Role of NleH, a type III secreted effector from attaching and effacing pathogens, in colonization of the bovine, ovine, and murine gut

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-kappaB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC DeltanleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-kappaB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-kappaB response elements, we found that NleH causes an increase in NF-kappaB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

Gut hormone GPCRs: structure, function, drug discovery

Crystallization and determination of the high resolution three-dimensional structure of the β2-adrenergic receptor in 2007 was followed by structure elucidation of a number of other receptors, including those for neurotensin and glucagon. These major advances foster the understanding of structure-activity relationship of these receptors and structure-based rational design of new ligands having more predictable activity. At present, structure determination of gut hormone receptors in complex with their ligands (natural, synthetic) and interacting signalling proteins, for example, G-proteins, arrestins, represents a challenge which promises to revolutionize gut hormone endocrinonology.

A' Magyar Urfi oder die Kunst in 46 Stunden gut ungarisch lesen, verstehen, sprechen und schreiben zu lernen : nebst einer Sammlung von Hauptwörtern und deren Leidensendungen von Bei- und Zeitwörtern zur Übung des Gedächtnisses : ferner: Gesprächen, Fabeln, u.s.w.

von J.T. Schuster

Gut Microbiota, Probiotics and Diabetes

Diabetes is a condition of multifactorial origin, involving several molecular mechanisms related to the intestinal microbiota for its development. In type 2 diabetes, receptor activation and recognition by microorganisms from the intestinal lumen may trigger inflammatory responses, inducing the phosphorylation of serine residues in insulin receptor substrate-1, reducing insulin sensitivity. In type 1 diabetes, the lowered expression of adhesion proteins within the intestinal epithelium favours a greater immune response that may result in destruction of pancreatic β cells by CD8+ T-lymphocytes, and increased expression of interleukin-17, related to autoimmunity. Research in animal models and humans has hypothesized whether the administration of probiotics may improve the prognosis of diabetes through modulation of gut microbiota. We have shown in this review that a large body of evidence suggests probiotics reduce the inflammatory response and oxidative stress, as well as increase the expression of adhesion proteins within the intestinal epithelium, reducing intestinal permeability. Such effects increase insulin sensitivity and reduce autoimmune response. However, further investigations are required to clarify whether the administration of probiotics can be efficiently used for the prevention and management of diabetes.

Factors affecting gut metabolism and bioavailabilty of orange juice flavanones in humans

Several studies have supported a beneficial role of dietary flavonoids in reducing the risk/progression of chronic diseases (including hypertension, cardiovascular disease, certain cancers, type-2-diabetes, cognitive dysfunction, age-related bone disease). Their beneficial properties are likely to be affected by their structure, distribution in foods, food matrix, life habits (physical activity). Most dietary polyphenols reach the colon where they are metabolized to phenolic acids by gut bacteria. Lack of knowledge of the factors affecting flavonoid metabolism and bioavailability hinders understanding of their health effects. Therefore, this thesis aimed to investigate the effect of factors on bioavailability and metabolism of dietary polyphenols from orange juices (OJ) in in vitro and in vivo studies. In chapter 3, the variability of orange juice polyphenolic content was assessed using in vitro models of the human gastrointestinal tract. Chapter 4 investigated the reduced urinary phenolic acids after OJ and yoghurt (Y) in humans compared to OJ alone using in vitro models of the human gut. In chapter 5, raftiline and glucose were tested for effects on metabolism of hesperidin (flavanone not OJ). In chapter 6, an intervention study of 4 weeks moderate intensity exercise determined whether exercise affected bioavailability and metabolism of OJ flavanones in healthy sedentary females. The studies in this thesis showed that food sources, food matrix and physical exercise may determine the significant variations in bioavailability and metabolism of flavonoids, seen in a number of studies. These factors could result in differences in bioactivity and bioefficacy of polyphenols, and need to be taken into account in further studies of the effects of flavanones on disease risk.

