<i>Escherichia coli</i> B2 strains prevalent in inflammatory bowel disease patients have distinct metabolic capabilities that enable colonization of intestinal mucosa

Abstract Background Escherichia coli is considered a leading bacterial trigger of inflammatory bowel disease (IBD). E. coli isolates from IBD patients primarily belong to phylogroup B2. Previous studies have focused on broad comparative genomic analysis of E. coli B2 isolates, and identified virulence factors that allow B2 strains to reside within human intestinal mucosa. Metabolic capabilities of E. coli strains have been shown to be related to their colonization site, but remain unexplored in IBD-associated strains. Results In this study, we utilized pan-genome analysis and genome-scale models (GEMs) of metabolism to study metabolic capabilities of IBD-associated E. coli B2 strains. The study yielded three results: i) Pan-genome analysis of 110 E. coli strains (including 53 isolates from IBD studies) revealed discriminating metabolic genes between B2 strains and other strains; ii) Both comparative genomic analysis and GEMs suggested that B2 strains have an advantage in degrading and utilizing sugars derived from mucus glycan, and iii) GEMs revealed distinct metabolic features in B2 strains that potentially allow them to utilize energy more efficiently. For example, B2 strains lack the enzymes to degrade amadori products, but instead rely on neighboring bacteria to convert these substrates into a more readily usable and potentially less sought after product. Conclusions Taken together, these results suggest that the metabolic capabilities of B2 strains vary significantly from those of other strains, enabling B2 strains to colonize intestinal mucosa.The results from this study motivate a broad experimental assessment of the nutritional effects on E. coli B2 pathophysiology in IBD patients

Metagenomics-Based, Strain-Level Analysis of Escherichia coli From a Time-Series of Microbiome Samples From a Crohn's Disease Patient

Dysbiosis of the gut microbiome, including elevated abundance of putative leading bacterial triggers such as E. coli in inflammatory bowel disease (IBD) patients, is of great interest. To date, most E. coli studies in IBD patients are focused on clinical isolates, overlooking their relative abundances and turnover over time. Metagenomics-based studies, on the other hand, are less focused on strain-level investigations. Here, using recently developed bioinformatic tools, we analyzed the abundance and properties of specific E. coli strains in a Crohns disease (CD) patient longitudinally, while also considering the composition of the entire community over time. In this report, we conducted a pilot study on metagenomic-based, strain-level analysis of a time-series of E. coli strains in a left-sided CD patient, who exhibited sustained levels of E. coli greater than 100X healthy controls. We: (1) mapped out the composition of the gut microbiome over time, particularly the presence of E. coli strains, and found that the abundance and dominance of specific E. coli strains in the community varied over time; (2) performed strain-level de novo assemblies of seven dominant E. coli strains, and illustrated disparity between these strains in both phylogenetic origin and genomic content; (3) observed that strain ST1 (recovered during peak inflammation) is highly similar to known pathogenic AIEC strains NC101 and LF82 in both virulence factors and metabolic functions, while other strains (ST2-ST7) that were collected during more stable states displayed diverse characteristics; (4) isolated, sequenced, experimentally characterized ST1, and confirmed the accuracy of the de novo assembly; and (5) assessed growth capability of ST1 with a newly reconstructed genome-scale metabolic model of the strain, and showed its potential to use substrates found abundantly in the human gut to outcompete other microbes. In conclusion, inflammation status (assessed by the blood C-reactive protein and stool calprotectin) is likely correlated with the abundance of a subgroup of E. coli strains with specific traits. Therefore, strain-level time-series analysis of dominant E. coli strains in a CD patient is highly informative, and motivates a study of a larger cohort of IBD patients

Metagenomics-Based, Strain-Level Analysis of <i>Escherichia coli</i> From a Time-Series of Microbiome Samples From a Crohn's Disease Patient

