Drosophila Gut Research Articles

Bacteria sensing mechanisms in Drosophila gut: Local and systemic consequences

All insects are colonized by microorganisms on their exoskeleton, their gut and even in some cases within their own somatic and germ line cells. This microbiota that can represent up to a few percent of the insect biomass may have a pervasive impact on many aspects of insect biology including physiology, nutrient acquisition, ageing, behaviour and resistance to infection. Mainly through ingestion of contaminated food, the mouth-gut axis represents the first and principal access of external bacteria to the host. Soon after ingestion, the feeding insect needs to rapidly and accurately identify the ingested microbes and decide whether to preserve them if beneficial or neutral, or to eliminate them if potentially harmful. We will review here the recent data acquired in Drosophila on the mechanisms that invertebrate enterocytes rely on to detect the presence of bacteria in the gut. We will compare these modes of bacteria sensing to those in other immune competent tissues and try to rationalize differences that may exist. We will also analyse the physiological consequences of bacteria detection not only locally for the gut itself but also for remote tissues. Finally, we will describe the physiological disorders that can occur due to inaccurate bacteria identification by the gut epithelium.

Roles of septins in prospore membrane morphogenesis and spore wall assembly inSaccharomyces cerevisiae

The highly conserved family of septin proteins has important functions in cytokinesis in mitotically proliferating cells. A different form of cytokinesis occurs during gametogenesis in Saccharomyces cerevisiae, in which four haploid meiotic products become encased by prospore membrane (PSMs) and specialized, stress-resistant spore walls. Septins are known to localize in a series of structures near the growing PSM, but previous studies noted only mild sporulation defects upon septin mutation. We report that directed PSM extension fails in many septin-mutant cells, and, for those that do succeed, walls are abnormal, leading to increased susceptibility to heating, freezing, and digestion by the Drosophila gut. Septin mutants mislocalize the leading-edge protein (LEP) complex required for normal PSM and wall biogenesis, and ectopic expression of the LEP protein Ssp1 perturbs mitotic septin localization and function, suggesting a functional interaction. Strikingly, extra copies of septin CDC10 rescue sporulation and LEP localization in cells lacking Sma1, a phospholipase D-associated protein dispensable for initiation of PSM assembly and PSM curvature but required for PSM extension. These findings point to key septin functions in directing efficient membrane and cell wall synthesis during budding yeast gametogenesis.

Got Lactobacillus? Commensals Power Growth.

Although Lactobacilli are generally considered probiotic agents in metazoans, the underlying molecular mechanisms are largely unknown. In this issue of Cell Host & Microbe, Erkosar et al. (2015) reveal that a Drosophila gut commensal, Lactobacillus plantarum(WJL), promotes animal growth by enhancing the host's capacity for protein degradation.

Genetic, molecular and physiological basis of variation in Drosophila gut immunocompetence.

Gut immunocompetence involves immune, stress and regenerative processes. To investigate the determinants underlying inter-individual variation in gut immunocompetence, we perform enteric infection of 140 Drosophila lines with the entomopathogenic bacterium Pseudomonas entomophila and observe extensive variation in survival. Using genome-wide association analysis, we identify several novel immune modulators. Transcriptional profiling further shows that the intestinal molecular state differs between resistant and susceptible lines, already before infection, with one transcriptional module involving genes linked to reactive oxygen species (ROS) metabolism contributing to this difference. This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines. This study provides novel insights into the determinants underlying population-level variability in gut immunocompetence, revealing how relatively minor, but systematic genetic and transcriptional variation can mediate overt physiological differences that determine enteric infection susceptibility.

P62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB

The selective autophagy receptor p62/sequestosome 1 (SQSTM1) interacts directly with LC3 and is involved in oxidative stress signaling in two ways in mammals. First, p62 is transcriptionally induced upon oxidative stress by the NF-E2-related factor 2 (NRF2) by direct binding to an antioxidant response element in the p62 promoter. Second, p62 accumulation, occurring when autophagy is impaired, leads to increased p62 binding to the NRF2 inhibitor KEAP1, resulting in reduced proteasomal turnover of NRF2. This gives chronic oxidative stress signaling through a feed forward loop. Here, we show that the Drosophila p62/SQSTM1 orthologue, Ref(2)P, interacts directly with DmAtg8a via an LC3-interacting region motif, supporting a role for Ref(2)P in selective autophagy. The ref(2)P promoter also contains a functional antioxidant response element that is directly bound by the NRF2 orthologue, CncC, which can induce ref(2)P expression along with the oxidative stress-associated gene gstD1. However, distinct from the situation in mammals, Ref(2)P does not interact directly with DmKeap1 via a KEAP1-interacting region motif; nor does ectopically expressed Ref(2)P or autophagy deficiency activate the oxidative stress response. Instead, DmAtg8a interacts directly with DmKeap1, and DmKeap1 is removed upon programmed autophagy in Drosophila gut cells. Strikingly, CncC induced increased Atg8a levels and autophagy independent of TFEB/MitF in fat body and larval gut tissues. Thus, these results extend the intimate relationship between oxidative stress-sensing NRF2/CncC transcription factors and autophagy and suggest that NRF2/CncC may regulate autophagic activity in other organisms too.

