Hamid Manyani

Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide

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.

Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide.

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.

Translational and Structural Requirements of the Early Nodulin Gene enod40, a Short-Open Reading Frame-Containing RNA, for Elicitation of a Cell-Specific Growth Response in the Alfalfa Root Cortex

ABSTRACT A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin geneenod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5′ and 3′ regions ofenod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

Characterization of Rhizobium tropici CIAT899 nodulation factors : The role of nodH and nodPQ genes in their sulfation

We have purified and characterized the nodulation factors produced by Rhizobium tropici CIAT899. This strain produces a large variety of nodulation factors, these being a mixture of sulfated or nonsulfated penta- or tetra-chito-oligosaccharides to which any of six different fatty acyl moieties may be attached to nitrogen of the nonreducing terminal residue. In this mixture we have also found methylated or nonmethylated lipo-chitin oligosaccharides. Here we describe a novel lipo-chitin-oligosaccharide consisting of a linear backbone of 4 N-acetylglucosamine residues and one mannose that is the reducing-terminal residue and bearing a C18:1 fatty acyl moiety on the nonreducing terminal residue. In addition, we have identified, cloned, and sequenced R. tropici nodH and nodPQ genes, generated mutations in the nodH and nodQ genes, and tested the mutant strains for nodulation in Phaseolus and Leucaena plants. Our results indicate that the sulfate group present in wild-type Nod factors plays a major role in nodulation of Leucaena plants by strain CIAT899 of R. tropici.

Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae

In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (deltahupG, deltahupH, deltahupI, and deltahupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The deltahupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the deltahupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from deltahupH, deltahupI, and deltahupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the deltahupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.

Different and new Nod factors produced by Rhizobium tropici CIAT899 following Na+ stress.

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.

A new generation of vectors with increased induction ratios by overimposing a second regulatory level by attenuation

A major drawback of regulated gene expression from vectors bearing strong promoters is the associated high basal expression level. Simple regulatory systems have an intrinsic limitation in the range of induction, and attempts to mutate promoters to reduce basal expression usually result in concomitant reduction of induced levels. We have explored the possibility of reducing basal levels of gene expression while keeping induced levels intact by incorporating an additional regulatory circuit controlling a different step of the expression process. We have integrated the nasFEDCBA transcriptional attenuation system of Klebsiella oxytoca into a cascade expression circuit based on different regulatory elements of Pseudomonas putida, and also into a system based on the tac promoter, to expand their regulatory capacity. Basal expression from the promoters of these circuits was reduced by more than 10-fold by the nasF attenuator sequence while keeping the induced levels intact in the presence of the antiterminator protein, thus increasing the induction ratio by up to 1700-fold. In addition, using different combinations of regulatory elements and inducing conditions, we were able to obtain a broad range of expression levels. These vectors and the concept of their design will be very useful in regulating overproduction of heterologous proteins both at laboratory and industrial scales.

High NaCl Concentrations Induce the nod Genes of Rhizobium tropici CIAT899 in the Absence of Flavonoid Inducers

The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors.

Dual role of HupF in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum

Background[NiFe] hydrogenases are enzymes that catalyze the oxidation of hydrogen into protons and electrons, to use H2 as energy source, or the production of hydrogen through proton reduction, as an escape valve for the excess of reduction equivalents in anaerobic metabolism. Biosynthesis of [NiFe] hydrogenases is a complex process that occurs in the cytoplasm, where a number of auxiliary proteins are required to synthesize and insert the metal cofactors into the enzyme structural units. The endosymbiotic bacterium Rhizobium leguminosarum requires the products of eighteen genes (hupSLCDEFGHIJKhypABFCDEX) to synthesize an active hydrogenase. hupF and hupK genes are found only in hydrogenase clusters from bacteria expressing hydrogenase in the presence of oxygen.ResultsHupF is a HypC paralogue with a similar predicted structure, except for the C-terminal domain present only in HupF. Deletion of hupF results in the inability to process the hydrogenase large subunit HupL, and also in reduced stability of this subunit when cells are exposed to high oxygen tensions. A ΔhupF mutant was fully complemented for hydrogenase activity by a C-terminal deletion derivative under symbiotic, ultra low-oxygen tensions, but only partial complementation was observed in free living cells under higher oxygen tensions (1% or 3%). Co-purification experiments using StrepTag-labelled HupF derivatives and mass spectrometry analysis indicate the existence of a major complex involving HupL and HupF, and a less abundant HupF-HupK complex.ConclusionsThe results indicate that HupF has a dual role during hydrogenase biosynthesis: it is required for hydrogenase large subunit processing and it also acts as a chaperone to stabilize HupL when hydrogenase is synthesized in the presence of oxygen.

The Nodulation of Alfalfa by the Acid-Tolerant Rhizobium sp. Strain LPU83 Does Not Require Sulfated Forms of Lipochitooligosaccharide Nodulation Signals

The induction of root nodules by the majority of rhizobia has a strict requirement for the secretion of symbiosis-specific lipochitooligosaccharides (nodulation factors [NFs]). The nature of the chemical substitution on the NFs depends on the particular rhizobium and contributes to the host specificity imparted by the NFs. We present here a description of the genetic organization of the nod gene cluster and the characterization of the chemical structure of the NFs associated with the broad-host-range Rhizobium sp. strain LPU83, a bacterium capable of nodulating at least alfalfa, bean, and Leucena leucocephala. The nod gene cluster was located on the plasmid pLPU83b. The organization of the cluster showed synteny with those of the alfalfa-nodulating rhizobia, Sinorhizobium meliloti and Sinorhizobium medicae. Interestingly, the strongest sequence similarity observed was between the partial nod sequences of Rhizobium mongolense USDA 1844 and the corresponding LPU83 nod genes sequences. The phylogenetic analysis of the intergenic region nodEG positions strain LPU83 and the type strain R. mongolense 1844 in the same branch, which indicates that Rhizobium sp. strain LPU83 might represent an early alfalfa-nodulating genotype. The NF chemical structures obtained for the wild-type strain consist of a trimeric, tetrameric, and pentameric chitin backbone that shares some substitutions with both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83 most of the NFs were sulfated in their reducing terminal residue, none of the NFs isolated from the nodH mutant LPU83-H were sulfated. The evidence obtained supports the notion that the sulfate decoration of NFs in LPU83 is not necessary for alfalfa nodulation.