ABSTRACT
Experimental mouse models are central to basic biomedical research; however, variability exists across genetically identical mice and mouse facilities making comparisons difficult. Whether specific indigenous gut bacteria drive immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 genetically identical laboratory mice from a single animal facility, designed to identify the causes of disease variability in the widely used dextran sulphate sodium mouse model of inflammatory bowel disease. Commonly used treatment endpoint measures-weight loss and intestinal pathology-showed limited correlation and varied across mouse lineages. Analysis of the gut microbiome, coupled with machine learning and targeted anaerobic culturing, identified and isolated two previously undescribed species, Duncaniella muricolitica and Alistipes okayasuensis, and demonstrated that they exert dominant effects in the dextran sulphate sodium model leading to variable treatment endpoint measures. We show that the identified gut microbial species are common, but not ubiquitous, in mouse facilities around the world, and suggest that researchers monitor for these species to provide experimental design opportunities for improved mouse models of human intestinal diseases.
Subject(s)
Colitis , Gastrointestinal Microbiome , Inflammatory Bowel Diseases , Animals , Bacteroidetes , Colitis/chemically induced , Colitis/microbiology , Disease Models, Animal , Inflammatory Bowel Diseases/microbiology , MiceABSTRACT
AIM: The aim was to evaluate the efficacy and effectiveness of tooth-supported fixed prostheses in partially edentulous patients with stage IV periodontitis. MATERIAL AND METHODS: Randomized and controlled clinical trials (RCTs and CCTs) and prospective/retrospective cohort studies or case series were searched in three databases. Survival rate of abutment teeth was considered the primary outcome. Meta-analyses were performed whenever possible. RESULTS: Twenty-four publications were included. No RCTs or CCTs compared the efficacy of tooth-supported fixed prostheses between patients with severe periodontitis or non-periodontitis patients. Most of the data retrieved were derived from case series. The incidence of abutment-tooth loss after a follow-up period from 2 to 35.4 years was low (n = 17 studies; weighted mean incidence (WMI) = 4.8%; confidence interval (CI) [3.2, 6.5]). The corresponding figure for prostheses failure was WMI = 6.9% (n = 18; 95% CI [4.1, 9.7]). Technical complications seemed to be more frequent than biological complications (caries, endodontic failure, root fracture, etc.). Periodontal outcomes tended to remain stable over time. CONCLUSION: Tooth-supported fixed prostheses seemed to be a valid treatment approach to restore masticatory function in patients with stage IV periodontitis once periodontal therapy has been accomplished. However, the comparative efficacy of this treatment among periodontitis and non-periodontitis patients is unclear due to the absence of clinical trials.
Subject(s)
Dental Implants , Mouth, Edentulous , Periodontitis , Dental Abutments , Dental Implants/adverse effects , Dental Prosthesis, Implant-Supported/adverse effects , Dental Restoration Failure , Denture, Partial, Fixed/adverse effects , Humans , Mouth, Edentulous/etiology , Periodontitis/complications , Periodontitis/therapy , Prospective Studies , Retrospective StudiesABSTRACT
By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.
Subject(s)
Enterobacteriaceae Infections/immunology , Genetic Variation/genetics , High-Throughput Screening Assays/methods , Immunophenotyping/methods , Salmonella Infections/immunology , Animals , Citrobacter/immunology , Enterobacteriaceae Infections/microbiology , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Salmonella/immunology , Salmonella Infections/microbiologyABSTRACT
OBJECTIVE: Western lifestyle and diet are major environmental factors playing a role in the development of IBD. Titanium dioxide (TiO2) nanoparticles are widely used as food additives or in pharmaceutical formulations and are consumed by millions of people on a daily basis. We investigated the effects of TiO2 in the development of colitis and the role of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome. DESIGN: Wild-type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis were orally administered with TiO2 nanoparticles. The proinflammatory effects of TiO2 particles in cultured human intestinal epithelial cells (IECs) and macrophages were also studied, as well as the ability of TiO2 crystals to traverse IEC monolayers and accumulate in the blood of patients with IBD using inductively coupled plasma mass spectrometry. RESULTS: Oral administration of TiO2 nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO2-administered mice. In vitro, TiO2 particles were taken up by IECs and macrophages and triggered NLRP3-ASC-caspase-1 assembly, caspase-1 cleavage and the release of NLRP3-associated interleukin (IL)-1ß and IL-18. TiO2 also induced reactive oxygen species generation and increased epithelial permeability in IEC monolayers. Increased levels of titanium were found in blood of patients with UC having active disease. CONCLUSION: These findings indicate that individuals with a defective intestinal barrier function and pre-existing inflammatory condition, such as IBD, might be negatively impacted by the use of TiO2 nanoparticles.
