abstract: Obesity is a worldwide epidemic accompanied by multiple comorbidities. Bariatric surgery is currently the most efficient treatment for morbid obesity and its comorbidities. The etiology of obesity is unknown, although genetic, environmental, and most recently, microbiome elements have been recognized as contributors to this rising epidemic. The role of the gut microbiome in weight-loss or weight-gain warrants investigation, and bariatric surgery provides a good model to study influences of the microbiome on host metabolism. The underlying goals of my research were to analyze (i) the factors that change the microbiome after bariatric surgery, (ii) the effects of different types of bariatric surgeries on the gut microbiome and metabolism, (iii) the role of the microbiome on the success of bariatric surgery, and (iv) temporal and spatial changes of the microbiome after bariatric surgery. Roux-en-Y gastric bypass (RYGB) rearranges the gastrointestinal tract and reduces gastric acid secretions. Therefore, pH could be one of the factors that change microbiome after RYGB. Using mixed-cultures and co-cultures of species enriched after RYGB, I showed that as small as 0.5 units higher gut pH can aid in the survival of acid-sensitive microorganisms after RYGB and alter gut microbiome function towards the production of weight loss-associated metabolites. By comparing microbiome after two different bariatric surgeries, RYGB and laparoscopic adjustable gastric banding (LAGB), I revealed that gut microbiome structure and metabolism after RYGB are remarkably different than LAGB, and LAGB change microbiome minimally. Given the distinct RYGB alterations to the microbiome, I examined the contribution of the microbiome to weight loss. Analyses revealed that Fusobacterium might lessen the success of RYGB by producing putrescine, which may enhance weight-gain and could serve as biomarker for unsuccessful RYGB. Finally, I showed that RYGB alters the luminal and the mucosal microbiome. Changes in gut microbial metabolic products occur in the short-term and persist over the long-term. Overall, the work in this dissertation provides insight into how the gut microbiome structure and function is altered after bariatric surgery, and how these changes potentially affect the host metabolism. These findings will be helpful in subsequent development of microbiome-based therapeutics to treat obesity. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

The availability of high-throughput sequencing technologies has enabled metagenomicinvestigations into complex bacterial communities with unprecedented resolution andthroughput. The production of dedicated data sets for metagenomic analyses is, however, acostly process and, frequently, the first research questions focus on the study species itself. Ifthe source material is represented by fecal samples, target capture of host-specific sequencesis applied to enrich the complex DNA mixtures contained within a typical fecal DNA extract.Yet, even after this enrichment, the samples still contain a large amount of environmentalDNA that is usually left unanalysed. In my study I investigate the possibility of using shotgunsequencing data that has been subjected to target enrichment for mtDNA from the hostspecies, Grauer’s gorilla (Gorilla beringei graueri), for further analysis of the microbialcommunity present in these samples. The purpose of these analyses is to study the differencesin the bacterial communities present within a high-altitude Grauer’s gorilla, low-altitudeGrauer’s gorilla, and a sympatric chimpanzee population. Additionally, I explore the adaptivepotential of the gut microbiota within these great ape populations.I evaluated the impact that the enrichment process had on the microbial community by usingpre- and post-capture museum preserved samples. In addition to this, I also analysed the effectof two different extraction methods on the bacterial communities.My results show that the relative abundances of the bacterial taxa remain relatively unaffectedby the enrichment process and the extraction methods. The overall number of taxa is,however, reduced by each additional capture round and is not consistent between theextraction methods. This means that both the enrichment and extraction processes introducebiases that require the usage of abundance-based distance measures for biological inferences.Additionally, even if the data cannot be used to study the bacterial communities in anunbiased manner, it provides useful comparative insights for samples that were treated in thesame fashion.With this background, I used museum and fecal samples to perform cluster analysis to explorethe relationships between the gut microbiota of the three great ape populations. I found thatpopulations cluster by species first, and only then group according to habitat. I further foundthat a bacterial taxon that degrades plant matter is enriched in the gut microbiota of all threegreat ape species, where it could help with the digestion of vegetative foods. Another bacterialtaxon that consumes glucose is enriched in the gut microbiota of the low-altitude gorilla andchimpanzee populations, where it could help with the modulation of the host’s mucosalimmune system, and could point to the availability of fruit in the animals diet. In addition, Ifound a bacterial taxon that is linked with diarrhea in humans to be part of the gut microbiotaof the habituated high-altitude gorilla population, which could indicate that this pathogen hasbeen transmitted to the gorillas from their interaction with humans, or it could be indicative ofthe presence of a contaminated water source.