<p>Dysbiosis of the gut microbiome, including elevated abundance of putative leading bacterial triggers such as E. coli in inflammatory bowel disease (IBD) patients, is of great interest. To date, most E. coli studies in IBD patients are focused on clinical isolates, overlooking their relative abundances and turnover over time. Metagenomics-based studies, on the other hand, are less focused on strain-level investigations. Here, using recently developed bioinformatic tools, we analyzed the abundance and properties of specific E. coli strains in a Crohns disease (CD) patient longitudinally, while also considering the composition of the entire community over time. In this report, we conducted a pilot study on metagenomic-based, strain-level analysis of a time-series of E. coli strains in a left-sided CD patient, who exhibited sustained levels of E. coli greater than 100X healthy controls. We: (1) mapped out the composition of the gut microbiome over time, particularly the presence of E. coli strains, and found that the abundance and dominance of specific E. coli strains in the community varied over time; (2) performed strain-level de novo assemblies of seven dominant E. coli strains, and illustrated disparity between these strains in both phylogenetic origin and genomic content; (3) observed that strain ST1 (recovered during peak inflammation) is highly similar to known pathogenic AIEC strains NC101 and LF82 in both virulence factors and metabolic functions, while other strains (ST2-ST7) that were collected during more stable states displayed diverse characteristics; (4) isolated, sequenced, experimentally characterized ST1, and confirmed the accuracy of the de novo assembly; and (5) assessed growth capability of ST1 with a newly reconstructed genome-scale metabolic model of the strain, and showed its potential to use substrates found abundantly in the human gut to outcompete other microbes. In conclusion, inflammation status (assessed by the blood C-reactive protein and stool calprotectin) is likely correlated with the abundance of a subgroup of E. coli strains with specific traits. Therefore, strain-level time-series analysis of dominant E. coli strains in a CD patient is highly informative, and motivates a study of a larger cohort of IBD patients.</p

Genetic, In Vitro Phenotypic, and Clinical Characterization of Atypical Enteropathogenic E. coli Infection and Pathogenesis

Enteropathogenic E. coli (EPEC) are enteric pathogens that are non-invasive, lack Shiga toxin, and use a type 3 secretion system encoded on the locus of enterocyte effacement (LEE) pathogenicity island to translocate bacterial effector proteins into host intestinal epithelial cells leading to diarrhea. Atypical EPEC (aEPEC), in contrast to typical EPEC (tEPEC), lack bundle-forming pili, likely altering adherence and pathogenicity. Detection of aEPEC with co-infecting pathogens and in some asymptomatic individuals leads to questions regarding aEPEC virulence, especially in adults. I aimed to characterize the clinical manifestations of aEPEC infection and the genetic and in vitro phenotypic factors that contribute to aEPEC virulence. aEPEC are associated with a wide array of symptoms, ranging from asymptomatic carriage to severe diarrhea with up to 10-40 bowel movements/day and persistent/chronic diarrhea in some. Co-infecting pathogens did not alter diarrhea severity. EPEC loads were higher in symptomatic individuals but did not predict diarrhea severity. aEPEC isolates from asymptomatic and symptomatic individuals originated from sporadic infections and diverse lineages. Translocated intimin receptor (Tir), an effector involved in intimate host attachment and actin accumulation under attached bacteria termed pedestals, was a major virulence determinant with Tir subtypes correlating with all examined genetic and in vitro phenotypic virulence factors. Principal component analyses revealed distinct clusters of aEPEC isolates based on virulence determinants. The most virulent aEPEC isolates were characterized by having the greatest homology to tEPEC/EHEC in EspA, an adhesin and needle protein, and the least homology in Tir and other LEE effectors. These isolates also had the greatest number of non-LEE effectors and adhesins, the greatest adherence and pedestal formation on intestinal epithelial cells, and most robust diarrheal symptoms and severity. The least virulent aEPEC isolates had the opposite genetic and phenotypic virulence factors. Those isolates with variable genetic virulence factors correspondingly had variability in phenotypic and clinical manifestations. A subset of aEPEC isolates originating from symptomatic individuals did not fit this trend and likely possess unique pathogenic mechanisms. This is the first study to correlate in vitro phenotypes and clinical manifestations with genetic virulence factors of aEPEC isolates from children and adults in the US

EVALUATION OF ESCHERICHIA COLI SPECIALIZED METABOLITES IMPLICATED IN INFLAMMATORY BOWEL DISEASE COMPLICATIONS

Inflammatory bowel disease (IBD) is defined by painful and chronic intestinal inflammation that can lead to life-threatening complications, including intestinal fibrosis and colorectal cancer (CRC). IBD is a rising global health threat with no cure and limited therapeutic options for affected patients. Gut resident adherent-invasive Escherichia coli (AIEC) are strongly linked to IBD development and are a key target for microbiota-directed IBD therapies. Despite decades of research, there is no genetic definition for AIEC – this suggests it is the functional response of AIEC to the host environment that alters disease development. E. coli impact IBD via production of pro-inflammatory and pro-carcinogenic molecules. Two key E. coli-derived specialized metabolites that drive IBD pathology are yersiniabactin (Ybt) and colibactin. Ybt promotes IBD-associated intestinal fibrosis and colibactin is a CRC-promoting genotoxin. Like many specialized metabolites, the production of Ybt and colibactin is finely tuned based on environmental signals, many of which remain undefined. In my dissertation, I define relevant host factors that regulate production of E. coli specialized metabolites. Further, I evaluate how Ybt and colibactin may drive IBD complications, including intestinal fibrosis and inflammation-associated CRC. Through this body of work, I illustrate some of the many diverse mechanisms for how seemingly innocuous intestinal microbes respond to stress and induce disease. My findings are expected to inform novel microbiota-targeted interventions for IBD and related complications, like intestinal fibrosis and CRC.Doctor of Philosoph

