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Mucin glycoproteins are essential components of the mucosal barrier, which protects the host from pathogens. Throughout evolution, bacteria have developed strategies to modulate and penetrate this barrier, and cause virulence by interacting with mucin O-glycans at the epithelial cell-surface. O-fucosylated glycan epitopes on mucins are key ligands of many bacterial lectins. Here, a chemoenzymatic synthesis strategy is described to prepare a library of fucosylated mucin core glycopeptides to enable studies of mucin-interacting and fucose-binding bacterial lectins. Glycan cores with biologically important Lewis and H-antigens were prepared decorating the peptide backbone at different sites and densities. The fucosylated mucin glycopeptides were applied in microarray binding studies to explore the importance of glycan core and peptide backbone presentation of these antigens in binding interactions with the P. aeruginosa lectin LecB and the C. difficile toxin A.

ChemCatChem 14(6), e202200037 (2022). doi:10.1002/cctc.202200037, Published by WILEY-VCH Verlag, Weinheim

Galectins are a family of carbohydrate-binding lectins modulating cell events such as cell proliferation, apoptosis, adhesion or migration by cross-linking the glycan structures of cell membranes and/or extracellular matrix components. In a diseased organism, galectins are upregulated and trigger the progression of diseases such as inflammation, cancerogenesis, fibrosis, cardiovascular and metabolic disorders. Targeting galectins with glycomaterials for the aims of diagnostics or therapy is, therefore, a focus of biotechnological and biomedicinal research, and already led to candidates for clinical trials. Testing and evaluation of galectin-glycomaterial interactions require informative and versatile analytical methods at several levels of knowledge, from basic inter-molecular interaction to complex cell-based assays. This review aims to classify and characterize a selection of the most promising methods to identify the prospective glycomaterials for translating galectin targeting from the molecular level to the level of tailored in vivo assays.

Mucin glycoproteins are essential components of the mucosal protective barrier, which constantly senses and clears the host from pathogens. Throughout evolution, bacteria and virus have developed strategies to modulate and penetrate the mucosal barrier and cause virulence by interacting with the glycans of membrane-bound mucins at the epithelial cell-surface. These interactions may promote bacteria cell-adhesion, biofilm formation, protein toxin delivery, or cause an inflammatory environment. O-fucosylated glycan epitopes are commonly found on mucin glycoproteins, and are key ligands of many bacterial and viral lectins (glycan binding proteins). Herein we describe a chemoenzymatic synthesis strategy to efficiently prepare an extensive library of fucosylated mucin core tandem repeats glycopeptides to elucidate the fine fucose-binding specificities of the Pseudomonas aeruginosa lectin LecB and the Clostridium difficile toxin A. Therefore, glycan core structures were decorated with terminal Lewis and H-antigens, which play critical roles in infection biology. The fucosylated mucin glycopeptides were applied in microarray binding studies to explore the importance of the glycan and peptide backbone presentation of these terminal antigens in binding interactions with the two bacterial lectins.

Clostridium difficile infections cause gastrointestinal disorders and can lead to life-threatening conditions. The symptoms can vary from severe diarrhea to the formation of pseudomembranous colitis and therefore trigger a need for new therapies. The initial step of disease is the binding of the bacterial enterotoxins toxin A and B to the cell surface of epithelial intestinal cells. Scavenging of the toxins is crucial to inhibit their fatal effect in the human body and circumvent the administration of antibiotics. Cell surface glycans are common as ligands for TcdA. Although crucial for carbohydrate-protein interactions, a multivalent presentation of glycans for binding has been hardly considered. Here, we establish a neo-glycoprotein-based glycan library to identify an effective multivalent glycan ligand for TcdA. It comprises 40 different glycan epitopes based on N-acetyllactosamine precursors. Nine structures exhibit strong binding of the receptor domain. Among them, the Lewisy-Lewisx-epitope shows the best performance for binding both the receptor domain and the holotoxin. Therefore, the glycan was synthesized de novo and coupled to BSA as a scaffold for multivalent presentation. The corresponding neo-glycoprotein facilitates the proper scavenging of TcdA in vitro and effectively protects HT29 cells from TcdA-induced cell damage.

Galectin-3 plays a crucial role in cancerogenesis; its targeting is a prospective pathway in cancer diagnostics and therapy. Multivalent presentation of glycans was shown to strongly increase the affinity of glycoconjugates to galectin-3. Further strengthening of interaction with galectin-3 may be accomplished using artificial glycomimetics with apt aryl substitutions. We established a new, as yet undescribed chemoenzymatic method to produce selective C-3-substituted N,N′-diacetyllactosamine glycomimetics and coupled them to human serum albumin. From a library of enzymes, only β-N-acetylhexosaminidase from Talaromyces flavus was able to efficiently synthesize the C-3-propargylated disaccharide. Various aryl residues were attached to the functionalized N,N′-diacetyllactosamine via click chemistry to assess the impact of the aromatic substitution. In ELISA-type assays with galectin-3, free glycomimetics exhibited up to 43-fold stronger inhibitory potency to Gal-3 than the lactose standard. Coupling to human serum albumin afforded multivalent neo-glycoproteins with up to 4209-fold increased inhibitory potency per glycan compared to the monovalent lactose standard. Surface plasmon resonance brought further information on the kinetics of galectin-3 inhibition. The potential of prepared neo-glycoproteins to target galectin-3 was demonstrated on colorectal adenocarcinoma DLD-1 cells. We investigated the uptake of neo-glycoproteins into cells and observed limited non-specific transport into the cytoplasm. Therefore, neo-glycoproteins primarily act as efficient scavengers of exogenous galectin-3 of cancer cells, inhibiting its interaction with the cell surface, and protecting T-lymphocytes against galectin-3-induced apoptosis. The present neo-glycoproteins combine the advantage of a straightforward synthesis, selectivity, non-toxicity, and high efficiency for targeting exogenous galectin-3, with possible application in the immunomodulatory treatment of galectin-3-overexpressing cancers.

Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called ‘Pig intestinal bacterial collection’ (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota., The authors present a public collection of 117 bacterial isolates from the pig gut, including the description of 38 novel taxa. Interesting functions discovered in these organisms include a new fucosyltransferease and sactipeptide-like molecules encoded by biosynthetic gene clusters.

Macromolecular bioscience 20(9), 2000163 (2020). doi:10.1002/mabi.202000163, Published by Wiley-VCH, Weinheim

RSC Advances 9(41), 23484-23497 (2019). doi:10.1039/C9RA05497A, Published by RSC Publishing, London

2-Mercaptosuccinate (MS) and 3,3´-ditiodipropionate (DTDP) were discussed as precursor substance for production of polythioesters (PTE). Therefore, degradation of MS and DTDP was investigated in Advenella mimigardefordensis strain DPN7T, applying differential proteomic analysis, gene deletion and enzyme assays. Protein extracts of cells cultivated with MS, DTDP or 3-sulfinopropionic acid (SP) were compared with those cultivated with propionate (P) and/or succinate (S). The chaperone DnaK (ratio DTDP/P 9.2, 3SP/P 4.0, MS/S 6.1, DTDP/S 6.2) and a Do-like serine protease (DegP) were increased during utilization of all organic sulfur compounds. Furthermore, a putative bacterioferritin (locus tag MIM_c12960) showed high abundance (ratio DTDP/P 5.3, 3SP/P 3.2, MS/S 4.8, DTDP/S 3.9) and is probably involved in a thiol-specific stress response. The deletion of two genes encoding transcriptional regulators (LysR (MIM_c31370) and Xre (MIM_c31360)) in the close proximity of the relevant genes of DTDP catabolism (acdA, mdo and the genes encoding the enzymes of the methylcitric acid cycle; prpC,acnD, prpF and prpB) showed that these two regulators are essential for growth of A. mimigardefordensis strain DPN7T with DTDP and that they most probably regulate transcription of genes mandatory for this catabolic pathway. Furthermore, proteome analysis revealed a high abundance (ratio MS/S 10.9) of a hypothetical cupin-2-domain containing protein (MIM_c37420). This protein shows an amino acid sequence similarity of 60% to a newly identified MS dioxygenase from Variovorax paradoxus strain B4. Deletion of the gene and the adjacently located transcriptional regulator LysR, as well as heterologous expression of MIM_c37420, the putative mercaptosuccinate dioxygenase (Msdo) from A. mimigardefordensis, showed that this protein is the key enzyme of MS degradation in A. mimigardefordensis strain DPN7T (KM 0.2 mM, specific activity 17.1 μmol mg-1 min-1) and is controlled by LysR (MIM_c37410).

We investigated the illness records and life stories of 17 leukaemia survivors using narrative autobiographical interviews. Audio tapes were transcribed and analysed according to qualitative methodology. Using the sociological concept of “trajectory of suffering” (TOS) as a means of analysis we focused on the survivors’ mechanisms of psychosocial adaptation including integration of disease-related experiences as part of their autobiographical narration. Verbal data show how the diagnosis pulls affected people out of their everyday life from healthy, strong and with plans for the future to seriously ill, weak and facing death, and thus suspends their self-confidence and social action competence. Analysing the interview transcripts we found six categories of coping with TOS: (1) personal meaningful nourishments, (2) challenging experience with significant others, (3), courage to persevere, (4) family support, (5) dramatic family events, and (6) dreams. The results of our study demonstrate that the cancer is still a dark shadow over the lives of all survivors. They are discussed in the context of coping theory postulating creativity as an up to now underestimated resource of coping behaviour. As a consequence it seems to be vital that medical staff should recognize and discuss these individual needs and feelings of their patients in daily clinical practice.

Studies concentrating on the temporal dependence of subjective concepts during oncological treatment are underrepresented. Subjective interpretation contexts develop in the course of illness. The study focuses on the ideal-typical gestalt of these contents.In a follow-up study on coping, 12 patients with acute leukaemia (AL) were interviewed using a semistructured interview at the end of initial inpatient treatment. Using qualitative methodology, we inductively developed categories and assigned them to formal main categories.The following categories were developed: causal uncertainty as burden; discrepancy between subjective and objective assessment of degree of threat; knowledge of disease: conflict between information-seeking and information-avoiding behavior; dominance of medical approach to treatment; pursuit of normality; defense of emotions; orientation to workflows on the ward; adjustment as a coping strategy; positive attitude as a resource; life between hope and fear; limited future; latent fear of death.Themes of coping with the disease become visible. Some of these contents are tacit and latent, although of high subjective relevance to the patient. Their consideration could improve the patient-physician relationship.