Biomedical subjects

Wim D'Haeze

Publications and source records attributed to Wim D'Haeze.

11 recordsLinked to original sources

PubMed2006

R104H may suppress transthyretin amyloidogenesis by thermodynamic stabilization, but not by the kinetic mechanism characterizing T119 interallelic trans-suppression.

The tetrameric protein transthyretin (TTR) forms amyloid fibrils upon dissociation and subsequent monomer misfolding, enabling misassembly. Remarkably, the aggregation of one of over 100 destabilized TTR variants leads to familial amyloid disease. It is known that trans-suppression mediated by the incorporation of T119M subunits into tetramers otherwise composed of the most common familial variant V30M, ameliorates disease by substantially slowing the rate of tetramer dissociation, a mechanism referred to as kinetic stabilization of the native state. R104H TTR has been reported to be non-pathogenic, and recently, this variant has been invoked as a trans-suppressor of amyloid fibril formation. Here, we demonstrate that the trans-suppression mechanism of R104H does not involve kinetic stabilization of the tetrameric structure, instead its modest trans-suppression most likely results from the thermodynamic stabilization of the tetrameric TTR structure. Thermodynamic stabilization increases the fraction of tetramer at the expense of the misfolding competent monomer decreasing the ability of TTR to aggregate into amyloid fibrils. As a consequence of this stabilization mechanism, R104H may be capable of protecting patients with modestly destabilizing mutations against amyloidosis by slightly lowering the overall population of monomeric protein that can misfold and form amyloid.

Amino Acid Substitution↗

PubMed2006

Exo-oligosaccharides of Rhizobium sp. strain NGR234 are required for symbiosis with various legumes.

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

Bacterial Proteins↗

PubMed2005

Identification and active expression of the Mycobacterium tuberculosis gene encoding 5-phospho-{alpha}-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase, the first enzyme committed to decaprenylphosphoryl-d-arabinose synthesis.

Decaprenylphosphoryl-d-arabinose, the lipid donor of mycobacterial d-arabinofuranosyl residues, is synthesized from phosphoribose diphosphate rather than from a sugar nucleotide. The first committed step in the process is the transfer of a 5-phosphoribosyl residue from phosphoribose diphosphate to decaprenyl phosphate to form decaprenylphosphoryl-5-phosphoribose via a 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phospho-ribosyltransferase. A candidate for the gene encoding this enzyme (Rv3806c) was identified in Mycobacterium tuberculosis, primarily via its homology to one of four genes responsible for d-arabinosylation of nodulation factor in Azorhizobium caulinodans. The resulting protein was predicted to contain eight or nine transmembrane domains. The gene was expressed in Escherichia coli, and membranes from the expression strain of E. coli but not from a control strain of E. coli were shown to convert phosphoribose diphosphate and decaprenyl phosphate into decaprenylphosphoryl-5-phosphoribose. Neither UDP-galactose nor GDP-mannose was active as a sugar donor. The enzyme favored polyprenyl phosphate with 50-60 carbon atoms, was unable to use C-20 polyprenyl phosphate, and used C-75 polyprenyl phosphate less efficiently than C-50 or C-60. It requires CHAPS detergent and Mg(2+) for activity. The Rv3806c gene encoding 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase is known to be essential for the growth of M. tuberculosis, and the tuberculosis drug ethambutol inhibits other steps in arabinan biosynthesis. Thus the Rv3806c-encoded enzyme appears to be a good target for the development of new tuberculosis drugs.

Antitubercular Agents↗

PubMed2005

Lipopolysaccharides as a communication signal for progression of legume endosymbiosis.

Establishment of a successful symbiosis between rhizobia and legumes results from an elaborate molecular dialogue between both partners. Bacterial nodulation (Nod) factors are indispensable for initiating plant responses, whereas bacterial surface polysaccharides are important for infection progression and nodule development. The mutant ORS571-oac2 of Azorhizobium caulinodans, affected in its surface polysaccharides, provokes a defective interaction with its host Sesbania rostrata. ORS571-oac2 induced structures with retarded development and continued generation of infection centers and organ primordia, leading to multilobed ineffective nodules. Bacterial development throughout the interaction occurred without major defects. A functional bidirectional complementation was obtained upon coinfection of ORS571-oac2 and a Nod factor-deficient mutant, indicating that the Fix- phenotype of ORS571-oac2-induced nodules resulted from the absence of a positive signal from ORS571-oac2. Indeed, the Fix- phenotype could be complemented by coinoculation of ORS571-oac2 with lipopolysaccharides (LPSs) purified from A. caulinodans. Our data show that Nod factors and LPSs are consecutive signals in symbiosis. Nod factors act first to trigger the onset of the nodulation and invasion program; LPSs inform the plant to proceed with the symbiotic interaction and to develop a functional fixation zone.

