Adhesive glycostrategies from opportunistic filamentous fungi

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High Resolution Genotyping of Clinical Aspergillus flavus Isolates from India Using Microsatellites

Contains fulltext : 124312.pdf (publisher's version ) (Open Access)BACKGROUND: Worldwide, Aspergillus flavus is the second leading cause of allergic, invasive and colonizing fungal diseases in humans. However, it is the most common species causing fungal rhinosinusitis and eye infections in tropical countries. Despite the growing challenges due to A. flavus, the molecular epidemiology of this fungus has not been well studied. We evaluated the use of microsatellites for high resolution genotyping of A. flavus from India and a possible connection between clinical presentation and genotype of the involved isolate. METHODOLOGY/PRINCIPAL FINDINGS: A panel of nine microsatellite markers were selected from the genome of A. flavus NRRL 3357. These markers were used to type 162 clinical isolates of A. flavus. All nine markers proved to be polymorphic displaying up to 33 alleles per marker. Thirteen isolates proved to be a mixture of different genotypes. Among the 149 pure isolates, 124 different genotypes could be recognized. The discriminatory power (D) for the individual markers ranged from 0.657 to 0.954. The D value of the panel of nine markers combined was 0.997. The multiplex multicolor approach was instrumental in rapid typing of a large number of isolates. There was no correlation between genotype and the clinical presentation of the infection. CONCLUSIONS/SIGNIFICANCE: There is a large genotypic diversity in clinical A. flavus isolates from India. The presence of more than one genotype in clinical samples illustrates the possibility that persons may be colonized by multiple genotypes and that any isolate from a clinical specimen is not necessarily the one actually causing infection. Microsatellites are excellent typing targets for discriminating between A. flavus isolates from various origins

Metabolic Variation during Development in Culture of Leishmania donovani Promastigotes

The genome sequencing of several Leishmania species has provided immense amounts of data and allowed the prediction of the metabolic pathways potentially operating. Subsequent genetic and proteomic studies have identified stage-specific proteins and putative virulence factors but many aspects of the metabolic adaptations of Leishmania remain to be elucidated. In this study, we have used an untargeted metabolomics approach to analyze changes in the metabolite profile as promastigotes of L. donovani develop during in vitro cultures from logarithmic to stationary phase. The results show that the metabolomes of promastigotes on days 3–6 of culture differ significantly from each other, consistent with there being distinct developmental changes. Most notable were the structural changes in glycerophospholipids and increase in the abundance of sphingolipids and glycerolipids as cells progress from logarithmic to stationary phase

Identification of Essential Amino Acids in the Azorhizobium caulinodans Fucosyltransferase NodZ

The nodZ gene, which is present in various rhizobial species, is involved in the addition of a fucose residue in an α1-6 linkage to the reducing N-acetylglucosamine residue of lipo-chitin oligosaccharide signal molecules, the so-called Nod factors. Fucosylation of Nod factors is known to affect nodulation efficiency and host specificity. Despite a lack of overall sequence identity, NodZ proteins share conserved peptide motifs with mammalian and plant fucosyltransferases that participate in the biosynthesis of complex glycans and polysaccharides. These peptide motifs are thought to play important roles in catalysis. NodZ was expressed as an active and soluble form in Escherichia coli and was subjected to site-directed mutagenesis to investigate the role of the most conserved residues. Enzyme assays demonstrate that the replacement of the invariant Arg-182 by either alanine, lysine, or aspartate results in products with no detectable activity. A similar result is obtained with the replacement of the conserved acidic position (Asp-275) into its corresponding amide form. The residues His-183 and Asn-185 appear to fulfill functions that are more specific to the NodZ subfamily. Secondary structure predictions and threading analyses suggest the presence of a “Rossmann-type” nucleotide binding domain in the half C-terminal part of the catalytic domain of fucosyltransferases. Site-directed mutagenesis combined with theoretical approaches have shed light on the possible nucleotide donor recognition mode for NodZ and related fucosyltransferases

Production of recombinant xenotransplantation antigen in <i>Escherichia coli</i>

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Synthesis of D-galactopyranosylphosphonic and (D-galactopyranosylmethyl)phosphonic acids as intermediates of inhibitors of galactosyltransferases

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The living factory: in vivo production of N-acetyllactosamine containing carbohydrates in E. coli.

International audienceScientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a beta(1,4)GlcNAc-oligosaccharide synthase), and the Neisseria beta(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as betaGal(1,4)[betaGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for betaGal(1,4)GlcNAc alpha(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a beta(1,4)GlcNAc-oligosaccharide synthase), and the Neisseria beta(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as betaGal(1,4)[betaGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for betaGal(1,4)GlcNAc alpha(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides

Title: Biochemical and Structural Analysis of Helix pomatia Agglutinin: A HEXAMERIC LECTIN WITH A NOVEL FOLD

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin found in the albumen gland of the roman snail. As a constituent of perivitelline fluid, HPA protects fertilized eggs from bacteria and is part of the innate immunity system of the snail. The peptide sequence deduced from gene cloning demonstrates that HPA belongs to a family of carbohydrate-binding proteins recently identified in several invertebrates. This domain is also present in discoidin from the slime mold Dictyostelium discoideum. Investigation of the lectin specificity was performed with the use of glycan arrays, demonstrating that several GalNAc-containing oligosaccharides are bound and rationalizing the use of this lectin as a cancer marker. Titration microcalorimetry performed on the interaction between HPA and GalNAc indicates an affinity in the 10(-4) m range with an enthalpy-driven binding mechanism. The crystal structure of HPA demonstrates the occurrence of a new beta-sandwich lectin fold. The hexameric quaternary state was never observed previously for a lectin. The high resolution structure complex of HPA with GalNAc characterizes a new carbohydrate binding site and rationalizes the observed preference for alphaGalNAc-containing oligosaccharide

β-Propeller crystal structure of <i>Psathyrella velutina</i> lectin. An integrin-like fungal protein interacting with monosaccharides and calcium

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Isolectins I-A and I-B of <i>Griffonia (Bandeiraea) simplicifolia</i>: Crystal structure of metal-free GS I-B4 and molecular basis for metal binding and monosaccharide specificity

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