Our gut microbiota is involved in human development, nutrition, and the pathogenesis of gut disorders, but has more recently been implicated as a possible mechanism in the pathophysiology of several brain disorders, including disorders of mood and affect, such as depression. Researchers have referred to this dynamic, bidirectional signaling pathway between the gut and the brain as the “gut-brain axis.” However, most research on this axis has been limited to rodent studies, and there has been little insight into the mechanism behind it. I propose that the kynurenine pathway, where tryptophan is converted to kynurenine, is a compelling mechanism mediating the gut microbiota’s influence on depression. Kynurenine is a metabolite associated with depression, and this pathway has been shown to be manipulated through probiotic (Lactobacillus reuteri) consumption. I propose to study a probiotic intervention in humans, which would assess tryptophan metabolism along the kynurenine pathway by measuring metabolites downstream of this pathway. Urine, feces and blood samples would be collected from two groups, control and probiotic treatment, on day zero and day thirty. Colonic biopsies would be obtained on day thirty, and various analyses would be run to measure metabolite concentrations from the collected samples. The results from this study will help clarify a mechanistic connection between gut microbes and depression via the kynurenine pathway. Additionally, findings could indicate that a probiotic intervention has the ability to influence depressive behavior via a two-pronged approach originating from the kynurenine pathway.

abstract: The Human Gut Microbiome (GM) modulates a variety of structural, metabolic, and protective functions to benefit the host. A few recent studies also support the role of the gut microbiome in the regulation of bone health. The relationship between GM and bone health was analyzed based on the data collected from a group of twenty-three adolescent boys and girls who participated in a controlled feeding study, during which two different doses (0 g/d fiber and 12 g/d fiber) of Soluble Corn Fiber (SCF) were added to their diet. This analysis was performed by predicting measures of Bone Mineral Density (BMD) and Bone Mineral Content (BMC) which are indicators of bone strength, using the GM sequence of proportions of 178 microbes collected from 23 subjects, by building a machine learning regression model. The model developed was evaluated by calculating performance metrics such as Root Mean Squared Error, Pearson’s correlation coefficient, and Spearman’s rank correlation coefficient, using cross-validation. A noticeable correlation was observed between the GM and bone health, and it was observed that the overall prediction correlation was higher with SCF intervention (r ~ 0.51). The genera of microbes that played an important role in this relationship were identified. Eubacterium (g), Bacteroides (g), Megamonas (g), Acetivibrio (g), Faecalibacterium (g), and Paraprevotella (g) were some of the microbes that showed an increase in proportion with SCF intervention. / Dissertation/Thesis / Masters Thesis Electrical Engineering 2020

Hajj, the annual Islamic pilgrimage to Makkah, Saudi Arabia, is a unique mass gathering event that brings more than 2 million individuals from around the world. Several public health considerations, such as the spread of infectious diseases, must be taken into account with this large temporary influx of people. Gastrointestinal diseases, such as diarrhea, are common at Hajj, yet little is known about the etiology. The human gut microbiome, collection of organisms residing within the intestinal tract, has been under intense study recently, since next generation DNA sequencing technologies allow for extensive surveying of genetic material found in complex biological samples, such as those containing many different organisms. Thus, using 16S rRNA and metagenomic shotgun sequencing, we have characterized the gut microbiome of over 612 pilgrims with and without diarrhea. Several metadata factors, such as hospitalization and different comorbidities, were found to have significant effects on the overall gut microbiome composition. Metagenomic shotgun sequencing efforts revealed the presence of antimicrobial resistance genes originating from disparate regions from around the world. This study provides a snapshot of information concerning the health status of the gut microbiome of Hajj pilgrims and provides more context to the investigation of how to best prepare for mass gathering events.

