Browsing by Author "Rawls, John F"

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The intestine is a multifunctional organ that must perform dichotomous roles in order to maintain health. While it is the primary site of absorption of dietary nutrients, it must also serve as a barrier to both the multitude of microorganisms which reside in the intestinal lumen (the microbiota) and foreign compounds (xenobiotics) which can be toxic to the host. Moreover, the microbiota are required for normal physiology, regulating immunological development, metabolism and behavior. Understanding how the intestine maintains homeostasis and responds to insult in the face of a chemically and microbially complex and dynamic environment is not only a fundamental question of biology, but has important implications for human health. We used zebrafish in order to better understand how the intestine responds to xenobiotics (Chapter 2) and transduces signals from the microbiota to the immune system (Chapter 3).

In Chapter 1, I introduce the complex and reciprocal interactions between xenobiotics, the microbiota, and the host. I highlight examples whereby the microbiota modulates the activity and toxicity of pharmaceuticals, with relevance to diseases of different organ systems. I also describe mechanisms by which the intestine responds to xenobiotic toxicity, and finally advocate for the use of novel model organisms to improve our understanding of these complex interactions.

In Chapter 2, I present our work using the NSAID Glafenine to explore how the intestine responds to xenobiotic challenge. Using transgenic zebrafish and high resolution in vivo imaging, we demonstrate loss epithelial cells in a live animal following xenobiotic challenge. Moreover, Glafenine causes intestinal inflammation, which is potentiated by microbial dysbiosis. We also show that Glafenine can directly alter microbiota composition. Glafenine treatment resulted in activation of the unfolded protein response (UPR), and while pharmacological inhibition of the UPR sensor Ire1a suppressed Glafenine-induced IEC loss, this was associated with increased inflammation and mortality. Ultimately, we demonstrate that Glafenine-induced intestinal toxicity is likely due to off-target inhibition of multidrug resistance (MDR) efflux pumps, as other MDR inhibitors were able to elicit similar phenotypes. Collectively, our findings revealed that (i) MDRs serve an evolutionarily conserved role in maintenance of intestinal homeostasis and (ii) IEC delamination is a protective mechanism which serves to limit inflammation and promote animal survival.

While studies in gnotobiotic mice and zebrafish have demonstrated that the microbiota are required for normal development of the innate immune system, the underlying host and microbial signals which mediate these effects remain largely unknown. We had previously demonstrated that motility of gut commensal bacteria in zebrafish was important for successful colonization of some strains and stimulation of the normal host innate immune response to colonization. In Chapter 3, we describe how microbiota colonization is associated with changes in the PMN transcriptome in addition to promoting systemic abundance and distribution of myeloid cells. Intriguingly, the only pattern recognition receptors found to be differentially expressed in PMNs were the Flagellin receptors tlr5a and tlr5b. Colonization of zebrafish larvae with bacteria lacking Flagellin resulted in attenuated PMN transcriptional activation compared to larvae colonized with isogenic wild type (WT) bacteria. We subsequently demonstrated that direct exposure to purified Flagellin can potently induce transcriptional activation in zebrafish PMNs. These findings identify how the presence of the microbe associated molecular pattern (MAMP) Flagellin serves as a bacterial cue from the microbiota which promotes PMN activation. In Chapter 4, I offer perspectives as to how the Glafenine-zebrafish model system can be used to more deeply investigate host-microbiota-xenobiotic interactions, and genetic, biochemical and computational analyses can help delineate mechanisms by which MDR efflux pumps function in the maintenance of intestinal homeostasis. Moreover, I propose the use of bacterial screens as well as inflammatory and infectious challenge assays in order to better understand the functional outcomes of PMN transcriptional activation elicited by microbiota-derived signals such as Flagellin.

