Chrysoula Pitsouli

Drosophila is an important model organism for studying epithelial barrier pathogenesis by Pseudomonas aeruginosa because it allows clinically important infectivity to be mimicked. Here, we present a Protocol Extension to introduce P. aeruginosa and other bacteria into the adult Drosophila intestine by feeding, further to our previously published protocol that used the needle-pricking method to impose a wound infection and the injector-pumping method of bacterial delivery directly into the fly hemocoel. We start with Drosophila preparation and priming for feeding, in parallel with bacterial growth and infection mix preparation. We continue with the feeding setup followed by the output measurements, which include, but are not limited to, fly survival, bacterial load and systemic spread, intestinal regeneration and gene expression. The protocol can be used to infect Drosophila with tens of different clinically important and insect-related bacterial species, one at a time or in combination; modify the inoculum consistency to control and even eliminate P. aeruginosa virulence; measure bacterial load in the hemolymph; assess intestinal histopathology of tumor-prone flies; and assess the host transcriptome. Preparation of adult flies for feeding lasts ≤1 week, and infection mixes can be prepared in 2 d, which require a minimum level of expertise in fly handling and microbiological techniques, respectively. All output measurements usually take ≤10 d or until all flies die from a virulent infection. Fly survival and bacterial load assessment require a short training, while intestinal histopathology and host gene expression assessment need up to a month of systematic training. Assessing bacterial load and gut measurements require 2 d of work per infection time point considered.

Dysbiosis, the imbalance of an organism’s commensal microbiota, may drive dysplasia and tumorigenesis via aberrant immune response, increased inflammation, and stem cell overactivation. The Drosophila gut is a useful model to study host-microbiota interactions leading to tumorigenesis due to its molecular, cellular, and physiological similarity with the human gut and its simplicity at the host and microbiome levels. A primary lesson learned from Drosophila gut studies is that microbiota and pathogenic microbes, such as bacteria and viruses, induce multiple innate immune and stress signaling pathways in enterocytes and other gut cells that, in turn, activate mitogens driving regenerative stem cell proliferation. A second important lesson learned from Drosophila gut studies is that wild-type and genetically predisposed intestinal epithelial cells are more likely to initiate tumorigenesis and tissue invasion upon interaction with intestinal microbes. Lastly, microbial nutrients may directly affect intestinal stem cell proliferation and tissue regeneration. This review discusses the mechanisms by which the microbiome influences directly and indirectly stem cell proliferation, tumorigenesis, and tumor cell dissemination, shedding light on microbiota-mediated cancer predisposition.

The intestine is populated by a complex and dynamic assortment of microbes, collectively called gut microbiota, that interact with the host and contribute to its metabolism and physiology. Diet is considered a key regulator of intestinal microbiota, as ingested nutrients interact with and shape the resident microbiota composition. Furthermore, recent studies underscore the interplay of dietary and microbiota‐derived nutrients, which directly impinge on intestinal stem cells regulating their turnover to ensure a healthy gut barrier. Although advanced sequencing methodologies have allowed the characterization of the human gut microbiome, mechanistic studies assessing diet–microbiota–host interactions depend on the use of genetically tractable models, such as Drosophila melanogaster. In this review, we first discuss the similarities between the human and fly intestines and then we focus on the effects of diet and microbiota on nutrient‐sensing signaling cascades controlling intestinal stem cell self‐renewal and differentiation, as well as disease. Finally, we underline the use of the Drosophila model in assessing the role of microbiota in gut‐related pathologies and in understanding the mechanisms that mediate different whole‐body manifestations of gut dysfunction.

