Conidium Specific Polysaccharides in .

Earlier studies have shown that the outer layers of the conidial and mycelial cell walls of are different. In this work, we analyzed the polysaccharidome of the resting conidial cell wall and observed major differences within the mycelium cell wall. Mainly, the conidia cell wall was characterized by (i) a smaller amount of α-(1,3)-glucan and chitin; (ii) a larger amount of β-(1,3)-glucan, which was divided into alkali-insoluble and water-soluble fractions, and (iii) the existence of a specific mannan with side chains containing galactopyranose, glucose, and N-acetylglucosamine residues.

Complement-Mediated Differential Immune Response of Human Macrophages to Species Through Interaction With Their Cell Wall Peptidorhamnomannans.

In this study, the human immune response mechanisms against and , two causative agents of human and animal sporotrichosis, were investigated. The interaction of and with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the yeast surfaces.

Structural and functional characterization of the PDZ domain of the human phosphatase PTPN3 and its interaction with the human papillomavirus E6 oncoprotein.

The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a PDZ (PSD-95/Dlg/ZO-1) domain-containing phosphatase with a tumor-suppressive or a tumor-promoting role in many cancers. Interestingly, the high-risk genital human papillomavirus (HPV) types 16 and 18 target the PDZ domain of PTPN3. The presence of a PDZ binding motif (PBM) on E6 confers interaction with a number of different cellular PDZ domain-containing proteins and is a marker of high oncogenic potential.

Target Engagement and Binding Mode of an Antituberculosis Drug to Its Bacterial Target Deciphered in Whole Living Cells by NMR.

Detailed information on hit-target interaction is very valuable for drug discovery efforts and indispensable for rational hit to lead optimization. We developed a new approach combining NMR in whole-cells in-cell NMR) and docking to characterize hit-target interaction at the atomic level. By using in-cell NMR, we validated target engagement of the antituberculosis imidazopyridine amide (IPA) series with the subunit b of the cytochrome bc:aa, the major respiratory terminal oxidase in mycobacteria.

Large-scale synthesis and structural analysis of a synthetic glycopeptide dendrimer as an anti-cancer vaccine candidate.

Herein, we report a new process that enables the gram-scale production of a fully synthetic anti-cancer vaccine for human use. This therapeutic vaccine candidate, named MAG-Tn3, is a high-molecular-weight tetrameric glycopeptide encompassing carbohydrate tumor-associated Tn antigen clusters and peptidic CD4 T-cell epitopes. The synthetic process involves (i) the stepwise solid-phase assembly of protected amino acids, including the high value-added Tn building blocks with only 1.

Structural basis of the signalling through a bacterial membrane receptor HasR deciphered by an integrative approach.

Bacteria use diverse signalling pathways to adapt gene expression to external stimuli. In Gram-negative bacteria, the binding of scarce nutrients to membrane transporters triggers a signalling process that up-regulates the expression of genes of various functions, from uptake of nutrient to production of virulence factors. Although proteins involved in this process have been identified, signal transduction through this family of transporters is not well understood.

Interaction of a partially disordered antisigma factor with its partner, the signaling domain of the TonB-dependent transporter HasR.

Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of a nutrient is sensed by an outer membrane transporter. This signal is then transmitted to an antisigma factor and subsequently to the cytoplasm where an ECF sigma factor induces expression of genes related to the acquisition of this nutrient.

The structure of HasB reveals a new class of TonB protein fold.

TonB is a key protein in active transport of essential nutrients like vitamin B12 and metal sources through the outer membrane transporters of Gram-negative bacteria. This inner membrane protein spans the periplasm, contacts the outer membrane receptor by its periplasmic domain and transduces energy from the cytoplasmic membrane pmf to the receptor allowing nutrient internalization. Whereas generally a single TonB protein allows the acquisition of several nutrients through their cognate receptor, in some species one particular TonB is dedicated to a specific system.

Interference with the PTEN-MAST2 interaction by a viral protein leads to cellular relocalization of PTEN.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) and MAST2 (microtubule-associated serine and threonine kinase 2) interact with each other through the PDZ domain of MAST2 (MAST2-PDZ) and the carboxyl-terminal (C-terminal) PDZ domain-binding site (PDZ-BS) of PTEN. These two proteins function as negative regulators of cell survival pathways, and silencing of either one promotes neuronal survival. In human neuroblastoma cells infected with rabies virus (RABV), the C-terminal PDZ domain of the viral glycoprotein (G protein) can target MAST2-PDZ, and RABV infection triggers neuronal survival in a PDZ-BS-dependent fashion.

(1)H, (13)C and (15)N resonance assignments of the periplasmic signalling domain of HasR, a TonB-dependent outer membrane heme transporter.

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that internalize nutrients such as vitamin B12, metal complexes, heme, some carbohydrates, etc. In addition to their transport activity, several TBDTs are also involved in a signalling cascade from the cell surface into the cytoplasm, via their periplasmic signalling domain. Here we report the backbone and side chain resonance assignments of the signalling domain of HasR, a TonB-dependent outer membrane heme transporter from Serratia marcescens as a first step towards its structural study.

Galactosaminogalactan, a new immunosuppressive polysaccharide of Aspergillus fumigatus.

A new polysaccharide secreted by the human opportunistic fungal pathogen Aspergillus fumigatus has been characterized. Carbohydrate analysis using specific chemical degradations, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance showed that this polysaccharide is a linear heterogeneous galactosaminogalactan composed of α1-4 linked galactose and α1-4 linked N-acetylgalactosamine residues where both monosacharides are randomly distributed and where the percentage of galactose per chain varied from 15 to 60%. This polysaccharide is antigenic and is recognized by a majority of the human population irrespectively of the occurrence of an Aspergillus infection.

