Glyco-Tools to Crack Unsolved Biomedical Needs

CA18132publishersversionpublishe

A chronic strain of the cystic fibrosis pathogen Pandoraeapulmonicola expresses a heterogenous hypo-acylated lipid A

Pandoraeasp. is an emerging Gram-negative pathogen in cystic fibrosis causing severe and persistent inflammation and damageof the lungs. The molecular mechanisms underlying the high pathogenicity ofPandoraeaspecies are still largely unknown. AsGram-negatives,Pandoraeasp. express lipopolysaccharides (LPS) whose recognition by the host immune system triggers aninflammatory response aimed at the bacterial eradication from the infected tissues. The degree of the inflammatory responsestrongly relies on the fine structure of the LPS and, in particular, of its glycolipid moiety, i.e. the lipid A. Here we report thestructure of the lipid A isolated from the LPS of a chronic strain ofP. pulmonicola(RL 8228), one of the most virulent identifiedso far among thePandoraeaspecies. Our data demonstrated that the examined chronic strain produces a smooth-type LPS with acomplex mixture of hypoacylated lipid A species displaying, among other uncommon characteristics, the 2-hydroxylation ofsome of the acyl chains and the substitution by an additional glucosamine on one or both the phosphate groups

Molecular Structure of Endotoxins from Gram-negative Marine Bacteria: An Update

Marine bacteria are microrganisms that have adapted, through millions of years, to survival in environments often characterized by one or more extreme physical or chemical parameters, namely pressure, temperature and salinity. The main interest in the research on marine bacteria is due to their ability to produce several biologically active molecules, such as antibiotics, toxins and antitoxins, antitumor and antimicrobial agents. Nonetheless, lipopolysaccharides (LPSs), or their portions, from Gram-negative marine bacteria, have often shown low virulence, and represent potential candidates in the development of drugs to prevent septic shock. Besides, the molecular architecture of such molecules is related to the possibility of thriving in marine habitats, shielding the cell from the disrupting action of natural stress factors. Over the last few years, the depiction of a variety of structures of lipids A, core oligosaccharides and O-specific polysaccharides from LPSs of marine microrganisms has been given. In particular, here we will examine the most recently encountered structures for bacteria belonging to the genera Shewanella, Pseudoalteromonas and Alteromonas, of the γ-Proteobacteria phylum, and to the genera Flavobacterium, Cellulophaga, Arenibacter and Chryseobacterium, of the Cytophaga-Flavobacterium-Bacteroides phylum. Particular attention will be paid to the chemical features expressed by these structures (characteristic monosaccharides, non-glycidic appendages, phosphate groups), to the typifying traits of LPSs from marine bacteria and to the possible correlation existing between such features and the adaptation, over years, of bacteria to marine environments

Hopanoids Producing Bacteria and Related Biofertilizers, Compositions, Methods and Systems

Hopanoids, hopanoids-producing nitrogen-fixing bacteria, and related formulations, systems and methods are described herein. In particular, hopanoids alone or in combination with hopanoid-producing nitrogen-fixing bacteria can be used as biofertilizer to stimulate plant growth and yield with enhanced tolerance to diverse stresses found in plant-microbe symbiotic microenvironments

Protective effect of Opuntia ficus-indica L. cladodes against UVA-induced oxidative stress in normal human keratinocytes

Opuntia ficus-indica L. is known for its beneficial effects on human health, but still little is known on cladodes as a potent source of antioxidants. Here, a direct, economic and safe method was set up to obtain water extracts from Opuntia ficus-indica cladodes rich in antioxidant compounds. When human keratinocytes were pre-treated with the extract before being exposed to UVA radiations, a clear protective effect against UVA-induced stress was evidenced, as indicated by the inhibition of stress-induced processes, such as free radicals production, lipid peroxidation and GSH depletion. Moreover, a clear protective effect against apoptosis in pre-treated irradiated cells was evidenced. We found that eucomic and piscidic acids were responsible for the anti-oxidative stress action of cladode extract. In conclusion, a bioactive, safe, low-cost and high value-added extract from Opuntia cladodes was obtained to be used for skin health/protection

Siglec-7 Mediates Immunomodulation by Colorectal Cancer-Associated Fusobacterium nucleatum ssp. animalis