Subject(s)
Colitis/immunology , Coloring Agents/adverse effects , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , Nanoparticles/adverse effects , Titanium/adverse effects , Animals , Caspase 1/metabolism , Colitis/chemically induced , Colitis/metabolism , Coloring Agents/administration & dosage , Dextran Sulfate/adverse effects , Disease Models, Animal , Epithelial Cells/metabolism , Humans , Interleukin-18/biosynthesis , Interleukin-1beta/metabolism , Intestines/cytology , Macrophages/metabolism , Mice, Inbred C57BL , Mice, Knockout , Nanoparticles/administration & dosage , Reactive Oxygen Species/metabolism , Spleen/pathology , Titanium/administration & dosage , Titanium/bloodABSTRACT
BACKGROUND: Spermidine is a dietary polyamine that is able to activate protein tyrosine phosphatase non-receptor type 2 (PTPN2). As PTPN2 is known to be a negative regulator of interferon-gamma (IFN-γ)-induced responses, and IFN-γ stimulation of immune cells is a critical process in the immunopathology of inflammatory bowel disease (IBD), we wished to explore the potential of spermidine for reducing pro-inflammatory effects in vitro and in vivo. METHODS: Human THP-1 monocytes were treated with IFN-γ and/or spermidine. Protein expression and phosphorylation were analyzed by Western blot, cytokine expression by quantitative-PCR, and cytokine secretion by ELISA. Colitis was induced in mice by dextran sodium sulfate (DSS) administration. Disease severity was assessed by recording body weight, colonoscopy and histology. RESULTS: Spermidine increased expression and activity of PTPN2 in THP-1 monocytes and reduced IFN-γ-induced phosphorylation of signal transducer and activator of transcription (STAT) 1 and 3, as well as p38 mitogen-activated protein kinase (MAPK) in a PTPN2 dependent manner. Subsequently, IFN-γ-induced expression/secretion of intracellular cell adhesion molecule (ICAM)-1 mRNA, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 was reduced in spermidine-treated cells. The latter effects were absent in PTPN2-knockdown cells. In mice with DSS-induced colitis, spermidine treatment resulted in ameliorated weight loss and decreased mucosal damage indicating reduced disease severity. CONCLUSIONS: Activation of PTPN2 by spermidine ameliorates IFN-γ-induced inflammatory responses in THP-1 cells. Furthermore, spermidine treatment significantly reduces disease severity in mice with DSS-induced colitis; hence, spermidine supplementation and subsequent PTPN2 activation may be helpful in the treatment of chronic intestinal inflammation such as IBD.
Subject(s)
Colitis/prevention & control , Monocytes/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism , Spermidine/pharmacology , Animals , Blotting, Western , Cell Line, Tumor , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Colitis/chemically induced , Colitis/metabolism , Dextran Sulfate , Enzyme Activation/drug effects , Enzyme-Linked Immunosorbent Assay , Female , Humans , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Interferon-gamma/pharmacology , Interleukin-6/genetics , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Monocytes/metabolism , Monocytes/pathology , Phosphorylation/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
OBJECTIVES: Histological examination of duodenal biopsies is the gold standard for assessing intestinal damage in celiac disease (CD). A noninvasive marker of disease status is necessary, because obtaining duodenal biopsies is invasive and not suitable for routine monitoring of CD patients. As the small intestine is a major site of cytochrome P450 3A4 (CYP3A4) activity and also the location of the celiac lesion, we investigated whether patients with active CD display abnormal pharmacokinetics of an orally administered CYP3A4 substrate, simvastatin (SV), which could potentially be used for noninvasive assessment of their small intestinal health. METHODS: Preclinical experiments were performed in CYP3A4-humanized mice to examine the feasibility of the test. Subsequently, a clinical trial was undertaken with 11 healthy volunteers, 18 newly diagnosed patients with CD, and 25 celiac patients who had followed a gluten-free diet (GFD) for more than 1 year. The maximum concentration (Cmax) of orally administered SV plus its major non-CYP3A4-derived metabolite SV acid (SV equivalent (SVeq)) was measured, and compared with clinical, histological, and serological parameters. RESULTS: In CYP3A4-humanized mice, a marked decrease in SV metabolism was observed in response to enteropathy. In the clinical setting, untreated celiac patients displayed a significantly higher SVeq Cmax (46±24 nM) compared with treated patients (21±16 nM, P<0.001) or healthy subjects (19±11 nM, P<0.005). SVeq Cmax correctly predicted the diagnosis in 16/18 untreated celiac patients, and also the recovery status of all follow-up patients that exhibited normal or near-normal biopsies (Marsh 0-2). All patients with abnormal SVeq Cmax showed a reduction in the value after 1 year of following a GFD. CONCLUSIONS: SVeq Cmax is a promising noninvasive marker for assessment of small intestinal health. Further studies are warranted to establish its clinical utility for assessing gut status of patients with CD.