Biogeographical distribution of Rimicaris exoculata resident gut epibiont communities along the Mid-Atlantic Ridge hydrothermal vent sites

Rimicaris exoculata is a deep-sea hydrothermal vent shrimp which enlarged gill chamber houses a complex trophic epibiotic community. Its gut harbours an autochthonous and distinct microbial community. This species dominates hydrothermal ecosystems megafauna along the Mid-Atlantic Ridge, regardless of contrasted geochemical conditions prevailing in them. Here, the resident gut epibiont community at four contrasted hydrothermal vent sites (Rainbow/TAG/Logatchev/Ashadze) was analysed and compiled with previous data to evaluate the possible influence of site location, using 16S rRNA surveys and microscopic observations (TEM, SEM and FISH analyses). Filamentous epibionts inserted between the epithelial cells microvilli were observed on all examined samples. Results confirmed resident gut community affiliation to Deferribacteres, Mollicutes, Epsilonproteobacteria and to a lesser extent Gammaproteobacteria lineages. Still a single Deferribacteres phylotype was retrieved at all sites. Four Mollicutes-related OTUs were distinguished, one being only identified on Rainbow specimens. The topology of ribotypes median-joining networks illustrated a community diversification possibly following demographic expansions, suggesting a more ancient evolutionary history and/or a larger effective population size at Rainbow. Finally, the gill chamber community distribution was also analysed through ribotypes networks based on sequences from R. exoculata collected at Rainbow/Snake Pit/TAG/Logatchev/Ashadze sites. Results allow refining hypotheses on the epibiont role and transmission pathways.

Ich hoffe dass alles da gut lauft. Eine Analyse von Aufsätzen finnischer Gymnasiasten

Diese Pro-Gradu-Arbeit basiert auf Deutschaufsätzen von finnischen Gymnasiasten. Es soll der Versuch unternommen werden, die Schreibkompetenz der Lernenden zu analysieren. Es wird betrachtet, welche Verbstrukturen sie in ihren Aufsätzen benutzen und wie sie sie anwenden. Auch die Stellung des Verbs wird untersucht. Diese werden mit den Inhalten, die im Unterricht gelehrt worden sind, verglichen. Das Beherrschen der Verbstrukturen hat einen starken Einfluss auf die Bestimmung der sprachlichen Kompetenz. Das Primärmaterial besteht aus zwölf Aufsätzen und neun Lehrbüchern. Die Aufsätze wurden während des dritten und sechsten B2-Deutschkurses geschrieben. Die SchülerInnen haben unterschiedliche Hintergründe: Einige lernen Deutsch erst seit ein Paar Jahren, während andere sogar deutsche Verwandte haben. Der deutsche Hintergrund hat die Sprachkompetenz positiv beeinflusst, aber auch z. B. die Motivation, sprachliche Begabung und die Lehrwerke tragen hierzu bei. Auch Transfer wurde untersucht. Die Aufsätze wurden Satz per Satz analysiert. Es wurde betrachtet, welche Strukturen vorkamen und wie vielseitig sie benutzt wurden. Die Lernenden hatten in den ersten Texten sehr einfache Sätze geschrieben, aber sie haben Variation z. B. durch die Inversion gebracht. In den näcshten Aufsätzen war der Satzbau komplexer, aber dadurch waren die Sätze manchmal nicht sofort zu verstehen. Der sprachliche Fortschritt wurde mit Hilfe des Analysemodells von Rösch (2011) analysiert und die Sprachkompetenz wurde in die Niveaus des gemeinsamen europäischen Referenzrahmens eingeordnet. Der finnische Lehrplan stellt eigene Ziele zum Fremdsprachenunterricht und die Lernenden dieser Untersuchungsgruppe haben die Ziele schon nach dem dritten Kurs erreicht. Zwischen dem dritten und sechsten Kurs haben sie das folgende Niveau nicht erreicht, aber die Schreibkompetenz hat sich vertieft. Mit ein bisschen Übung würden sie bestimmt das folgende Niveau erreichen. Die Kompetenzunterschiede zwischen den Lernenden waren deutlich zu sehen.

Diet, microbes and gut immune responses in the pathogenesis of experimental type 1 diabetes