Puberty is a critical period of development characterized by rapid physiological changes and significant brain reorganizing and remodeling. These rapid changes render the developing brain particularly vulnerable to stress and immune challenge. In mice, exposure to an immune challenge (lipopolysaccharide; LPS) during puberty causes enduring effects on stress reactivity, cognitive functioning, and depression- and anxiety-like behaviors later in life. However, the mechanisms underlying these effects are unknown. The gut microbiome can profoundly influence the immune system. There is also close bidirectional communication between the gut microbiome and the central nervous system (CNS) through neural, endocrine and immune signaling pathways, which can alter brain chemistry and emotional behaviour. Thus, we hypothesized that altering microbial composition during puberty could mitigate acute immune responses and prevent enduring outcomes later in life. The current thesis examined the effect of gut manipulation with probiotics during puberty on LPS-induced immune responses and enduring anxiety- and depression-like behaviours, and stress-reactivity in adulthood, in male and female CD1 mice (Article 1). Next, we examined age and sex differences in gut microbial composition before and after exposure to an immune challenge. We also examined the effects of consuming a single strain probiotic bacterium (Lactobacillus Reuteri) during puberty on the immune response and the long-term changes in memory, anxiety-like behavior, and stress reactivity in adulthood (Article 2). Lastly, we examined how microbial colonization between pubertal and adult mice can alter acute peripheral and central inflammatory responses to LPS (Article 3). The current dissertation has addressed sex-specific vulnerabilities to an immune challenge during pubertal development and the moderating influence of the gut microbiome. These studies have demonstrated that manipulating the gut microbiome during puberty can mitigate acute immune responses and prevent enduring neurobehavioural outcomes later in life.