Bacterial uracil modulates Drosophila DUOX-dependent gut immunity via Hedgehog-induced signaling endosomes.

Genetic studies in Drosophila have demonstrated that generation of microbicidal reactive oxygen species (ROS) through the NADPH dual oxidase (DUOX) is a first line of defense in the gut epithelia. Bacterial uracil acts as DUOX-activating ligand through poorly understood mechanisms. Here, we show that the Hedgehog (Hh) signaling pathway modulates uracil-induced DUOX activation. Uracil-induced Hh signaling is required for intestinal expression of the calcium-dependent cell adhesion molecule Cadherin 99C (Cad99C) and subsequent Cad99C-dependent formation of endosomes. These endosomes play essential roles in uracil-induced ROS production by acting as signaling platforms for PLCβ/PKC/Ca2+-dependent DUOX activation. Animals with impaired Hh signaling exhibit abolished Cad99C-dependent endosome formation and reduced DUOX activity, resulting in high mortality during enteric infection. Importantly, endosome formation, DUOX activation, and normal host survival are restored by genetic reintroduction of Cad99C into enterocytes, demonstrating the important role for Hh signaling in host resistance to enteric infection.

The role of Drosophila antifungal immune response genes in intestinal homeostasis

Abstract The signaling pathways that control intestinal development, regeneration and disease show a high degree of conservation between Drosophila and mammals. The gut epithelia of Drosophila provide protection against invasion of microorganisms through production of reactive oxygen species (ROS) and antimicrobial peptides (AMPs). Although Drosophila gut immunity has been extensively studied, the specific responses to Gram-positive bacteria, fungi and toxic compounds are not fully understood. To identify the physiological role of genes involved in host defense we studied Drosophila mutants in antifungal genes identified previously and tested their survival upon feeding with various pathogens and toxic compounds. The results showed that several mutants displayed decreased viability compared with wild-type flies, and the lower survival rates were attributed to morphological change and excessive cell death in mutant guts. Thus, we identified several new Drosophila genes (spen, jumeau, inv, DDB1 and shg) required for intestinal homeostasis or stress responses.

Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster

Orcokinins are neuropeptides that have been identified in diverse arthropods. In some species, an orcokinin gene encodes two isoforms of mature orcokinin peptide through alternative mRNA splicing. The existence of two orcokinin isoforms was predicted in Drosophila melanogaster as well, but the expression pattern of both isoforms has not been characterized. Here, we use in situ hybridization, antibody staining, and enhancer fusion GAL4 transgenic flies to examine the expression patterns of the A and B forms of orcokinin, and provide evidence that they are expressed differentially in the central nervous system (CNS) and the intestinal enteroendocrine system. The orcokinin A isoform is mainly expressed in the CNS of both larvae and adults. The A form is expressed in 5 pairs of neurons in abdominal neuromeres 1–5 of the larval CNS. In the adult brain, the A form is expressed in one pair of neurons in the posteriorlateral protocerebrum, and an additional four pairs of neurons located near the basement of the accessory medulla. Orcokinin A expression is also observed in two pairs of neurons in the ventral nerve cord (VNC). The orcokinin B form is mainly expressed in intestinal enteroendocrine cells in the larva and adult, with additional expression in one unpaired neuron in the adult abdominal ganglion. Together, our results provide elucidation of the existence and differential expression of the two orcokinin isoforms in the Drosophila brain and gut, setting the stage for future functional studies of orcokinins utilizing the genetically amenable fly model.

A Protocol to Generate Germ Free Drosophila for Microbial Interaction Studies

Drosophila (fly) gut is used as a model system to study the host microbe interaction. Various developmental, physiological, functional and immunological similarities exist between the gut of the vertebrate and the fly. These similarities advocates their utility as a model system for the study of various gut related diseases. Wild type flies found in its natural environment and those raised in the enclosed laboratory conditions have their own natural microbiota colonizing the gut. This natural gut microbiota may interfere with orally administered microbe-Drosophila interaction studies. To overcome this problem various labs are in the process of raising germ free flies. Germ free Drosophila is completely devoid of intestinal microbiota or any kind of microbial colonization. These germ free fly would be helpful as powerful model in exploring the role of a particular microbial strain or a specific microbial genera interacting with the host in isolation either in the mono- or poly-associated state unravelling the complex interplay between bacteria and the host. The current paper describes a protocol to generate a germ free fly for use in microbial interaction studies.

The multilayered innate immune defense of the gut.

