Engineering of CHO cells for the production of vertebrate recombinant sialyltransferases

Background Sialyltransferases (SIATs) are a family of enzymes that transfer sialic acid (Sia) to glycan chains on glycoproteins, glycolipids, and oligosaccharides. They play key roles in determining cell–cell and cell-matrix interactions and are important in neuronal development, immune regulation, protein stability and clearance. Most fully characterized SIATs are of mammalian origin and these have been used for in vitro and in vivo modification of glycans. Additional versatility could be achieved by the use of animal SIATs from other species that live in much more variable environments. Our aim was to generate a panel of stable CHO cell lines expressing a range of vertebrate SIATs with different physicochemical and functional properties. Methods The soluble forms of various animal ST6Gal and ST3Gal enzymes were stably expressed from a Gateway-modified secretion vector in CHO cells. The secreted proteins were IMAC-purified from serum-free media. Functionality of the protein was initially assessed by lectin binding to the host CHO cells. Activity of purified proteins was determined by a number of approaches that included a phosphate-linked sialyltransferase assay, HILIC-HPLC identification of sialyllactose products and enzyme-linked lectin assay (ELLA). Results A range of sialyltransferase from mammals, birds and fish were stably expressed in CHO Flp-In cells. The stable cell lines expressing ST6Gal1 modify the glycans on the surface of the CHO cells as detected by fluorescently labelled lectin microscopy. The catalytic domains, as isolated by Ni Sepharose from culture media, have enzymatic activities comparable to commercial enzymes. Sialyllactoses were identified by HILIC-HPLC on incubation of the enzymes from lactose or whey permeate. The enzymes also increased SNA-I labelling of asialofetuin when incubated in a plate format. Conclusion Stable cell lines are available that may provide options for the in vivo sialylation of glycoproteins. Proteins are active and should display a variety of biological and physicochemical properties based on the animal source of the enzyme

Maximum depth sequencing reveals an ON/OFF replication slippage switch and apparent in vivo selection for bifidobacterial pilus expression

The human gut microbiome, of which the genus Bifidobacterium is a prevalent and abundant member, is thought to sustain and enhance human health. Several surface-exposed structures, including so-called sortase-dependent pili, represent important bifidobacterial gut colonization factors. Here we show that expression of two sortase-dependent pilus clusters of the prototype Bifidobacterium breve UCC2003 depends on replication slippage at an intragenic G-tract, equivalents of which are present in various members of the Bifidobacterium genus. The nature and extent of this slippage is modulated by the host environment. Involvement of such sortase-dependent pilus clusters in microbe-host interactions, including bacterial attachment to the gut epithelial cells, has been shown previously and is corroborated here for one case. Using a Maximum Depth Sequencing strategy aimed at excluding PCR and sequencing errors introduced by DNA polymerase reagents, specific G-tract sequences in B. breve UCC2003 reveal a range of G-tract lengths whose plasticity within the population is functionally utilized. Interestingly, replication slippage is shown to be modulated under in vivo conditions in a murine model. This in vivo modulation causes an enrichment of a G-tract length which appears to allow biosynthesis of these sortase-dependent pili. This work provides the first example of productive replication slippage influenced by in vivo conditions. It highlights the potential for microdiversity generation in “beneficial” gut commensals

Glycobiology of commensal bacteria with emphasis on cell surface adhesins and exopolysaccharides

The human gastro-intestinal tract (GIT) is an elaborate ecosystem specific to an individual and his physiological stage that involves inter- and intra-relationships between the bacteria composing the microbiota and between the host and the microbes. The idea of a communication system or a \u27crosstalk\u27 between the host and the microbiota has emerged after bacterial-derived molecules, such as polysaccharide A in Bacteroides fragilis, were shown to be essential for the maturation of the host immune system (Mazmanian et al., 2007). Understanding the molecular nature of the interaction between host gut epithelial cells and commensal microbes is essential for promotion of the mutualistic relationship with all its associated benefits for the host and prevention of the deleterious effects of infection by pathogens. Considerable efforts have been directed at the exploration of the lectin - adhesins of pathogenic bacteria and their relevant glycan receptors, with a view to identification of potential candidates for vaccines. To date, very little is known about adhesion mechanisms in gut commensal species. In particular, the lectin-like adhesins and their associated carbohydrate ligands have not been described. Recent technological advances in glycomics (omic databases, synthesis of glyco-conjugates and glycan microarrays) have provided new tools to study lectin/adhesin interactions with glycans. In this thesis, an in silico approach was used to identify surface-exposed proteins and putative adhesins of human commensal species, from the genera, Faecalibacterium, Bifidobacterium and Lactobacillus, that could represent potential probiotic strains. In vitro expression studies supported the in silico work, especially for the Lactobacillus species. Selected strains and their extracted pili were also incubated on mucin and neo-glycoconjugate microarrays and glycan ligands identified in F. prausnitzii, B. bifidum PRL2010 and L. paracasei subsp. paracasei. The potential of these novel approaches for the study of adhesins was thus demonstrated. The beneficial effects attributed to commensals in the GIT are in many cases exerted through exopolysaccharides (EPSs). Bacteria assemble a variety of glycoconjugates and polysaccharides as part of their cell walls. These include anchored molecules such as bacterial peptidoglycan, capsules and EPSs. Several functions, including health benefits for the host, have been attributed to EPSs. However, their isolation and analysis is a lengthy process that requires several analytical steps. The study of mammalian glycosylation has benefited significantly from recent developments in microarray technology, which enables the simultaneous analysis of the interactions between glycans and immobilised carbohydrate-binding proteins, such as plant lectins. Here, lectin microarray technology was successfully used for the first time to profile and differentiate fluorescently-labelled EPSs from commensal strains, as confirmed by monosaccharide analyses. Thus will help to move our understanding of EPS molecules ahead more quickly and promote their exploitation.2019-02-2

