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.

Identification of putative adhesins and carbohydrate ligands of Lactobacillus paracasei using a combinatorial in silico 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 â†’ 3)-linked sialic acid and α-(1 â†’ 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.

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.

The MSHA pilus of Vibrio parahaemolyticus has lectin functionality and enables TTSS-mediated pathogenicity.

Vibrio parahaemolyticus is a seafood-borne pathogen which causes acute inflammatory gastroenteritis--a process which is mediated by the translocation of type three secretion system effector proteins. The molecular interactions governing colonization of the intestinal epithelium by this pathogen remain poorly understood. The mannose-sensitive haemagglutinin (MSHA) pilus was identified in this study as a significant factor in bacterial-host cell adherence and subsequent pathogenesis towards Caco-2 human intestinal epithelial cells. Deletion of essential components of the MSHA pilus resulted in a 60% decrease in adherence and a similar reduction in bacterial uptake by human intestinal cells. The diminished adherence of MSHA mutants correlated with significant decreases in V. parahaemolyticus-induced Caco-2 cell lysis, cell rounding and IL-8 secretion. Glycan array comparison between the V. parahaemolyticus wild type and MSHA deficient mutants identified lectin functionality for the MSHA pilus with specificity towards the fucosylated blood group oligosaccharide antigens Lewis A and X and blood groups A and B. The MSHA pilus also exhibited high affinity for the structurally related asialo-GM1 ganglioside, lacto-N-fucopentaose I and lacto-N-difucohexaose I. We hypothesize that these glycans act as receptors for the MSHA pilus in the gastrointestinal tract, thereby facilitating efficient colonization of the intestinal epithelium by V. parahaemolyticus.

Molecular profiling of the gilthead sea bream (Sparus aurata L.) response to chronic exposure to the myxosporean parasite Enteromyxum leei.

The aim of the present work was to investigate the transcriptome response of gilthead sea bream (Sparus aurata) after challenge with the myxosporean Enteromyxum leei, a wide-spread enteric parasite causing heavy economic losses in Mediterranean sparid farms. This parasite causes severe desquamative enteritis which usually leads to death of the fish, and there are no preventative or curative treatments for this enteromyxosis. After 113 days of exposure to parasite-contaminated effluent, fish were classified into three cohorts: control fish not exposed to parasite, those that were exposed and infected, and those that were exposed but not infected. In order to detect target genes that may be candidates for infective status or resistance, a cDNA microarray containing 18,490 cDNA clones enriched in genes differentially expressed after infection was hybridised with head kidney and intestine samples. In infected fish, 371 and 373 genes were differentially regulated at the >1.5-fold level in intestine and head kidney respectively, whereas in non-infected fish 175 and 501 genes were differentially regulated in these tissues, respectively. A global marked gene down-regulation was evident in infected fish, mainly in genes involved in the immune and acute phase response particularly complement and mannose binding lectin. Microarray analysis demonstrated a complex interplay between host and/or parasite derived proteases and protease inhibitors, apoptosis, cell proliferation and antioxidant defence genes in exposed fish. In the head kidney of non-infected fish a marked depression of genes involved in the acute phase response was evident. By contrast, in the intestine of non-infected fish, interferon-stimulated and MHC class II genes involved in antigen processing and presentation were up-regulated, possibly indicating that an active immune response at the local level is important to avoid infection with or proliferation of the parasite.

Testes and brain gene expression in precocious male and adult maturing Atlantic salmon (Salmo salar).

BACKGROUND: The male Atlantic salmon generally matures in fresh water upon returning after one or several years at sea. Some fast-growing male parr develop an alternative life strategy where they sexually mature before migrating to the oceans. These so called 'precocious' parr or 'sneakers' can successfully fertilise adult female eggs and so perpetuate their line. We have used a custom-built cDNA microarray to investigate gene expression changes occurring in the salmon gonad and brain associated with precocious maturation. The microarray has been populated with genes selected specifically for involvement in sexual maturation (precocious and adult) and in the parr-smolt transformation. RESULTS: Immature and mature parr collected from a hatchery-reared stock in January were significantly different in weight, length and condition factor. Changes in brain expression were small - never more than 2-fold on the microarray, and down-regulation of genes was much more pronounced than up-regulation. Significantly changing genes included isotocin, vasotocin, cathepsin D, anamorsin and apolipoprotein E. Much greater changes in expression were seen in the testes. Among those genes in the testis with the most significant changes in expression were anti-Mullerian hormone, collagen 1A, and zinc finger protein (Zic1), which were down-regulated in precocity and apolipoproteins E and C-1, lipoprotein lipase and anti-leukoproteinase precursor which were up-regulated in precocity. Expression changes of several genes were confirmed in individual fish by quantitative PCR and several genes (anti-Mullerian hormone, collagen 1A, beta-globin and guanine nucleotide binding protein (G protein) beta polypeptide 2-like 1 (GNB2L1) were also examined in adult maturing testes. Down-regulation of anti-Mullerian hormone was judged to be greater than 160-fold for precocious males and greater than 230-fold for November adult testes in comparison to July testes by this method. For anti-Mullerian hormone and guanine nucleotide binding protein beta polypeptide 2-like 1 expression changes in precocious males mirrored mature adults (November) but for collagen 1A and beta-globin the pattern was more complex. CONCLUSIONS: Expression changes in the fish brain during the process of precocious sexual maturation were small compared to those in the testes. Microarray analysis suggested down-regulation of housekeeping functions and up-regulation of a small number of specific processes. Transcriptional changes in the testes were much more pronounced with anti-Mullerian hormone playing a major role. Expression profiles for mature parr and maturing adult testes indicate subtle differences in gene expression between these two related groups.

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 < 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.