Type 1diabetes (T1D) is an autoimmune disease, which is influenced by a variety of environmental factors including diet and microbes. These factors affect the homeostasis and the immune system of the gut. This thesis explored the altered regulation of the immune system and the development of diabetes in non-obese diabetic (NOD) mice. Inflammation in the entire intestine of diabetes-prone NOD mice was studied using a novel ex-vivo imaging system of reactive oxygen and nitrogen species (RONS), in relation to two feeding regimens. In parallel, gut barrier integrity and intestinal T-cell activation were assessed. Extra-intestinal manifestations of inflammation and decreased barrier integrity were sought for by studying peritoneal leukocytes. In addition, the role of pectin and xylan as dietary factors involved in diabetes development in NOD mice was explored. NOD mice showed expression of RONS especially in the distal small intestine, which coincided with T-cell activation and increased permeability to macromolecules. The introduction of a casein hydrolysate (hydrolysed milk protein) diet reduced these phenomena, altered the gut microbiota and reduced the incidence of T1D. Extra-intestinally, macrophages appeared in large numbers in the peritoneum of NOD mice after weaning. Peritoneal macrophages (PM) expressed high levels of interleukin-1 receptor associated kinase M (IRAK-M), which was indicative of exposure to ligands of toll-like receptor 4 (TLR-4) such as bacterial lipopolysaccharide (LPS). Intraperitoneal LPS injections activated T cells in the pancreatic lymph nodes (PaLN) and thus, therefore potentially could activate islet-specific T cells. Addition of pectin and xylan to an otherwise diabetes-retarding semisynthetic diet affected microbial colonization of newly-weaned NOD mice, disturbed gut homeostasis and promoted diabetes development. These results help us to understand how diet and microbiota impact the regulation of the gut immune system in a way that might promote T1D in NOD mice.

Implication of gut microbiota in nonalcoholic fatty liver disease

International audience

A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10-5. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.

Antibiotic resistance in the gut microbiota

Antibiotic resistance is an increasing threat to our ability to treat infectious diseases. Thus, understanding the effects of antibiotics on the gut microbiota, as well as the potential for such populations to act as a reservoir for resistance genes, is imperative. This thesis set out to investigate the gut microbiota of antibiotic treated infants compared to untreated controls using high-throughput DNA sequencing. The results demonstrated the significant effects of antibiotic treatment, resulting in increased proportions of Proteobacteria and decreased proportions of Bifidobacterium. The species diversity of bifidobacteria was also reduced. This thesis also highlights the ability of the human gut microbiota to act as an antibiotic resistance reservoir. Using metagenomic DNA extracted from faecal samples from adult males, PCR was employed to demonstrate the prevalence and diversity of aminoglycoside and β-lactam resistance genes in the adult gut microbiota and highlighted the merits of the approach adopted. Using infant faecal samples, we constructed and screened a second fosmid metagenomic bank for the same families of resistance genes and demonstrated that the infant gut microbiota is also a reservoir for resistance genes. Using in silico analysis we highlighted the existence of putative aminoglycoside and β-lactam resistance determinants within the genomes of Bifidobacterium species. In the case of the β- lactamases, these appear to be mis-annotated. However, through homologous recombination-mediated insertional inactivation, we have demonstrated that the putative aminoglycoside resistance proteins do contribute to resistance. In additional studies, we investigated the effects of short bowel syndrome on infant gut microbiota, the immune system and bile acid metabolism. We also sequenced the microbiota of the human vermiform appendix, highlighting its complexity. Finally, this thesis demonstrated the strain specific nature of 2 different probiotic CLA-producing Bifidobacterium breve on the murine gut microbiota.

The impact of whey protein consumption and exercise on the composition and diversity of the gut microbiota: a high through-put DNA sequencing approach

Advances in culture independent technologies over the last decade have highlighted the pivotal role which the gut microbiota plays in maintaining human health. Conversely, perturbations to the composition or actions of the ‘normal/functioning’ microbiota have been frequently associated with the pathogenesis of several disease states. Therefore the selective modulation of enteric microbial communities represents a viable target for the development of novel treatments for such diseases. Notably, while bovine whey proteins and exercise have been shown to positively influence several physiological processes, such as energy balance, their effect on the composition or functionality of the gut microbiota remains largely unknown. In this thesis, a variety of ex vivo, murine and human models are used in conjunction with high-throughput DNA sequencing-based analysis to provide valuable and novel insights into the impact of both whey proteins and exercise on enteric microbial communities. Overall the results presented in this thesis highlight that the consumption both whey protein isolate (WPI), and individual component proteins of whey such as bovine serum albumin (BSA) and lactoferrin, reduce high fat diet associated body weight gain and are associated with beneficial alterations within the murine gut microbiota. Although the impact of exercise on enteric microbial communities remains less clear, it may be that longer term investigations are required for the true effect of exercise on the gut microbiota to be fully elucidated.