Regulation of digestive tract and progenitor cell homeostasis by intestinal bacteria in Drosophila melanogaster

• Author / Creator

  Fast, David

• The digestive tract facilitates nutrient uptake in the presence of a heterogenous cohort of symbiotic microbes. These microbes along with their collective genetic material form the intestinal microbiome and contribute to animal phenotypes. Similar to mammals, the microbiome of insects forms a barrier that rebuffs invasive bacteria and activates the intestinal immune response. In the fruit fly, Drosophila melanogaster, intestinal immunity couples the bactericidal action of antimicrobial peptides and reactive oxygen species with epithelial repair programs to effectively eliminate toxic bacteria and regenerate the epithelium. Intestinal renewal is accomplished by the proliferation of multipotent intestinal stem cells (ISCs), which divide and differentiate to generate new epithelial cells. Signalling through a complex network of conserved mitogenic pathways regulates ISC division. Epithelial damage, ingestion of cytotoxic compounds, or the presence of gut bacteria activates these pathways in ISCs to stimulate proliferation. Conventionally reared (CR) flies, which host a normal intestinal microbiome, overtime accumulate a population of miss-differentiated cells that leads to the gradual onset of intestinal tissue dysplasia via activation of ISC proliferation. Removal of the microbiome to generate a germ free (GF) organism slows the frequency of epithelial turnover, preserves tissue organization, and extends adult fly lifespan. To explore the relationship between symbiotic bacteria and Drosophila longevity, I examined the contributions of individual symbiotic species to adult fly lifespan. Individual populations of GF flies were re-associated with monocultures of bacteria to repopulate the intestine with a single symbiotic species. Association with the widely reported fly commensal Lactobacillus plantarum (L. plantarum) shortened the lifespan of GF flies. Contrary to expectations, L. plantarum monoassociation did not promote the rapid onset of tissue dysplasia characteristic of aged CR flies. Instead, guts associated with L. plantarum had diminished expression of growth promoting ligands and reduced epithelial turnover, characterized by a disruption to posterior midgut architecture.

  In addition to symbiotic bacteria, the intestine is frequently exposed to pathogens that compete with the microbiome within the digestive tract. One mechanism employed by enteric pathogens to compete with other bacteria in the niche is the type VI secretion system (T6SS). The T6SS of V. cholerae and other Gram-negative bacteria is an injection apparatus that translocates toxic effector molecules into adjacent prokaryotic or eukaryotic cells. As the T6SS is active in vivo and mediates interactions with other bacterial cells, I examined the contribution of the T6SS to V. cholerae pathogenesis in the guts of CR adult Drosophila. I demonstrated that ablation of T6SS function extends the viability of flies infected with V. cholerae, relative to infection with T6SS functional Vibrio. T6SS mediated reduction in viability was dependent on the microbiome as the T6SS was dispensable for V. cholerae pathogenesis in a GF host. The reintroduction of symbionts vulnerable to T6SS mediated competition, sensitized the host to T6SS killing via the activation of putative host secondary responses. Given the effect of T6SS mediated interactions on host viability, I examined how these bacteria-bacteria interactions impact intestinal immune responses.
  Loss of damaged epithelial cells is complemented by the proliferation of ISCs. However, despite significant intestinal damage, infection with T6SS functional V. cholerae did not activate compensatory ISC growth. Instead, challenge with T6SS functional V. cholerae impaired proliferation and downregulated the transcription of signaling components required for epithelial renewal. T6SS-dependent arrest of intestinal repair was the result of interactions between the microbiome and the T6SS, as ablation of the microbiome restored epithelial regeneration in response to T6SS functional V. cholerae. This inhibition of renewal was not the result of a bilateral interaction between V. cholerae and a single symbiotic species, but required interactions between V. cholerae and a multi-species consortium of intestinal symbionts. Together, the findings in this thesis define the impact of individual species on intestinal homeostasis and examine how interactions between bacteria in the digestive tract influence host viability and intestinal regeneration.

• Subjects / Keywords

  • Bacteria
  • progenitor cells
  • Microbiome
  • Drosophila
  • T6SS

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-4btv-tb51

• License

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