Azorhizobium caulinodans↗

PubMed2005

Decaprenylphosphoryl arabinofuranose, the donor of the D-arabinofuranosyl residues of mycobacterial arabinan, is formed via a two-step epimerization of decaprenylphosphoryl ribose.

The major cell wall polysaccharide of mycobacteria is a branched-chain arabinogalactan in which arabinan chains are attached to the 5 carbon of some of the 6-linked galactofuranose residues; these arabinan chains are composed exclusively of D-arabinofuranose (Araf) residues. The immediate precursor of the polymerized Araf is decaprenylphosphoryl-D-Araf, which is derived from 5-phosphoribose 1-diphosphate (pRpp) in an undefined manner. On the basis of time course, feedback, and chemical reduction experiment results we propose that decaprenylphosphoryl-Araf is synthesized by the following sequence of events. (i) pRpp is transferred to a decaprenyl-phosphate molecule to form decaprenylphosphoryl-beta-D-5-phosphoribose. (ii) Decaprenylphosphoryl-beta-D-5-phosphoribose is dephosphorylated to form decaprenylphosphoryl-beta-D-ribose. (iii) The hydroxyl group at the 2 position of the ribose is oxidized and is likely to form decaprenylphosphoryl-2-keto-beta-D-erythro-pentofuranose. (iv) Decaprenylphosphoryl-2-keto-beta-D-erythro-pentofuranose is reduced to form decaprenylphosphoryl-beta-D-Araf. Thus, the epimerization of the ribosyl to an arabinosyl residue occurs at the lipid-linked level; this is the first report of an epimerase that utilizes a lipid-linked sugar as a substrate. On the basis of similarity to proteins implicated in the arabinosylation of the Azorhizobium caulidans nodulation factor, two genes were cloned from the Mycobacterium tuberculosis genome and expressed in a heterologous host, and the protein was purified. Together, these proteins (Rv3790 and Rv3791) are able to catalyze the epimerization, although neither protein individually is sufficient to support the activity.

Arabinose↗

PubMed2004

A gfp reporter plasmid to visualize Azorhizobium caulinodans during nodulation of Sesbania rostrata.

Compared with other labeling techniques, the use of the green fluorescent protein (GFP) is advantageous to visualize bacteria because observations can be performed in real time. This feature is particularly interesting to study invasion events of rhizobia during nodule development on their legume host plant. To investigate the symbiotic interaction between Azorhizobium caulinodans ORS571 and Sesbania rostrata, we constructed two plasmids, pMP220-hem-gfp5 and pBBR5-hem-gfp5-S65T, that carry a modified gfp gene, the expression of which is controlled by the constitutive hem promoter. Introduction of either of these plasmids into A. caulinodans allowed the visualization of single bacteria. Determination of the plasmid stability in cultured bacteria and in nodules demonstrated that pBBR5-hem-gfp5-S65T is more stable than pMP220-hem-gfp5. The plasmid pBBR5-hem-gfp5-S65T can be used to study early invasion events during nodule development on hydroponic roots of S. rostrata.

Azorhizobium caulinodans↗

PubMed2004

Surface polysaccharides enable bacteria to evade plant immunity.

Plants have an immune system to perceive pathogenic or potentially beneficial bacteria. Aspects of perception, signal transduction and the responses that the plant produces resemble features of innate immunity observed in animals. Plant reactions are various and include the production of antimicrobial compounds. Bacteria that are successful in establishing pathogenic or symbiotic interactions have developed multiple ways to protect themselves. We review the general importance of bacterial surface polysaccharides in the evasion of plant immune responses and elaborate on their role in protecting symbiotic bacteria against toxic reactive oxygen species during invasion of the host plant.