Alterations in composition and function of human gut microbiota can affect physiological processes and are known to be associated with many diseases including inflammatory bowel disease, hypertension, asthma and colon cancer. Complex interactions between gut microbiota, environmental toxins, nutrients and host genetics may result in an increased permeability of the gut, which is closely linked to the presence of adverse metabolic conditions. As a consequence, translocation of bacterial DNA into the blood circulation increases in patients with obesity. Obesity is a growing health problem worldwide and often paired with severe secondary complications, such as type 2 diabetes or cardiovascular problems. A main feature of disease progression is a chronic low-grade inflammation of adipose tissue which contributes to the development and aggravation of insulin resistance and many of the underlying mechanisms are still unknown. Although data from mice studies suggest that the presence of bacterial components in adipose tissue can support these processes, human studies on this subject are lacking. In my thesis entitled ``Bacterial translocation to adipose tissue in metabolic diseas'' I provide evidence supporting the initial hypothesis, namely that bacterial DNA is present in adipose tissue, even after stringent controlling for contaminants. To this end, I established a wet lab routine protocol to eliminate contamination as well as a bioinformatics pipeline accounting for contamination by subtracting negative controls. Briefly, this included the use of lab ware and reagents UV-treated for at least 90 minutes, the use of breath protection, extra-long gloves and single-use lab coats as well as working under a sterile laminar flood hood in a clean lab free of any PCR products. The bioinformatics pipeline employed commonly used 16S rRNA gene analysis tools including qiime2, phyloseq and DESeq2 as well as decontam, a novel tool to extract negative controls. Observed quantity of bacterial DNA was in the range of 1 to 10 pg/µg total isolated genomic DNA, which is equivalent to about 0.01 to 0.7% of bacterial cells per human cell. The highest quantity was present in subcutaneous adipose tissue, followed by mesenteric adipose tissue. Bacterial amount correlated with adipose tissue macrophages and PPARG expression in omental and with IL1B and TNF expression in subcutaneous adipose tissue. Mesenteric adipose tissue showed the highest diversity of the observed genera. The most commonly observed phyla in all tissues were Proteobacteria and Firmicutes, which is in line with previously published data on blood bacterial DNA. Still, many genera were predominantly found in specific tissues, e.g. Enterobacter in subcutaneous and Acinetobacter in omental adipose tissue. I further showed that the distribution of observed features could partially be explained by markers of insulin resistance (HOMA-IR, HbA1c) and inflammation (IL-6, TNFa, macrophages) and that certain genera, such as Rhodoferax or Lactobacillus are associated with type 2 diabetes status. In first functional approaches I demonstrated that concentrations of bacterial DNA in the observed range are sufficient to stimulate an inflammatory response in immortalized subcutaneous adipocytes derived from a healthy donor. The effect was most prominent after four hours of treatment and increased in a dose-dependent matter. One of the aims in the present study was to determine levels of gut leakage by measuring zonulin, the most commonly used biomarker for intestinal permeability in humans, and analyze possible associations with adipose tissue bacterial signature. As there are few well-conducted studies on circulating zonulin levels in patients with metabolic diseases, I first performed a correlation study in the available and metabolically well-characterized Sorbs cohort. Circulating zonulin correlated significantly positive with BMI, fasting glucose, triglycerides and cholesterol and negatively with HOMA-IS, high density lipoprotein and circulating adiponectin levels. Albeit these strong correlations with markers of glucose and lipid metabolism supported previously reported findings, the results pointed to some inconsistencies. As zonulin is reported to be pre-haptoglobin 2 (preHP2), and about 15% percent of a typical western European population are homozygous for haptoglobin 1, they should not express zonulin at all. I confirmed in the Sorbs cohort previously reported distributions of haptoglobin genotypes and showed that the target of the only commercially available zonulin ELISA kit was not related to haptoglobin genotype, therefore presumably not measuring zonulin/ preHP2. Subsequently, I identified properdin as a possible target by employing mass spectrometry approaches. Properdin is structurally related to haptoglobin, as both proteins belong to the mannose-associated serine protease family, however further experiments are needed to validate a possible functional resemblance. In regard to bacterial translocation two adipose tissue depots were of notable interest due to their close proximity to the gastrointestinal tract. Mesenteric adipose tissue is located around the small intestine and the adipose tissue of the appendices epiploicae, small chunks of fat also called epiploic adipose tissue, are directly attached to the colon. After a thorough literature research I could also assert that both adipose tissues were rarely analyzed in the context of obesity. Therefore they were extensively investigated by measuring gene expression of adipo(cyto)kines, circulating inflammatory markers and analyzing adipocyte size and adipose tissue macrophages. Furthermore, a ``multiomics'' characterization was conducted and by analyzing transcriptome and methylome data I could identify epiploic adipose tissue as a tissue of interest in regard to type 2 diabetes and insulin resistance, which was further confirmed by untargeted proteomics data. Contrary to initial assumptions, I observed only a slight increase in translocation of bacterial DNA and no increased inflammation, as measured by tissue specific TNF and IL6 expression as well as adipose tissue macrophage infiltration. However, both transcriptome and proteome profiles allowed a clear discrimination of patients with and without insulin resistance which was most distinct in epiploic adipose tissue. Compared to other fat depots, epiploic adipose tissue exhibited a discriminable metabolic profile whereas mesenteric adipose tissue was more similar to omental-visceral adipose tissue. Most strikingly, epiploic adipose tissue showed a strong increase in leptin expression and, in general, the upregulation of various metabolic pathways involved in sugar, amino acid or sphingolipid metabolism. In accord with the leaky gut hypothesis high expression of lipopolysaccharide binding protein and various pathways involved in chemokine signaling were observed. In summary, I did not observe an increase in bacterial DNA or adipose tissue macrophages, but demonstrated elevated inflammatory signals such as increased chemokine or IL-8 signaling which are linked with an overall increase of metabolic processes and an increased expression of various adipokines. Epiploic adipose tissue might have a watch dog function by being the first adipose tissue sensing and forwarding certain (microbial) stimuli from the large intestine to the host. In the last part of my thesis I addressed a possible role of the HLA genomic region on the development of type 2 diabetes. The influence of HLA genetics on type 2 diabetes has been under debate for several decades, since HLA was recognized to largely contribute to type 1 diabetes heritability. However, studies remained inconclusive due to lacking cohorts with sample sizes providing sufficient statistical power for association analyses. More recently, animal studies suggested MHC class II proteins as crucial factors mediating adipose tissue inflammation and insulin resistance. The sample size of the leaky gut cohort was insufficient to determine any correlation between HLA class II genotypes and the presence or type of bacteria in adipose tissue due to the high variability in the observed genomic region. Yet, I had access to three large population-based cohorts which allowed me to analyze associations between HLA class II alleles and type 2 diabetes. Therefore HLA genotypes of the LIFE-Adult (N=4649), LIFE-Heart (N=4815) and Sorbs (N=949) cohort were imputed from SNP genotyping data and analyzed for association with type 2 diabetes. In a meta-analysis including all three cohorts, I identified a protective effect for the well-established type 1 diabetes protective haplotype DRB1*15:01~DQA1*01:02~DQB1*06:02 and confirmed DRB1*07:01~DQA1*02:01~DQB1*03:03 as a risk haplotype in non-insulin treated diabetes. These results suggest that the genetic foundation of both type 1 and 2 diabetes shares common elements involving the HLA class II locus. In conclusion, to the best of my knowledge, I provide in my work the first contaminant-aware identification of bacterial DNA in human adipose tissue and highlight the importance of analyzing novel adipose tissue depots by showing that fat of the appendices epiploicae, previously only considered to have a cushioning function, is metabolically active and possibly involved in the development of insulin resistance.