Dynamic aspects of antibody:oligosaccharide complexes characterized by molecular dynamics simulations and saturation transfer difference nuclear magnetic resonance.

Carbohydrates are likely to maintain significant conformational flexibility in antibody (Ab):carbohydrate complexes. As demonstrated herein for the protective monoclonal Ab (mAb) F22-4 recognizing the Shigella flexneri 2a O-antigen (O-Ag) and numerous synthetic oligosaccharide fragments thereof, the combination of molecular dynamics simulations and nuclear magnetic resonance saturation transfer difference experiments, supported by physicochemical analysis, allows us to determine the binding epitope and its various contributions to affinity without using any modified oligosaccharides. Moreover, the methods used provide insights into ligand flexibility in the complex, thus enabling a better understanding of the Ab affinities observed for a representative set of synthetic O-Ag fragments.

Effects of backbone substitutions on the conformational behavior of Shigella flexneri O-antigens: implications for vaccine strategy.

The O-antigen (O-Ag), the polysaccharide part of the lipopolysaccharide, is the major target of the serotype-specific protective humoral response elicited upon host infection by Shigella flexneri, the main causal agent of the endemic form of bacillary dysentery. The O-Ag repeat units (RUs) of 12 S. flexneri serotypes share the tetrasaccharide backbone →2)-α-l-Rhap-(1 → 2)-α-l-Rhap-(1 → 3)-α-l-Rhap-(1 → 3)-β-d-GlcpNAc-(1→, with site-selective glucosylation(s) and/or O-acetylation defining the serotypes.

Characterization of glucuronic acid containing glycolipid in Aspergillus fumigatus mycelium.

A glucuronic acid containing glycerolipid was isolated from the filamentous fungi Aspergillus fumigatus. This acidic glycolipid was extracted from the membrane of mycelium and purified by two successive chromatographic steps on DEAE-Sephadex and Silica columns. Chemical structural analysis was performed using methylation, gas-chromatography, gas-chromatography-mass spectrometry, nano-electrospray mass spectrometry and (1)H/(13)C NMR spectra.

1H, 13C and 15N resonance assignments of the PDZ of microtubule-associated serine/threonine kinase 205 (MAST205) in complex with the C-terminal motif from the rabies virus glycoprotein.

Most of microbes hijack the cellular machinery to their advantage by interacting with specific target of the host cell. Glycoprotein of rabies virus is a key factor controlling the homeostasis of infected neuronal cells and proteins belonging to the human microtubule associated serine threonine kinase family have been identified as potential cellular partners. As a first step towards its structural study, we have assigned the backbone and side chain nuclei resonances of the PDZ domain (PSD-95, Discs Large, ZO-1) of MAST205 in complex with the C-terminal residues of the glycoprotein of rabies virus.

Cell wall beta-(1,6)-glucan of Saccharomyces cerevisiae: structural characterization and in situ synthesis.

Despite its essential role in the yeast cell wall, the exact composition of the beta-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant beta-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of approximately 38 kDa and could be hydrolyzed only by beta-(1,6)-glucanase.

(1)H, (13)C and (15)N resonance assignments of the C-terminal domain of HasB, a specific TonB like protein, from Serratia marcescens.

The backbone and side chain resonance assignments of the periplasmic domain of HasB, the energy transducer for heme active transport through the specific receptor HasR of Serratia marcescens, have been determined as a first step towards its structural study. The BMRB accession code is 15440.

Glycosylinositolphosphoceramides in Aspergillus fumigatus.

Fungal glycosylinositolphosphoceramides (GIPCs) are involved in cell growth and fungal-host interactions. In this study, six GIPCs from the mycelium of the human pathogen Aspergillus fumigatus were purified and characterized using Q-TOF mass spectrometry and 1H, 13C, and 31P NMR. All structures have the same inositolphosphoceramide moiety with the presence of a C(18:0)-phytosphingosine conjugated to a 2-hydroxylated saturated fatty acid (2-hydroxy-lignoceric acid).

Toward a better understanding of the basis of the molecular mimicry of polysaccharide antigens by peptides: the example of Shigella flexneri 5a.

Protein conjugates of oligosaccharides or peptides that mimic complex bacterial polysaccharide antigens represent alternatives to the classical polysaccharide-based conjugate vaccines developed so far. Hence, a better understanding of the molecular basis ensuring appropriate mimicry is required in order to design efficient carbohydrate mimic-based vaccines. This study focuses on the following two unrelated sets of mimics of the Shigella flexneri 5a O-specific polysaccharide (O-SP): (i) a synthetic branched pentasaccharide known to mimic the average solution conformation of S.

Conformational studies of the O-specific polysaccharide of Shigella flexneri 5a and of four related synthetic pentasaccharide fragments using NMR and molecular modeling.

As part of a program for the development of synthetic vaccines against the pathogen Shigella flexneri, we used a combination of NMR and molecular modeling methods to study the conformations of the O-specific polysaccharide (O-SP) of S. flexneri 5a and of four related synthetic pentasaccharide fragments. The NMR study, based on the analysis of 1H and 13C chemical shifts, the evaluation of inter-residue distances, and the measurement of one- and three-bond heteronuclear coupling constants, showed that the conformation of one of the four pentasaccharides is similar to that of the native O-SP in solution.

Histidine pK(a) shifts and changes of tautomeric states induced by the binding of gallium-protoporphyrin IX in the hemophore HasA(SM).

The HasA(SM) hemophore, secreted by Serratia marcescens, binds free or hemoprotein bound heme with high affinity and delivers it to a specific outer membrane receptor, HasR. In HasA(SM), heme is held by two loops and coordinated to iron by two residues, His 32 and Tyr 75. A third residue His 83 was shown recently to play a crucial role in heme ligation.