Subject(s)
Celiac Disease/drug therapy , Celiac Disease/metabolism , Cytochrome P-450 CYP3A/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacokinetics , Intestine, Small/drug effects , Intestine, Small/metabolism , Simvastatin/pharmacokinetics , Adolescent , Adult , Animals , Biomarkers/metabolism , Diet, Gluten-Free , Feasibility Studies , Female , Humans , Immunohistochemistry , Male , Mice , Mice, Transgenic , Middle Aged , ROC CurveABSTRACT
BACKGROUND AND AIMS: Celiac sprue is a life-long disease characterized by an intestinal inflammatory response to dietary gluten. A gluten-free diet is an effective treatment for most patients, but accidental ingestion of gluten is common, leading to incomplete recovery or relapse. Food-grade proteases capable of detoxifying moderate quantities of dietary gluten could mitigate this problem. METHODS: We evaluated the gluten detoxification properties of two food-grade enzymes, aspergillopepsin (ASP) from Aspergillus niger and dipeptidyl peptidase IV (DPPIV) from Aspergillus oryzae. The ability of each enzyme to hydrolyze gluten was tested against synthetic gluten peptides, a recombinant gluten protein, and simulated gastric digests of whole gluten and whole-wheat bread. Reaction products were analyzed by mass spectrometry, HPLC, ELISA with a monoclonal antibody that recognizes an immunodominant gluten epitope, and a T cell proliferation assay. RESULTS: ASP markedly enhanced gluten digestion relative to pepsin, and cleaved recombinant alpha2-gliadin at multiple sites in a non-specific manner. When used alone, neither ASP nor DPPIV efficiently cleaved synthetic immunotoxic gluten peptides. This lack of specificity for gluten was especially evident in the presence of casein, a competing dietary protein. However, supplementation of ASP with DPPIV enabled detoxification of moderate amounts of gluten in the presence of excess casein and in whole-wheat bread. ASP was also effective at enhancing the gluten-detoxifying efficacy of cysteine endoprotease EP-B2 under simulated gastric conditions. CONCLUSIONS: Clinical studies are warranted to evaluate whether a fixed dose ratio combination of ASP and DPPIV can provide near-term relief for celiac patients suffering from inadvertent gluten exposure. Due to its markedly greater hydrolytic activity against gluten than endogenous pepsin, food-grade ASP may also augment the activity of therapeutically relevant doses of glutenases such as EP-B2 and certain prolyl endopeptidases.
Subject(s)
Dipeptidyl Peptidase 4/metabolism , Food , Glutens/metabolism , Amino Acid Sequence , Chromatography, High Pressure Liquid , Enzyme-Linked Immunosorbent Assay , Glutens/chemistry , Hydrolysis , Mass Spectrometry , Molecular Sequence DataABSTRACT
The root nodule bacterium Rhizobium tropici strain CIAT899 is highly stress resistant. It grows under acid conditions, in large amounts of salt, and at high osmotic pressure. An earlier study reported a substantial qualitative and quantitative effect of acid stress on the biosynthesis of Nod factors. The aim of the present work was to investigate the effect of high salt (NaCl) concentrations, another common stress factor, on Nod factor production. For this purpose, thin-layer chromatography, HPLC and MS analyses were carried out. The expression of nodulation genes was also studied using a nodP:lacZ fusion. High concentrations of sodium enhanced nod gene expression and Nod factor biosynthesis. The effect is sodium specific because high potassium or chloride concentrations did not have this effect. Under salt stress conditions, 46 different Nod factors were identified in a CIAT899 culture, compared with 29 different Nod factors under control conditions. Only 15 Nod factor structures were common to both conditions. Under salt stress conditions, 14 different new Nod factor structures were identified that were not observed as being produced under neutral or acid conditions. The implications of our results are that stress has a great influence on Nod factor biosynthesis and that new, very interesting regulatory mechanisms, worth investigating, are involved in controlling Nod factor biosynthesis.