Mineralocorticoid receptor in fish: appraisal of its osmoregulatory role

International audienc

Identification of putative adhesins and carbohydrate ligands of <em>Lactobacillus paracasei</em> using a combinatorial <em>in silico</em> and glycomics microarray profiling approach

Commensal bacteria must colonize host mucosal surfaces to exert health-promoting properties, and bind to gastrointestinal tract (GIT) mucins via their cell surface adhesins. Considerable effort has been directed towards discovery of pathogen adhesins and their ligands to develop anti-infective strategies; however, little is known about the lectin-like adhesins and associated carbohydrate ligands in commensals. In this study, an in silico approach was used to detect surface exposed adhesins in the human commensal Lactobacillus paracasei subsp. paracasei, a promising probiotic commonly used in dairy product fermentation that presents anti-microbial activity. Of the 13 adhesin candidates, 3 sortase-dependent pili clusters were identified in this strain and expression of the adhesin candidate genes was confirmed in vitro. Mass spectrometry analysis confirmed the presence of surface adhesin elongation factor Tu and the chaperonin GroEL, but not pili expression. Whole cells were subsequently incubated on microarrays featuring a panel of GIT mucins from nine different mammalian species and two human-derived cell lines and a library of carbohydrate structures. Binding profiles were compared to those of two known pili-producing lactobacilli, L. johnsonii and L. rhamnosus and all Lactobacillus species displayed overlapping but distinct signatures, which may indicate different abilities for regiospecific GIT colonization. In addition, L. paracasei whole cells favoured binding to α-(2 → 3)-linked sialic acid and α-(1 → 2)-linked fucose-containing carbohydrate structures including blood groups A, B and O and Lewis antigens x, y and b. This study furthers our understanding of host-commensal cross-talk by identifying potential adhesins and specific GIT mucin and carbohydrate ligands and provides insight into the selection of colonization sites by commensals in the GIT

Mineralocorticoid receptor in fish: appraisal of its osmoregulatory role

International audienc

Use of microarray technology to assess the time course of liver stress response after confinement exposure in gilthead sea bream (sparus aurata l.)

Background: Selection programs for growth and stress traits in cultured fish are fundamental to the improvement of aquaculture production. The gilthead sea bream ( Sparus aurata) is the main aquacultured species in the Mediterranean area and there is considerable interest in the genetic improvement of this species. With the aim of increasing the genomic resources in gilthead sea bream and identifying genes and mechanisms underlying the physiology of the stress response, we developed a cDNA microarray for gilthead sea bream that is enriched by suppression substractive hybridization with stress and immunorelevant genes. This microarray is used to analyze the dynamics of gilthead sea bream liver expression profile after confinement exposure. Results: Groups of confined and control juvenile fish were sampled at 6, 24, 72 and 120 h post exposure. GeneSpring analyses identified 202 annotated genes that appeared differentially expressed at least at one sampling time (P &amp;lt; 0.05). Gene expression results were validated by quantitative PCR of 10 target genes, and K-means clustering of differently expressed genes identified four major temporal gene expression profiles. Set 1 encompassed a rapid metabolic readjustment with enhanced uptake and intracellular transport of fatty acids as metabolic fuels. Set 2 was associated with a wide variety of tissue repair and remodeling processes that were mostly mediated by the stress response of the endoplasmic reticulum ( ER). Sets 3 and 4 encompassed the re-establishment of cellular homeostasis with increased intracellular trafficking and scavenging of reactive oxygen species (ROS), accompanied by a bidirectional regulation of the immune system and a general decline of ROS production. Conclusions: Collectively, these findings show the complex nature of the adaptive stress response with a clear indication that the ER is an important control point for homeostatic adjustments. The study also identifies metabolic pathways which could be analyzed in greater detail to provide new insights regarding the transcriptional regulation of the stress response in fish