Over the last few decades, there has been an increasingly large body of research focused on the ecology and function of the gut bacteria, collectively known as the gut microbiome. This work has focused on the human gut microbiome as well as other animals including livestock and model organisms that can be genetically and experimentally manipulated. These organisms include laboratory rodents, fruit flies, worms, and pigs, to name a few. The background and future directions of this field are reviewed in Chapter 1 of this thesis. My work in the laboratory of Dr. John Rawls has focused on several aspects of the gut microbiome in different contexts, including how the gut microbiome is affected by nutritional challenges, host diseases, and lifestyle interventions, as well as the factors affecting microbiome composition, which might inform how we can develop strategies to manipulate the microbiome.In Chapter 2, I focus on the question of what genetic factors affect microbiome composition. To do so, I focus on a specific gut microbe known as Phocaeicola (Previously Bacteroides) vulgatus, or Pvu. Pvu is among the most abundant Bacteroidaceae species. Pvu also has myriad health associations in human studies, is an early life colonizer, and an efficient long-term colonizer in both humans and mice. However, the genes required for Pvu to establish itself in a complex microbiome are unknown. To address this gap in knowledge, I present experiments using transposon mutagenesis and insertion sequencing (INSeq) to understand Pvu colonization of the mammalian gut. This reverse genetics approach identifies several potential pathways that Pvu might use to colonize and persist in a complex microbiome. I further elucidate the functions of a hypothetical secreted protein, Pvu777, that is required for competition in vivo in a complex microbiome. In vivo competition experiments using genetically engineered Pvu strains recapitulate these findings in Pvu777 as well as the downstream putative fatty acid transporter Pvu776. Comparative genomics suggests that the operon containing Pvu777, which consists of the predicted DNA Binding/Histone-like protein Pvu778, Pvu777, and Pvu776 may be unique to Pvu and closely related gut Bacteroides and Phocaeicola species. RNA Seq approaches link Pvu777 to outer membrane and envelope functions. In conclusion, we identify a variety of pathways required for Pvu to colonize and persist in a complex microbiome using an INSeq screen, and elucidate the potential functions of one of the genes emerging from this screen using a range of experimental approaches. These findings could be used to inform further fitness-based studies of Pvu, but could also be used to inform methods to control its in vivo abundance, in addition to suggesting mechanisms that could be used to design efficiently colonizing engineered gut bacteria. In Chapter 3, I focus on the question of how nutritional challenges affect the gut microbiome using a zebrafish model of starvation. Starvation is a widespread nutritional challenge for which animals possess many physiological adaptations. However, current research into animal starvation has focused mainly on tissue histopathologies associated with starvation, excluding the physiological changes in the GI tract as well as the gut microbiome. In Chapter 3, we used RNA sequencing and 16S rRNA gene sequencing to uncover changes in the intestinal transcriptome and microbiome of zebrafish subjected to long-term starvation and refeeding compared to continuously fed controls. Starvation over 21 days led to increased diversity and altered composition in the intestinal microbiome compared to fed controls, including relative increases in Vibrio and reductions in Plesiomonas bacteria. Starvation also led to significant alterations in host gene expression in the intestine, with distinct pathways affected at early and late stages of starvation. This included increases in the expression of ribosome biogenesis genes early in starvation, followed by decreased expression of genes involved in antiviral immunity and lipid transport at later stages. These effects of starvation on the host transcriptome and microbiome were almost completely restored within 3 days after refeeding. Comparison with published datasets identified host genes responsive to starvation as well as high-fat feeding or microbiome colonization, and predicted host transcription factors that may be involved in starvation response. Overall, the results presented in Chapter 3 demonstrate that there may be distinct stages of starvation that lead to specific changes in gut microbial ecology and host GI tract transcriptome. These stages of starvation are largely reversible upon refeeding and the ensuing changes in host gene expression and microbiome composition may be an adaptive response to recover from starvation. This work could thus inform future research investigating the roles of specific bacterial taxa in host starvation, as well as mechanistic studies looking at the roles of specific host genes in starvation and refeeding using genetically modified hosts. In Chapter 4, I suggest studies that could extend from the work presented in Chapter 2, focusing on the role of individual genes in the Pvu 777 operon in vivo as well as within Pvu. I also suggest potential roles for the predicted DNA-Binding/Histone-like protein Pvu778 in the regulation of Pvu gene expression and Pvu fitness. I conclude by considering the evolutionary conservation of the 777 operon among Pvu and close relatives, and methods to investigate the fitness requirements of the 777 operon in these related bacterial species as well. Structural studies of Pvu777 are also proposed, which would help clarify the function and potential binding partners for the Pvu777 hypothetical protein. Thus, the studies proposed in Chapter 4 would help provide a clearer picture of the functions, regulation, and evolutionary history of the 777 operon, which would underscore its importance as a potentially conserved operon involved in Pvu fitness in vivo.

