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

Funding This work was supported by Fundação de Apoio à Pesquisa do Distrito Federal (FAP-DF)/CNPq, PRONEX grant ID: (FAP-DF, 0193.001.200/2016). VA is supported by the Centre Franco-Indien pour la Promotion de la Recherche Avancée (CEFIPRA) grant No. 5403-1 and ANR-DFG AfuINF grant. IG, VA, and CS were supported by the ANR-FUNHYDRO (ANR-16S-CE110020-01) grant. NG, GB and JW are supported by the Welcome Trust (102705, 097377, 101873, 215599 and 200208) and the Medical Research Council Centre for Medical Mycology (MR/N006364/2). Acknowledgments The authors acknowledge Dr. Lars Erwig, Dr. Jude Bain, and Dr. Kevin MacKenzie of University of Aberdeen for the scientific and technical support in the video microscopy experiments. LMLB was a research fellow of Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We acknowledge Fundação Carlos Chagas Filho de Amparo a Pesquisa do estado do Rio de Janeiro (Faperj) and Pasteur-Roux-Cantarini postdoctoral fellowship for the research fellowships given to GWPN and SSWW, respectively.Peer reviewedPublisher PD

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. GalNAc oligosaccharides are an essential epitope of the galactosaminogalactan that explains the universal antibody reaction due to cross reactivity with other antigenic molecules containing GalNAc stretches such as the N-glycans of Campylobacter jejuni. The galactosaminogalactan has no protective effect during Aspergillus infections. Most importantly, the polysaccharide promotes fungal development in immunocompetent mice due to its immunosuppressive activity associated with disminished neutrophil infiltrates

1H NMR studies of the FeS4 center in ferredoxin I from desulfovibrio desulfuricans norway: sequence-specific assignment of the cluster liganted cysteines

International audienceThe oxidized and reduced forms of the [Fe4-S4] ferredoxin I from Desulfovibrio desulfuricans Norway were investigated by 1H NMR spectroscopy with the aim of obtaining the complete assignment of the cysteines ligating the cluster. A combination of TOCSY and NOESY measurements together with information from the x-ray crystallographic structure of related ferredoxins provided the sequence-specific assignment of the four cysteines coordinated to the cluster. Through EXSY experiments, the hyperfine shifted resonance signals in the reduced ferredoxin were also assigned. The temperature dependence of the contact-shifted cysteinyl residues of the reduced ferredoxin reveals that two cysteines exhibit anti-Curie behavior whereas the other two cysteines display Curie behavior; that identifies Cys 9 (I) and Cys 15 (III) as ligated to the mixed-valence iron ions

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

International audienceThe 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. To investigate the conformational basis of serotype specificity, we sampled conformational behaviors during 60 ns of molecular dynamic simulations for oligosaccharides representing three RUs of each one of the O-Ags corresponding to serotypes 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X and Y, respectively. The calculated trajectories were checked by nuclear magnetic resonance (NMR) for 1a, 2a, 3a and 5a O-Ags. The simulations predict that in all O-Ags, but 1a and 1b, serotype-specific substitutions of the backbone do not induce any new backbone conformations compared with the linear type O-Ag Y, although they restrain locally the accessible conformational space. Moreover, the influence of any given substituent on the backbone is independent of the eventual presence of other substituents. Finally, only slight differences in conformational behavior between terminal and inner RUs were observed. These results suggest that the reported serotype-specificity of the protective immune response is not due to recognition of distinct backbone conformations, but to binding of the serotype-defining substituents in the O-Ag context. The gained knowledge is discussed in terms of impact on the development of a broad-serotype coverage vaccine

Glycosylinositolphosphoceramides in Aspergillus fumigatus.

International audienceFungal 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). The carbohydrate moiety defines two types of GIPC. The first, a mannosylated zwitterionic glycosphingolipid contains a glucosamine residue linked in alpha1-2 to an inositol ring that has been described in only two other fungal pathogens. The second type of GIPC presents an alpha-Manp-(1-->3)-alpha-Manp-(1-->2)-IPC common core. A galactofuranose residue is found in four GIPC structures, mainly at the terminal position via a beta1-2 linkage. Interestingly, this galactofuranose residue could be substituted by a choline-phosphate group, as observed only in the GIPC of Acremonium sp., a plant pathogen

Structural requirements for thymosin beta4 in its contact with actin. An NMR-analysis of thymosin beta4 mutants in solution and correlation with their biological activity.

International audienceWe examined the conformational preferences of mutants of thymosin beta4, an actin monomer sequestering protein by NMR spectroscopy in 60% (v/v) trifluoroethanol. Under these conditions, the wild-type thymosin beta4 conformation consists of an alpha-helix (helix I) extending from residues 5-16 with a more stable fragment from lysine 11 to lysine 16 and a second alpha-helix (helix II) encompassing residues 31-39. The point mutations studied here are located in helix I or in the LKKTET segment (residues 17-22) that form the two main entities of interaction with the actin molecule. The alpha-1H conformational shifts allow us to investigate the helicity of the polypeptides at the residue level and to correlate these structures with their biological activity. We determine that an extension of helix I at its C-terminal end over the LKK-segment results in loss of activity. The correct termination of this helix is connected to a specific orientation of the polypeptide essential for a cooperative action of the thymosin beta4 binding entities required for full activity

Sequence of a staphylococcal gene, vat, encoding an acetyltransferase inactivating the A-type compounds of virginiamycin-like antibiotics.

International audienceThe Staphylococcus aureus plasmids, pIP680 and pIP1156, which confer resistance to A-type compounds of virginiamycin-like antibiotics (Vml: streptogramin A, pristinamycin IIA, virginiamycin M) and to synergistic mixtures of the A and B compounds of Vml antibiotics, were shown to direct the modification of A-type compounds by acetylation. The vat gene, encoding the acetyltransferase modifying A-type compounds, was isolated from plasmid pIP680 and sequenced. This gene potentially encodes a 219-amino-acid (aa) protein, VAT, of 24 330 Da showing at least 38% aa identity with two chloramphenicol acetyltransferases encoded by cat genes isolated from Escherichia coli and Agrobacterium tumefaciens. Resistance to A-type compounds of Vml antibiotics conferred to S. aureus by vat was not expressed in E. coli, although a protein having a M(r) similar to that encoded by this gene was detected in E. coli minicells. The vat gene was detected by the polymerase chain reaction in two chromosomally located staphylococcal conjugative elements and in the conjugative plasmid, pIP1156, conferring resistance to A-type compounds

On the Knorr synthesis of 6-bromo-4-methylquinolin-2(1H)-one

International audienceIn the course of our work on infectious diseases, we were led to prepare 6-bromo-2-chloro-4-methylquinoline as a starting material. Since surprisingly little has been reported in the literature, the two synthetic steps to this compound were investigated. The synthesis involves a condensation between -ketoesters and 4-bromoaniline and the cyclization of the resulting anilides into 6-bromoquinolin-2(1H)-one otherwise known as the Knorr reaction. The 1H NMR monitoring of the first step allowed us to optimize the conditions leading specifically to the anilide without the occurrence of the alternative crotonate. To illustrate the scope of our finding, few additional anilides featuring electron attracting groups were prepared. The study of their cyclization revealed some unsuspected steric effect governing this second step. Aside from rectifying few claims in this chemistry, this study led to a three-step preparation of 6-bromo-2-chloro-4-methylquinoline in a 48 % overall yield from 4-bromoaniline