiety at a membrane-water interface. Database searching revealed a candidate gene (renamed lmtA) in L. interrogans showing distant homology to the yeast isoprenylcysteine carboxyl methyltransferase, encoded by sterile-14, which methylates the a-type mating factor. Orthologues of lmtA were not present in E. coli, the lipid A of which normally lacks the 1-phosphomethyl group, or in other spirochetes, which do not synthesize lipid A. Expression of the lmtA gene behind the lac promoter on a low copy plasmid resulted in the appearance of SAM-dependent methyltransferase activity in E. coli inner membranes and methylation of about 30% of the endogenous E. coli lipid A. Inactivation of the ABC transporter MsbA did not inhibit methylation of newly synthesized lipid A. Methylated E. coli lipid A was analyzed by mass spectrometry and NMR spectroscopy to confirm the location of the phosphomethyl group at the 1-position. In human cells, engineered to express the individual TLR subtypes, 1-phosphomethyl-lipid A purified from lmtA-expressing E. coli potently activated TLR4 but not TLR2.