A larval zebrafish (Danio rerio) model of adherent-invasive Escherichia coli infections

Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is a broad term for chronic intestinal disorders that severely impact patient morbidity and quality of life. The global prevalence of IBD is rising, with over one million patients affected in the US alone. Adherent-invasive E. coli (AIEC) is a pathobiont frequently found in IBD biopsies. AIEC adhere to and invade epithelial cells, and can survive inside macrophages in vitro. However, how AIEC contributes to IBD in vivo remains unclear. Here a larval zebrafish (Danio rerio) model of AIEC was established, which facilitates the study of the role of pre-existing inflammation, and host- and pathogen- genetic factors during IBD pathogenesis. Paramecium caudatum, a natural prey of zebrafish larvae, was used as a vehicle for AIEC delivery to the gastrointestinal tract, and dextran sulfate sodium (DSS) pharmacologically induced colitis. AIEC colonized the zebrafish gut in higher numbers and persisted for longer compared to non- pathogenic E. coli in the absence of chronic inflammation. Further, bacterial burden and persistence in the host were higher in fish with pre-existing DSS colitis. The proinflammatory response was further exacerbated by AIEC, resulting in higher neutrophil recruitment to the gut and increased relative expression of the genes that encode proinflammatory cytokines. In addition, we showed that two AIEC virulence factors, FimH and IbeA, play a role in AIEC colonization and contribute to intestinal inflammation in larval zebrafish, similarly to what has been observed in mice. In conclusion, we established a high-throughput, genetically tractable model to study AIEC–host interactions in the context of chronic inflammation

Decoding Gut Microbial Metabolites through G-Protein Coupled Receptor (GPCR) Activation

The microbiome encodes for a complex web of metabolites of which scientists are just starting to deconvolute. While a lot of focus has been on investigating the implications of the microbial metabolome on health and disease physiologies, we have merely uncovered the tip of the interactome of microbes and host G-Protein Coupled Receptors (GPCRs). Early literature has reported a plethora of short chain fatty acids fermented by dietary fibers acting as GPCR agonists. A few other studies have showcased that gut microbes produced N-acyl amides and secondary bile acids mimicking host ligands and therefore interacting with these GPCRs. Chapter 2 and 3 showcases the different strategies to mine GPCR agonists from the commensal microbiota

The interplay between inflammation and microbial activities in colorectal cancer

The microbiota affects host immune health by influencing immune system development and promoting tolerogenic immune responses, effects that have the potential to influence vaccine and cancer immunotherapy efficacy. Disruption of the delicate homeostatic balance between the host and microbiota can lead to intestinal diseases such as inflammatory bowel diseases (IBD) and colorectal cancer (CRC) and also extra-intestinal pathologies such as metabolic syndrome and autoimmune diseases. This dissertation focuses on the impact of the microbiota on host intestinal immune responses in relation to inflammation and carcinogenesis. The aim of the first project was to evaluate the role of the microbiota in modulating systemic neutrophil numbers and function in the developing zebrafish. Using a gnotobiotic approach we demonstrated colonization of germ-free (GF) zebrafish with a conventional microbiota increased neutrophil numbers and myeloperoixidase expression, altered neutrophil localization and migratory behavior and improved neutrophil recruitment to extra-intestinal injury. We showed that neutrophil migratory behavior was mediated through the acute phase response protein serum amyloid A (SAA), which was also induced by the microbiota. In vitro experiments revealed SAA exposure activated nuclear factor (NF)-κB in zebrafish cells, and NF-κB was also required within neutrophils for SAA-dependent migration. The goal of the second project was to evaluate the ability of CRC-associated microbes to induce inflammation and CRC in genetically susceptible gnotobiotic mice. Fusobacterium nucleatum and Escherichia coli that contain the genotoxic island, polyketide synthase (pks) are part of the altered microbiota that is associated with human CRC. We mono-associated ApcMin/+;Il10-/- mice with either F. nucleatum or E. coli and found only pks+ E. coli had the capacity to induce inflammation and tumorigenesis. Next, we examined the functional role of human biofilm associated microbes in CRC development using ApcMin/+;Il10-/- mice. We found that biofilm forming microbes promoted tumorigenesis, suggesting bacterial organization also plays a role in CRC pathogenesis. Taken together these studies stress the importance of balance in host-microbiota interactions. Elucidating host and microbial factors that contribute to disease states has the potential to transform how diseases are prevented and treated.Doctor of Philosoph