Subject(s)
Anti-Bacterial Agents/pharmacology , Lipopolysaccharides/biosynthesis , Osmotic Pressure , Rhizobium tropici/drug effects , Sodium Chloride/pharmacology , Stress, Physiological , Artificial Gene Fusion , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Gene Expression Profiling , Genes, Reporter , Mass Spectrometry , Rhizobium tropici/chemistry , Rhizobium tropici/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concentration) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clinical relevance. Through two rounds of iterative mutagenesis and analysis, variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, especially in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and analytical cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacologically significant improvements in gluten detoxification activity.
Subject(s)
Celiac Disease/drug therapy , Protein Engineering/methods , Serine Endopeptidases/genetics , Serine Endopeptidases/therapeutic use , Sphingomonas/enzymology , Algorithms , Amino Acid Sequence , Artificial Intelligence , Glutens/metabolism , Models, Molecular , Mutation , Prolyl Oligopeptidases , Protein Conformation , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolismABSTRACT
BACKGROUND AND AIMS: Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied. METHODS: Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin. RESULTS: The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease. CONCLUSIONS: The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.
Subject(s)
Celiac Disease/physiopathology , Food/toxicity , Glutens/toxicity , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Base Sequence , DNA Primers , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Gliadin/immunology , HumansABSTRACT
BACKGROUND: Celiac disease is an immune-mediated enteropathy caused by the ingestion of gluten, a protein fraction found in certain cereals. Immunotoxic gluten peptides that are recalcitrant to degradation of digestive enzymes appear to trigger celiac syndromes. A 33-mer peptide from alpha-2 gliadin has been identified as a principal contributor to gluten immunotoxicity. A gluten-free diet is the usual first therapy for celiac disease patients; therefore, the characterization and quantification of the toxic portion of the gluten in foodstuffs is crucial to avoid celiac damage. OBJECTIVE: We aimed to develop immunologic assays as a novel food analysis tool for measuring cereal fractions that are immunotoxic to celiac disease patients. DESIGN: The design focused on the production of monoclonal antibodies against the gliadin 33-mer peptide and the development of enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis with the use of novel antibodies. RESULTS: A sandwich ELISA method showed a detection limit for wheat, barley, and rye of <1 ppm prolamine. However, the method required a sample that was > or =1 order of magnitude greater for the detection of low-toxic oats, and there was no signal with the safe cereals maize and rice. A competitive ELISA method was also developed for detection of the toxic peptide in hydrolyzed food, which had a detection limit of <0.5 ppm gliadin. CONCLUSIONS: Both ELISAs designed for use with the toxic gliadin 33-mer peptide suggested a high correlation between the presence of the peptide and the amount of cereal that was toxic to celiac disease patients. The sensitivity was significantly higher than that of equivalent methods recognizing other gluten epitopes.
Subject(s)
Antibodies, Monoclonal , Celiac Disease/chemically induced , Edible Grain/chemistry , Edible Grain/immunology , Food Analysis/methods , Gliadin/analysis , Gliadin/toxicity , Avena/immunology , Celiac Disease/immunology , Enzyme-Linked Immunosorbent Assay/methods , Epitopes , Hordeum/immunology , Humans , Oryza/immunology , Peptide Fragments/analysis , Peptide Fragments/toxicity , Recombinant Proteins , Secale/immunology , Sensitivity and Specificity , Triticum/immunology , Zea mays/immunologyABSTRACT
Main nodulation signal molecules in the peanut-bradyrhizobia interaction were examined. Flavonoids exuded by Arachis hypogaea L. cultivar Tegua were genistein, daidzein and chrysin, the latest being released in lower quantities. Thin layer chromatography analysis from genistein-induced bacterial cultures of three peanut bradyrhizobia resulted in an identical Nod factor pattern, suggesting low variability in genes involved in the synthesis of these molecules. Structural study of Nod factor by mass spectrometry and NMR analysis revealed that it shares a variety of substituents with the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium spp. Nodulation assays in legumes nodulated by these rhizobia demonstrated differences between them and the three peanut bradyrhizobia. The three isolates were classified as Bradyrhizobium sp. Their fixation gene nifD and the common nodulation genes nodD and nodA were also analyzed.