Bacteria↗

PubMed2004

Structural characterization of extracellular polysaccharides of Azorhizobium caulinodans and importance for nodule initiation on Sesbania rostrata.

During lateral root base nodulation, the microsymbiont Azorhizobium caulinodans enters its host plant, Sesbania rostrata, via the formation of outer cortical infection pockets, a process that is characterized by a massive production of H(2)O(2). Infection threads guide bacteria from infection pockets towards nodule primordia. Previously, two mutants were constructed that produce lipopolysaccharides (LPSs) similar to one another but different from the wild-type LPS, and that are affected in extracellular polysaccharide (EPS) production. Mutant ORS571-X15 was blocked at the infection pocket stage and unable to produce EPS. The other mutant, ORS571-oac2, was impaired in the release from infection threads and was surrounded by a thin layer of EPS in comparison to the wild-type strain that produced massive amounts of EPS. Structural characterization revealed that EPS purified from cultured and nodule bacteria was a linear homopolysaccharide of alpha-1,3-linked 4,6-O-(1-carboxyethylidene)-D-galactosyl residues. In situ H(2)O(2) localization demonstrated that increased EPS production during early stages of invasion prevented the incorporation of H(2)O(2) inside the bacteria, suggesting a role for EPS in protecting the microsymbiont against H(2)O(2). In addition, ex planta assays confirmed a positive correlation between increased EPS production and enhanced protection against H(2)O(2).

Azorhizobium caulinodans↗

PubMed2003

Reactive oxygen species and ethylene play a positive role in lateral root base nodulation of a semiaquatic legume.

Lateral root base nodulation on the tropical, semiaquatic legume Sesbania rostrata results from two coordinated, Nod factor-dependent processes: formation of intercellular infection pockets and induction of cell division. Infection pocket formation is associated with cell death and production of hydrogen peroxide. Pharmacological experiments showed that ethylene and reactive oxygen species mediate Nod factor responses and are required for nodule initiation, whereby induction of division and infection could not be uncoupled. Application of purified Nod factors triggered cell division, and both Nod factors and ethylene induced cavities and cell death features in the root cortex. Thus, in S. rostrata, ethylene and reactive oxygen species act downstream from the Nod factors in pathways that lead to formation of infection pockets and initiation of nodule primordia.

Ethylenes↗

PubMed2002

pMH11, A tool for gene disruption and expression analysis in Azorhizobium caulinodans.

Tools for mutagenesis and expression analyses are needed to study the role of bacterial genes. Here, we report the construction of pMH11, a small, mobilizable plasmid that replicates in Escherichia coli, but not in Azorhizobium caulinodans, a nodulating microsymbiont of Sesbania rostrata, and that contains a unique BamHI restriction site upstream of a promoterless lacZ gene. pMH11 and two derivatives with the multiple cloning site of pBluescript (KS(II)) are useful for mutagenesis by gene disruption and for expression analyses after selection for cointegration by kanamycin resistance. Weakly constitutive promoter activity from the vector allowed transcription of genes downstream of the integration site, so that no polar effects were caused by gene disruption.

Azorhizobium caulinodans↗

PubMed2002

Nod factor structures, responses, and perception during initiation of nodule development.

The onset of nodule development, the result of rhizobia-legume symbioses, is determined by the exchange of chemical compounds between microsymbiont and leguminous host plant. Lipo-chitooligosaccharidic nodulation (Nod) factors, secreted by rhizobia, belong to these signal molecules. Nod factors consist of an acylated chitin oligomeric backbone with various substitutions at the (non)reducing-terminal and/or nonterminal residues. They induce the formation and deformation of root hairs, intra- and extracellular alkalinization, membrane potential depolarization, changes in ion fluxes, early nodulin gene expression, and formation of nodule primordia. Nod factors play a key role during nodule initiation and act at nano- to picomolar concentrations. A correct chemical structure is required for induction of a particular plant response, suggesting that Nod factor-receptor interaction(s) precede(s) a Nod factor-induced signal transduction cascade. Current data on Nod factor structures and Nod factor-induced responses are highlighted as well as recent advances in the characterization of proteins, possibly involved in recognition of Nod factors by the host plant.

Animals↗