Browsing by Subject "Microbiota"

Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli and significant players in the ecology and physiology of lichens. Despite recent interest in this topic, the phylogeny, ecology, and function of these bacteria remain largely unknown. The experiments presented in this dissertation employ culture-free methods to examine the bacteria housed in these unique environments to ultimately inform an assessment of their status with regard to the lichen symbiosis. Microbiotic surveys of lichen thalli using new oligonucleotide-primers targeting the 16S SSU rRNA gene (developed as part of this study to target Bacteria, but exclude sequences derived from chloroplasts and Cyanobacteria) revealed the identity of diverse bacterial associates, including members of an undescribed lineage in the order Rhizobiales (Lichen-Associated Rhizobiales 1; `LAR1'). It is shown that the LAR1 bacterial lineage, uniquely associated with lichen thalli, is widespread among lichens formed by distantly related lichen-forming fungi and is found in lichens collected from the tropics to the arctic. Through extensive molecular cloning of the 16S rRNA gene and 454 16S amplicon sequencing, ecological trends were inferred based on mycobiont, photobiont, and geography. The implications for using lichens as microcosms to study larger principles of ecology and evolution are discussed. In addition to phylogenetic and ecological studies of lichen-associated bacterial communities, this dissertation provides a first assessment of the functions performed by these bacteria within the lichen microbiome in nature through 454 sequencing of two different lichen metatranscriptomes (one from a chlorolichen, Cladonia grayi, and one from a cyanolichen, Peltigera praetextata). Non-photobiont bacterial genes for nitrogen fixation were not detected in the Cladonia thallus (even though transcripts of cyanobacterial nitrogen fixation genes from two different pathways were detected in the cyanolichen thallus), implying that the role of nitrogen fixation in the maintenance of chlorolichens might have previously been overstated. Additionally, bacterial polyol dehydrogenases were found to be expressed in chlorolichen thalli (along with fungal polyol dehydrogenases and kinases from the mycobiont), suggesting the potential for bacteria to begin the process of breaking down the fixed carbon compounds secreted by the photobiont for easier metabolism by the mycobiont. This first look at the group of functional genes expressed at the level of transcription provides initial insights into the symbiotic network of interacting genes within the lichen microbiome.

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.

Despite a largely static DNA sequence, our genomes are incredibly malleable. Comparative studies of chromatin features between different cell types, tissues, and species have revealed tremendous differences in how the genome is accessed, transcribed, and replicated. However, how the dynamics of chromatin accessibility contribute to development, environmental response, and disease status has only begun to be appreciated. In this work we identified chromatin accessibility changes by DNase-seq in three diverse processes: in granule neurons of the developing cerebellum, with intestinal epithelial cells in the absence of a normal microbiota, and with myelogenous leukemia cells in response to histone deacetylase inhibitor treatments. In all cases, we coupled these analyses with RNA-seq assays to identify concurrent transcriptional changes. By mapping the changes to these genome-wide signals we defined the contribution of local chromatin structure to the transcriptional programs underlying these processes, and improved our understanding of their relation to other chromatin changes like histone modifications. Furthermore we demonstrated use of the strongest accessibility changes to identify transcription factors critical for these processes by finding enrichment of their binding motifs. For a few of these key factors, depletion or overexpression of the protein was sufficient to regulate the expression of predicted target genes or exert limited chromatin accessibility changes, demonstrating the functional significance of these proteins in these processes. Together these studies have informed our understanding of the role chromatin accessibility changes play in development and environmental responses while also proving their utility for key regulator identification.

Dendritic cells (DCs) are potent antigen presenting cells (APC) that sense microbes and induce T cell activation and functional differentiation. The APC function of DCs is upregulated by the signaling pathway downstream of the microbial sensing receptor, a process well studied during pathogen infection and immunization. Multiple lines of evidence suggested that DCs in the intestine lamina propria (LP-DCs) frequently interact with the innocuous microbiota, and through these interactions LP-DCs support intestinal immune homeostasis. However, DC responses to microbiota, if not regulated, can give rise to inflammatory T cells and trigger inflammatory bowel disease (IBD). The DC subsets, DC functions and signaling pathways that induce inflammatory T cells remain incompletely characterized. Here, we demonstrated that mice lacking signaling attenuator A20 (A20cko mice) in DCs develop spontaneous small intestine inflammation that is dependent of microbiota, DCs and T cells. LP-DCs induce inflammatory T cells and that the signals perceived and APC functions are unique for three distinct LP-DC subsets. Thus, while CD103+CD11b- DCs exclusively upregulate their ability to instruct IFNγ+ T cells, CD103+CD11b+ DCs exclusively upregulate their ability to instruct IL-17+ T cells. Of note, APC functions of both DC subsets are upregulated in a MyD88-independent fashion. In contrast, CD103-CD11b+ DCs instruct both IFNγ+ and IL-17+ T cells, and only the IL-17-inducing APC functions require MyD88. In disease pathogenesis, both CD103-CD11b+ and CD103+CD11b+ DCs expand pathologic Th17 cells.

Although MyD88 pathways are potent inducer of intestinal inflammation in the colitis of IL-10 knockout mice and upon transferring of naïve T cells into Rag-deficient hosts, MyD88 pathways are not required for the inflammation of small intestine in A20cko mice. Among the MyD88-independent signaling pathways that could mediate host interaction with microbiota, Dectin-1 pathway is of particular interest because both the receptor Dectin-1 and the downstream signaling molecule CARD9 are IBD-associated genes. Additionally, the defect in either molecule influences the severity of the intestinal inflammation in mouse. We established that the production of inflammatory cytokines downstream of the Dectin-1 pathway is restricted by A20. Mechanistically, A20 inhibits TRAF6 ubiquitination downstream of the Dectin-1 pathway, thereby controlling NFκB and Jnk activation. Although we showed that CD103-CD11b+ and CD103+CD11b+ DCs express Dectin-1 and CARD9, the Dectin-1 pathway is not required for the upregulation of DC function and expansion of inflammatory T cells in the intestine of A20cko mice. Thus, our studies have unveiled a critical role of MyD88-independent pathways in mediating the interaction of the microbiota and LP-DCs. MyD88-independent pathway is capable of driving functional maturation of LP-DCs, pathological expansion of CD4 T cells, and the inflammatory disease in the small intestine.