Cholesterol is necessary for all cells to function. The intracellular cholesterol transporters Npc1 and Npc2 control sterol trafficking and their malfunction leads to Neimann–Pick Type C disease, a rare disorder affecting the nervous system and the intestine. Unlike humans that encode single Npc1 and Npc2 transporters, flies encompass two Npc1 (Npc1a-1b) and eight Npc2 (Npc2a-2h) members, and most of the Npc2 family genes remain unexplored. Here, we focus on the intestinal function of Npc2c in the adult. We find that Npc2c is necessary for intestinal stem cell (ISC) mitosis, maintenance of the ISC lineage, survival upon pathogenic infection, as well as tumor growth. Impaired mitosis of Npc2c-silenced midguts is accompanied by reduced expression of Cyclin genes, and genes encoding ISC regulators, such as Delta, unpaired1 and Socs36E. ISC-specific Npc2c silencing induces Attacin-A expression, a phenotype reminiscent of Gram-negative bacteria overabundance. Metagenomic analysis of Npc2c-depleted midguts indicates intestinal dysbiosis, whereby decreased commensal complexity is accompanied by increased gamma-proteobacteria. ISC-specific Npc2c silencing also results in increased cholesterol aggregation. Interestingly, administration of the non-steroidal ecdysone receptor agonist, RH5849, rescues mitosis of Npc2c-silenced midguts and increases expression of the ecdysone response gene Broad, underscoring the role of Npc2c and sterols in ecdysone signaling. Assessment of additional Npc2 family members indicates potential redundant roles with Npc2c in ISC control and response to ecdysone signaling. Our results highlight a previously unidentified essential role of Npc2c in ISC mitosis, as well as an important role in ecdysone signaling and microbiome composition in the Drosophila midgut.

Host-microbiota interactions are key modulators of host physiology and behavior. Accumulating evidence suggests that the complex interplay between microbiota, diet and the intestine controls host health. Great emphasis has been given on how gut microbes have evolved to harvest energy from the diet to control energy balance, host metabolism and fitness. In addition, many metabolites essential for intestinal homeostasis are mainly derived from gut microbiota and can alleviate nutritional imbalances. However, due to the high complexity of the system, the molecular mechanisms that control host-microbiota mutualism, as well as whether and how microbiota affects host intestinal stem cells (ISCs) remain elusive. Drosophila encompasses a low complexity intestinal microbiome and has recently emerged as a system that might uncover evolutionarily conserved mechanisms of microbiota-derived nutrient ISC regulation. Here, we review recent studies using the Drosophila model that directly link microbiota-derived metabolites and ISC function. This research field provides exciting perspectives for putative future treatments of ISC-related diseases based on monitoring and manipulating intestinal microbiota.

The Drosophila trachea, as the functional equivalent of mammalian blood vessels, senses hypoxia and oxygenates the body. Here, we show that the adult intestinal tracheae are dynamic and respond to enteric infection, oxidative agents and tumours with increased terminal branching. Increased tracheation is necessary for efficient damage-induced intestinal stem cell (ISC)-mediated regeneration and is sufficient to drive ISC proliferation in undamaged intestines. Gut damage or tumours induce HIF-1α (Sima in Drosophila), which stimulates tracheole branching via the FGF (Branchless (Bnl))–FGFR (Breathless (Btl)) signalling cascade. Bnl–Btl signalling is required in the intestinal epithelium and the trachea for efficient damage-induced tracheal remodelling and ISC proliferation. Chemical or Pseudomonas-generated reactive oxygen species directly affect the trachea and are necessary for branching and intestinal regeneration. Similarly, tracheole branching and the resulting increase in oxygenation are essential for intestinal tumour growth. We have identified a mechanism of tracheal–intestinal tissue communication, whereby damage and tumours induce neo-tracheogenesis in Drosophila, a process reminiscent of cancer-induced neoangiogenesis in mammals.