The gut microbiome and its metabolic processes are dynamic systems. Surprisingly, our understanding of gut microbiome dynamics is limited. Here we report a metaproteomic workflow that involves protein stable isotope probing (protein-SIP) and identification/quantification of partially labeled peptides. We also developed a package, which we call MetaProfiler, that corrects for false identifications and performs phylogenetic and time series analysis for the study of microbiome dynamics. From the stool sample of five mice that were fed with 15-N hydrolysate from Ralstonia eutropha, we identified 15,297 non-redundant unlabeled peptides of which 10,839 of their heavy counterparts were quantified. These peptides revealed incorporation profiles over time that were different between and within taxa, as well as between and within clusters of orthologous groups (COGs). Our study helps unravel the complex dynamics of protein synthesis and bacterial dynamics in the mouse gut microbiome.

Synthetic biology, a field that sits between Biology and Engineering disciplines, has come into its own in the last decade. The decreasing cost of DNA synthesis has lead to the creation of larger and more complex synthetic gene networks, engineered with functional goals rather than simple demonstration. While many methods have been developed to reduce the time required to produce complex networks, none focus upon the considerable tuning needed to turn structurally correct networks into functional gene networks. To this end, we created a Plug-and-Play synthetic gene network assembly that emphasizes character-driven iteration for producing functional synthetic gene networks. This platform enables post-construction modification and easy tuning of networks through its ability to swap individual parts. To demonstrate this system, we constructed a functional bistable genetic toggle and transformed it into two functionally distinct synthetic networks. Once these networks have been created and tuned at the bench, they next must be delivered to bacteria in their target environment. While this is easy for industrial applications, delivering synthetic networks as medical therapeutics has a host of problems, such as competing microbes, the host immune system, and harsh microenvironments. Therefore, we employed bacteriophage technologies to deliver functional synthetic gene networks to specific bacterial strains in various microenvironments. We first sought to deliver functional genetic networks to bacteria present in the gut microbiome. This allows for functionalization of these bacteria to eventually sense disease states and secrete therapeutics. As a proof of concept a simple circuit was created using the Plug-and-Play platform and tested before being moved into the replicative form plasmid of the M13 bacteriophage. Bacteriophage particles carrying this network were used to infect gut bacteria of mice. Infection and functionality of the synthetic network was monitored from screening fecal samples. Next, we employed phagemid technologies to deliver high copy plasmids expressing antibacterial networks to target bacteria. This allows for sustained expression of antibacterial genes that cause non-lytic bacterial death without reliance upon traditional small molecule antibiotics. Phagemid particles carrying our antibacterial networks were then tested against wild type and antibiotic-resistant bacteria in an in vitro and in vivo environment.

The composition of the gut microbiome can be affected by environmental factors, such as diet. The Western dietary pattern is associated with microbiome dysbiosis and adverse health outcomes, including obesity and metabolic disorders. The objective of this study was to examine the effect of gut microbiota from lean or obese human donors on metabolism and weight gain in recipient mice fed one of three basal diets: 1) the standard AIN93G diet, which promotes rodent health; 2) the total Western diet (TWD), which mimics the American dietary pattern and promotes inflammation-associated colorectal carcinogenesis; and 3) a 45% high fat diet-induced obesity (DIO) diet, which promotes excessive weight gain and symptoms of metabolic syndrome. We hypothesized that fecal microbiome transfer (FMT)from obese human donors would lead to an obese phenotype with symptoms of metabolic syndrome in recipient mice, and that consumption of TWD or DIO diets would further exacerbate the metabolic syndrome phenotype. The experiment design consisted of two main factors: body type of the human donor (obese or lean) and experimental diet (AIN, DIO or TWD), which was fed to mice for 22 weeks. Prior to FMT, the resident gut microbiome in mice was depleted using an established broad spectrum antibiotic/antifugal oral dosing regimen. Interestingly, human donor body type did not significantly affect final body weight or body composition in recipient