Subject(s)
Arachis/chemistry , Arachis/microbiology , Bradyrhizobium/chemistry , Soil Microbiology , Symbiosis , Arachis/physiology , Bradyrhizobium/classification , Bradyrhizobium/genetics , Bradyrhizobium/physiology , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Flavonoids/chemistry , Flavonoids/metabolism , Lipopolysaccharides/chemistry , Lipopolysaccharides/metabolism , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Root Nodules, Plant/chemistry , Root Nodules, Plant/metabolism , Root Nodules, Plant/microbiology , Sequence Analysis, DNA , Species SpecificityABSTRACT
Rhizobium gallicum is a fast-growing bacterium found in European, Australian and African soils; it was first isolated in France. It is a microsymbiont which is able to nodulate plants of the genus Phaseolus. Rhizobium gallicum bv. gallicum R602 produces four extracellular signal molecules consisting of a linear backbone of N-acetyl glucosamine, bearing on the nonreducing terminal residue an N-methyl group and different N-acyl substituents. The four acyloligosaccharides terminate with a sulfated N-acetylglucosaminitol. This unit may be also acetylated. These structures were determined using carbohydrate and methylation analysis, mass spectrometric analysis and one-dimensional- and two-dimensional-nuclear magnetic resonance experiments. This work establishes the common structure that a lipochito-oligosaccharide must have so that the Rhizobium that produces and excretes it is able to nodulate plants of Phaseolus vulgaris. The substituents common to all the molecules are an N-methyl group and a C(18:1) fatty acid on the nonreducing terminal residue.
Subject(s)
Lipopolysaccharides/chemistry , Phaseolus/microbiology , Rhizobium/chemistry , Glucosamine/analogs & derivatives , Glucosamine/analysis , Lipopolysaccharides/isolation & purification , Magnetic Resonance Spectroscopy , Mass Spectrometry , Rhizobium/metabolismABSTRACT
Rhizobium tropici CIAT899 has been cataloged as a nodulator of bean, a plant often growing in areas characterized by highly acidic soils. The purpose of this work was to explore the effects of acidity on the production of Nod factors by this strain and their impact on the establishment of effective symbioses. We report that acidity increases rhizobial Nod factors production, and we exhaustively study the nodulation factor structures produced under abiotic stress. Significant differences were observed between the structures produced at acid and neutral pH: 52 different molecules were produced at acid pH, 29 at neutral pH, and only 15 are common to bacteria grown at pH 7.0 or 4.5. The results indicate that R. tropici CIAT899 has successfully adapted to life in acidic soils and is a good inoculant for the bean under these conditions.
Subject(s)
Hydrogen-Ion Concentration , Lipopolysaccharides/biosynthesis , Rhizobium tropici/metabolism , Adaptation, Physiological , Gene Expression , Lipopolysaccharides/isolation & purification , Mass Spectrometry , Methylation , Nitrogen Fixation/genetics , Phaseolus/growth & development , Phaseolus/microbiology , Rhizobium tropici/physiologyABSTRACT
Rhizobium giardinii bv. giardinii is a microsymbiont of plants of the genus Phaseolus and produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis. We report here the structures of seven lipochitooligosaccharide (LCO) signal molecules secreted by R. giardinii bv. giardinii H152. Six of them are pentamers of GlcNAc carrying C 16:0, C 18:0, C 20:0 and C 18:1 fatty acyl chains on the non-reducing terminal residue. Four are sulfated at C-6 of the reducing terminal residue and one is acetylated in the same position. Six of them are N-methylated on the non-reducing GlcN residue and all the nodulation factors are carbamoylated on C-6 of the non-reducing terminal residue. The structures were determined using monosaccharide composition and methylation analyses, 1D- and 2D-NMR experiments and a range of mass spectrometric techniques. The position of the carbamoyl substituent on the non-reducing glucosamine residue was determined using a CID-MSMS experiment and an HMBC experiment.