Fusobacterium nucleatum is involved in the development of colorectal cancer (CRC) through innate immune cell modulation. However, the receptors of the interaction between F. nucleatum ssp. and immune cells remain largely undetermined. Here, we showed that F. nucleatum ssp. animalis interacts with Siglecs (sialic acid–binding immunoglobulin-like lectins) expressed on innate immune cells with highest binding to Siglec-7. Binding to Siglec-7 was also observed using F. nucleatum-derived outer membrane vesicles (OMVs) and lipopolysaccharide (LPS). F. nucleatum and its derived OMVs or LPS induced a pro-inflammatory profile in human monocyte-derived dendritic cells (moDCs) and a tumour associated profile in human monocyte-derived macrophages (moMϕs). Siglec-7 silencing in moDCs or CRISPR-cas9 Siglec-7-depletion of U-937 macrophage cells altered F. nucleatum induced cytokine but not marker expression. The molecular interaction between Siglec-7 and the LPS O-antigen purified from F. nucleatum ssp. animalis was further characterised by saturation transfer difference (STD) NMR spectroscopy, revealing novel ligands for Siglec-7. Together, these data support a new role for Siglec-7 in mediating immune modulation by F. nucleatum strains and their OMVs through recognition of LPS on the bacterial cell surface. This opens a new dimension in our understanding of how F. nucleatum promotes CRC progression through the generation of a pro-inflammatory environment and provides a molecular lead for the development of novel cancer therapeutic approaches targeting F. nucleatum-Siglec-7 interaction

Pseudomonas aeruginosa Exploits Lipid A and Muropeptides Modification as a Strategy to Lower Innate Immunity during Cystic Fibrosis Lung Infection

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1β cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF

Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-L-glyc-ero-L-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing b-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its a-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.Fil: Di Lorenzo, Flaviana. Università degli Studi di Napoli Federico II; ItaliaFil: Speciale, Immacolata. Università degli Studi di Napoli Federico II; ItaliaFil: Silipo, Alba. Università degli Studi di Napoli Federico II; ItaliaFil: Alías Villegas, Cynthia. Universidad de Sevilla; EspañaFil: Acosta Jurado, Sebastián. Universidad de Sevilla; EspañaFil: Rodríguez Carvajal, Miguel Ángel. Universidad de Sevilla; EspañaFil: Dardanelli, Marta Susana. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Biotecnología Ambiental y Salud - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Palmigiano, Angelo. Consiglio Nazionale delle Ricerche; ItaliaFil: Garozzo, Domenico. Consiglio Nazionale delle Ricerche; ItaliaFil: Ruiz Sainz, José Enrique. Universidad de Sevilla; EspañaFil: Molinaro, Antonio. University of Naples Federico II; ItaliaFil: Vinardell, José María. Universidad de Sevilla; Españ

Unveiling Molecular Recognition of Sialoglycans by Human Siglec-10

29 p.-6 fig.-2 tab.-7 fig. supl.-2 tab. supl.-1 graph. abst.Siglec-10 is an inhibitory I-type lectin selectively recognizing sialoglycans exposed on cell surfaces, involved in several patho-physiological processes. The key role Siglec-10 plays in the regulation of immune cell functions has made it a potential target for the development of immunotherapeutics against a broad range of diseases. However, the crystal structure of the protein has not been resolved for the time being and the atomic description of Siglec-10 interactions with complex glycans has not been previously unraveled. We present here the first insights of the molecular mechanisms regulating the interaction between Siglec-10 and naturally occurring sialoglycans. We used combined spectroscopic, computational and biophysical approaches to dissect glycans' epitope mapping and conformation upon binding in order to afford a description of the 3D complexes. Our outcomes provide a structural perspective for the rational design and development of high-affinity ligands to control the receptor functionality.This study was supported by the project ‘‘GLYTUNES’’ funded by MIUR Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN 2017) (2017XZ2ZBK, 2019–2022) to A.S.; by progetto POR SATIN and Progetto POR CampaniaOncoterapia to A.M.; by the European Commission (H2020-MSCA- 814102-SWEET CROSSTALK project) to A.M., R.M., and A.S.. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under grant agreement No 851356 to R.M. FSE,PON Ricerca e Innovazione 2014–2020, Azione I.1 ‘‘Dottorati Innovativi con caratterizzazione Industriale’’ is acknowledged for funding the PhD grant to R.E.F. Grants by the Spanish Ministry of Science MICINN (CTQ2017-88353-R and fellowship BES 2015–071588 to J.G.-C.) and Wellcome Trust 103744/Z/14/Z to P.R.C. are acknowledged.Peer reviewe