The inflammatory bowel diseases (IBD) occur in genetically susceptible individuals that mount inappropriate immune responses to their microbiota leading to chronic intestinal inflammation. The natural history of IBD progression includes early subclinical stages of disease occurring before disease is diagnosed in the clinic. There is evidence in first degree relatives of IBD patients and members of the general population who go on to develop IBD, that these stages are characterized by increased gut barrier permeability, increased levels of inflammation biomarkers, detection of microbiota-specific antibodies in sera, and changes in microbiota composition. Mouse models can be a useful tool in studying disease dynamics during these early stages. The transcription factor Hepatocyte nuclear factor 4 alpha (HNF4A) has been associated with human IBD, and deletion of Hnf4a in intestinal epithelial cells (IEC) in mice (Hnf4aΔIEC) leads to spontaneous colonic inflammation by 6-12 months of age. However, early stages of disease in this mouse model were not well defined, and the role of microbiota in promoting disease was also unclear. Here I tested if pathology in Hnf4aΔIEC mice begins earlier in life and if microbiota contribute to that process, as well as later inflammatory stages of disease. Longitudinal analysis revealed that Hnf4aΔIEC mice reared in specific pathogen-free (SPF) conditions develop episodically elevated fecal lipocalin 2 (Lcn2) and episodic loose stools beginning by 4-5 weeks of age. Lifetime cumulative Lcn2 levels correlated with histopathological features of colitis at 12 months of age. Antibiotic and gnotobiotic tests showed that these phenotypes in Hnf4aΔIEC mice were dependent on microbiota. Fecal 16S rRNA gene sequencing in SPF Hnf4aΔIEC and control mice disclosed that genotype significantly contributed to differences in microbiota composition by 12 months, and longitudinal analysis of the Hnf4aΔIEC mice with the highest lifetime cumulative Lcn2 revealed that microbial community differences emerged early in life when elevated fecal Lcn2 was first detected. These microbiota differences included enrichment of a novel phylogroup of Akkermansia muciniphila in Hnf4aΔIEC mice. I conclude that HNF4A functions in IEC to shape composition of the gut microbiota, and protect against episodic inflammation induced by microbiota throughout the lifespan. Lastly, I discuss future directions for this work, including using single cell RNA sequencing of the colonic epithelium to identify genes regulated by HNF4A in distinct colonic epithelial cell types, gnotobiotic studies using the strain of Akkermansia muciniphila we isolated to test the hypothesis that it can promote disease in Hnf4aΔIEC mice, testing clinically relevant disease triggers using this mouse IBD model, and further immune cell profiling.

Distinct microbial communities colonize diverse vertebrate mucosal surfaces including the intestinal tract. These microorganisms, termed the intestinal microbiota, impact many aspects of host physiology including metabolism, behavior, and immune development. A majority of gut associated microbes are genetically intractable and thus the mechanisms that mediate their colonization and influence on the host remain undefined. To define genetic mechanisms of bacterial colonization and their subsequent impact on host innate immune development and function, we utilized a gnotobiotic zebrafish model. Using germ free zebrafish, devoid of microbiota, compared to zebrafish colonized with either large isogenic mutant pools or complex microbial communities, we identify bacterial mechanisms of colonization (Chapter 2) and mechanisms of microbial control of host innate immune function (Chapter 3).

In Chapter 1, I introduce the intestinal microbiota and experimental strategies to interrogate their influence on host immunity. I highlight mechanisms by which animals recognize microbiota derived signals and products. Further I detail the cellular responses that lead to phenotypic alterations of host epithelial and innate immune cells following microbiota colonization. In Chapter 2 we use gnotobiotic zebrafish to investigate the mechanisms of intestinal colonization of a gut bacterial commensal, Exiguobacterium acetylicum. We performed parallel in vitro and in vivo competitions of large pools of E. acetylicum mutants. These experiments identified several mutations that are differentially enriched specifically in vivo. We also elucidated the ability of different E. acetylicum strains to colonize the zebrafish intestine utilizing high resolution live imaging of labeled strains. Our data indicate that traits, such as motility, are not always the major drivers of successful colonization.

The intestinal microbiota influences the development and function of myeloid lineages such as neutrophils, but the underlying molecular mechanisms are unresolved. In Chapter 3, we identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization in gnotobiotic zebrafish. Saa is a conserved secreted protein produced in the intestine and liver with enigmatic in vivo functions. We engineered saa mutant zebrafish to test requirements for Saa on innate immunity in vivo. Zebrafish mutant for saa displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. Saa’s effects on neutrophils further depend on microbiota colonization, suggesting this protein mediates the microbiota’s effects on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we over-expressed saa in the intestine or liver and found that sufficient to partially complement neutrophil phenotypes observed in saa mutants. These results indicate Saa produced by the intestine in response to microbiota serves as a systemic signal to neutrophils to restrict aberrant activation, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to wounds. These findings identify a host molecular mechanism that promotes innate immune tolerance to the commensal microbiota. In Chapter 4, I offer perspectives as to how Saa may function at the crossroads between host metabolism and immunity. I posit that Saa, and other microbiota-induced host factors from IECs, are the molecular underpinnings of animal-microbiota symbiosis, orchestrating systemic alterations in host metabolism and immune function.

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