A Gnotobiotic Mouse Model for Studying the Effect of Human Gut Community Ecology on a Pathobiont, Bacteroides fragilis

ABSTRACT OF THE DISSERTATION A Gnotobiotic Mouse Model for Studying the Effect of Human Gut Community Ecology on a Pathobiont, Bacteroides fragilis by Vitas Wagner Doctor of Philosophy in Biology and Biomedical Sciences Evolution, Ecology, and Population Biology Washington University in St. Louis, 2015 Professor Jeffrey I. Gordon, Chair Childhood undernutrition represents a pressing global health challenge. Epidemiologic studies have shown that undernutrition is not due to food insecurity alone, but rather represents a complex set of interactions between intra- and intergenerational factors. The gut microbiota has been implicated as one such factor. This thesis tested the hypothesis that enteropathogen burden affects the structure and expressed functions of the gut microbiota, and reciprocally, the gut microbiota affects susceptibility to the effects of enteropathogen invasion. To examine this hypothesis, groups of adult germ-free C57Bl/6 mice were colonized with fecal microbiota sampled from two 24 month-old members of a birth-cohort living in an urban slum in the Mirpur district of Dhaka, Bangladesh: one child had a healthy growth phenotype as judged by anthropometry, and the undernourished child was severely stunted and underweight and exhibited relative microbiota immaturity. The microbiota of both children contained Bacteroides fragilis, a pathobiont. Both groups of colonized mice were fed three diets that embodied the diets consumed by the population from which the microbiota donors were selected. Mice harboring the intact uncultured microbiota from the stunted donor exhibited severe weight-loss, while those receiving the healthy donor’s microbiota maintained weight on these diets. Clonally-arrayed, sequenced collections of cultured anaerobic bacteria strains, generated from the donors’ fecal microbiota, transmitted (i) the discordant weight phenotypes within and across generations of animals (in a diet-dependent fashion), as well as (ii) distinct host metabolic phenotypes (manifest by marked differences in tissue organic acid, amino acid and ceramide profiles as defined by mass spectrometry). The B. fragilis strain in the stunted donor’s culture collection was enterotoxigenic (ETBF), while the two B. fragilis strains in the healthy donor’s culture collection were non-toxigenic (NTBF). Through a series of experiments in which mice were colonized with either the stunted or healthy culture collection ± ETBF or ± NTBF, I demonstrated that ETBF was associated with weight loss as a member of the stunted donor’s community, but not the healthy donor’s community. Microbial RNA-Seq analysis revealed marked differences in ETBF gene expression in the two different community contexts, and as a function of the presence or absence of NTBF. Strikingly, ETBF induced expression of a large repertoire of virulence factor genes encoded in the genomes of the healthy culture collection members; these effects were mitigated when NTBF was present. The effects of ETBF on host metabolism were also community context-dependent. These results provide preclinical evidence that enteropathogen effects on host physiology and metabolism are greatly impacted by gut community ecology and illustrate the value of combining gnotobiotic mouse models, human diet embodiments, and ‘personal’ culture collections for dissecting microbial-microbial and microbial-host interactions. A parallel study using gnotobiotic mice and subsets of the culture collection from the healthy donor revealed how turmeric, a culturally relevant spice in the Bangladeshi diet, and microbial bile acid production/metabolism interact to impact gut motility

Gene Expression Patterns in Myelodyplasia Underline the Role of Apoptosis and Differentiation in Disease Initiation and Progression

The myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective and dysplastic hematopoiesis. The genetic and epigenetic pathways that determine disease stage and progression are largely unknown. In the current study we used gene expression microarray methodology to examine the gene expression differences between normal hematopoietic cells and hematopoietic cells from patients with MDS at different disease stages, using both unselected and CD34+ selected cells. Significant differences between normal and MDS hematopoietic cells were observed for several genes and pathways. Several genes promoting or opposing apoptosis were dysregulated in MDS cases, most notably MCL1 and EPOR. Progression from RA to RAEB(T) was associated with increased expression of several histone genes. In addition, the RAR-RXR pathway, critical for maintaining a balance between self-renewal and differentiation of hematopoietic stem cells, was found to be deregulated in hematopoietic cells from patients with advanced MDS compared to patients with refractory anemia. These findings provide new insights into the understanding of the pathophysiology and progression of MDS, and may guide to new targets for therapy. Taken together with previous published data, the present results also underscore the considerable complexity of the regulation of gene expression in MDS

Regulators of Gut Motility Revealed by a Gnotobiotic Model of Diet-Microbiome Interactions Related to Travel

To understand how different diets, the consumers' gut microbiota, and the enteric nervous system (ENS) interact to regulate gut motility, we developed a gnotobiotic mouse model that mimics short-term dietary changes that happen when humans are traveling to places with different culinary traditions. Studying animals transplanted with the microbiota from humans representing diverse culinary traditions and fed a sequence of diets representing those of all donors, we found that correlations between bacterial species abundances and transit times are diet dependent. However, the levels of unconjugated bile acids-generated by bacterial bile salt hydrolases (BSH)-correlated with faster transit, including during consumption of a Bangladeshi diet. Mice harboring a consortium of sequenced cultured bacterial strains from the Bangladeshi donor's microbiota and fed a Bangladeshi diet revealed that the commonly used cholekinetic spice, turmeric, affects gut motility through a mechanism that reflects bacterial BSH activity and Ret signaling in the ENS. These results demonstrate how a single food ingredient interacts with a functional microbiota trait to regulate host physiology

Occurrence et survie de Listeria monocytogenes au sein de divers types de fromage - Une review

Since the publication of Regulation (EC) N°2073/2005, ready-to-eat (RTE) food allowing the devel-opment of Listeria monocytogenes, including cheese, has to be free of this pathogen in 25 g of pro-duct. This review was carried out to gather studies on the prevalence of the pathogen in varioustypes of cheese in Europe, while also including data from other continents. Given that Regulation(EC) N° 2073/2005 distinguishes cheeses allowing or not the survival of L. monocytogenes basedon the food’s pH and water activity (aw), the review also focuses on the determi nants of thisgrowth/no growth in the same types of cheese