The Drosophila tracheal system, as the functional equivalent of mammalian blood vessels, responds to hypoxia and transports oxygen throughout the body. Although the signaling pathways involved in tracheal development and the hypoxic response are well-studied, how adult tracheae interact with their target tissues is largely unknown. Here we show that the tracheae that serve the adult intestine are dynamic and respond to enteric infection, oxidative agents, and the development of gut tumors with increased terminal branching. Increased tracheation is necessary for efficient damage-induced intestinal stem cell (ISC)-mediated midgut regeneration and sufficient to drive ISC proliferation in the absence of damage. Gut damage or tumors induce Hif-1α/Sima, which, in turn, stimulates tracheole branching via the FGF(Brachless/Bnl)/FGFR(Breathless/Btl) signaling cascade. Bnl/Btl signaling is required both in the intestinal epithelium and the tracheal system for efficient damage-induced tracheal remodeling and ISC proliferation. We show that chemical or Pseudomonas- generated ROS directly affect the trachea and are necessary for branching and intestinal regeneration. Similarly, tracheole branching and the resulting increase in oxygen supply are essential for tumor growth in the midgut. Thus, we have identified a novel mechanism of visceral tracheal-intestinal tissue communication, whereby oxidative damage and tumors induce neo-tracheogenesis in adult Drosophila. This process is reminiscent of cancer-induced neo-angiogenesis in mammals.

Systemic and stem cell niche-emanating cytokines and growth factors can promote regeneration, through mitosis. High mitosis, however, predisposes for all types of cancer and, thus, a trade-off exists between regeneration capacity and tissue homeostasis. Here, we study the role of tissue-intrinsic regenerative signaling in stem cell mitosis of adult Drosophila midgut of different genetic backgrounds. We provide evidence of two naturally occurring types of balance between mitosis and enterocyte nucleus growth: one based mostly on stem cell mitosis producing new cells and the other based mostly on the degree of young enterocyte nucleus size increase. Mitosis promotes intestinal host defense to infection, but predisposes for dysplasia in the form of stem cell-like clusters. Enterocyte nucleus growth also promotes host defense, without the drawback of promoting dysplasia. Through quantitative genetics, we identified eiger as an autocrine and paracrine inducer of stem cell mitosis. eiger expression in immature epithelial cells tilts the balance towards mitosis and dysplasia via a positive-feedback loop of highly mitotic stem cells sustaining more small nucleus enterocytes, which in turn supply more Eiger.

Physiology, fitness and disease phenotypes are complex traits exhibiting continuous variation in natural populations. To understand complex trait gene functions transgenic lines of undefined genetic background are often combined to assess quantitative phenotypes ignoring the impact of genetic polymorphisms. Here, we used inbred wild-type strains of the Drosophila Genetics Reference Panel to assess the phenotypic variation of six physiological and fitness traits, namely, female fecundity, survival and intestinal mitosis upon oral infection, defecation rate and fecal pH upon oral infection, and terminal tracheal cell branching in hypoxia. We found continuous variation in the approximately 150 strains tested for each trait, with extreme values differing by more than four standard deviations for all traits. In addition, we assessed the effects of commonly used DrosophilaUAS-RNAi transgenic strains and their backcrossed isogenized counterparts, in the same traits plus baseline intestinal mitosis and tracheal branching in normoxia, in heterozygous conditions, when only half of the genetic background was different among strains. We tested 20 non-isogenic strains (10 KK and 10 GD) from the Vienna Drosophila Resource Center and their isogenized counterparts without Gal4 induction. Survival upon infection and female fecundity exhibited differences in 50% and 40% of the tested isogenic vs. non-isogenic pairs, respectively, whereas all other traits were affected in only 10-25% of the cases. When 11 isogenic and their corresponding non-isogenic UAS-RNAi lines were expressed ubiquitously with Gal4, 4 isogenic vs. non-isogenic pairs exhibited differences in survival to infection. Furthermore, when a single UAS-RNAi line was crossed with the same Gal4 transgene inserted in different genetic backgrounds, the quantitative variations observed were unpredictable on the basis of pure line performance. Thus, irrespective of the trait of interest, the genetic background of commonly used transgenic strains needs to be considered carefully during experimentation.