In the wild, the fruit fly Drosophila melanogaster thrives on rotten fruit. The digestive tract maintains a powerful gut immune barrier to regulate the ingested microbiota, including entomopathogenic bacteria. This gut immune barrier includes a chitinous peritrophic matrix that isolates the gut contents from the epithelial cells. In addition, the epithelial cells are tightly sealed by septate junctions and can mount an inducible immune response. This local response can be activated by invasive bacteria, or triggered by commensal bacteria in the gut lumen. As with chronic inflammation in mammals, constitutive activation of the gut innate immune response is detrimental to the health of flies. Accordingly, the Drosophila gut innate immune response is tightly regulated to maintain the endogenous microbiota, while preventing infections by pathogenic microorganisms.

In vivo function and comparative genomic analyses of the Drosophila gut microbiota identify candidate symbiosis factors.

Symbiosis is often characterized by co-evolutionary changes in the genomes of the partners involved. An understanding of these changes can provide insight into the nature of the relationship, including the mechanisms that initiate and maintain an association between organisms. In this study we examined the genome sequences of bacteria isolated from the Drosophila melanogaster gut with the objective of identifying genes that are important for function in the host. We compared microbiota isolates with con-specific or closely related bacterial species isolated from non-fly environments. First the phenotype of germ-free Drosophila (axenic flies) was compared to that of flies colonized with specific bacteria (gnotobiotic flies) as a measure of symbiotic function. Non-fly isolates were functionally distinct from bacteria isolated from flies, conferring slower development and an altered nutrient profile in the host, traits known to be microbiota-dependent. Comparative genomic methods were next employed to identify putative symbiosis factors: genes found in bacteria that restore microbiota-dependent traits to gnotobiotic flies, but absent from those that do not. Factors identified include riboflavin synthesis and stress resistance. We also used a phylogenomic approach to identify protein coding genes for which fly-isolate sequences were more similar to each other than to other sequences, reasoning that these genes may have a shared function unique to the fly environment. This method identified genes in Acetobacter species that cluster in two distinct genomic loci: one predicted to be involved in oxidative stress detoxification and another encoding an efflux pump. In summary, we leveraged genomic and in vivo functional comparisons to identify candidate traits that distinguish symbiotic bacteria. These candidates can serve as the basis for further work investigating the genetic requirements of bacteria for function and persistence in the Drosophila gut.

Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut.

Homeostasis of the intestine is maintained by dynamic regulation of a pool of intestinal stem cells. The balance between stem cell self-renewal and differentiation is regulated by the Notch and insulin signaling pathways. Dependence on the insulin pathway places the stem cell pool under nutritional control, allowing gut homeostasis to adapt to environmental conditions. Here we present evidence that miR-305 is required for adaptive homeostasis of the gut. miR-305 regulates the Notch and insulin pathways in the intestinal stem cells. Notably, miR-305 expression in the stem cells is itself under nutritional control via the insulin pathway. This link places regulation of Notch pathway activity under nutritional control. These findings provide a mechanism through which the insulin pathway controls the balance between stem cell self-renewal and differentiation that is required for adaptive homeostasis in the gut in response to changing environmental conditions.

Identification of host defenses in the Drosophila gut using genome-scale RNAi

Identification of host defenses in the Drosophila gut using genome-scale RNAi

Cholera Toxin Disrupts Barrier Function by Inhibiting Exocyst-Mediated Trafficking of Host Proteins to Intestinal Cell Junctions

Cholera toxin (CT), a virulence factor elaborated by Vibrio cholerae, is sufficient to induce the severe diarrhea characteristic of cholera. The enzymatic moiety of CT (CtxA) increases cAMP synthesis in intestinal epithelial cells, leading to chloride ion (Cl(-)) efflux through the CFTR Cl(-) channel. To preserve electroneutrality and osmotic balance, sodium ions and water also flow into the intestinal lumen via a paracellular route. We find that CtxA-driven cAMP increase also inhibits Rab11/exocyst-mediated trafficking of host proteins including E-cadherin and Notch signaling components to cell-cell junctions in Drosophila, human intestinal epithelial cells, and ligated mouse ileal loops, thereby disrupting barrier function. Additionally, CtxA induces junctional damage, weight loss, and dye leakage in the Drosophila gut, contributing to lethality from live V. cholerae infection, all of which can be rescued by Rab11 overexpression. These barrier-disrupting effects of CtxA may act in parallel with Cl(-) secretion to drive the pathophysiology of cholera.

Bacterial-Derived Uracil as a Modulator of Mucosal Immunity and Gut-Microbe Homeostasis in Drosophila

All metazoan guts are subjected to immunologically unique conditions in which an efficient antimicrobial system operates to eliminate pathogens while tolerating symbiotic commensal microbiota. However, the molecular mechanisms controlling this process are only partially understood. Here, we show that bacterial-derived uracil acts as a ligand for dual oxidase (DUOX)-dependent reactive oxygen species generation in Drosophila gut and that the uracil production in bacteria causes inflammation in the gut. The acute and controlled uracil-induced immune response is required for efficient elimination of bacteria, intestinal cell repair, and host survival during infection of nonresident species. Among resident gut microbiota, uracil production is absent in symbionts, allowing harmonious colonization without DUOX activation, whereas uracil release from opportunistic pathobionts provokes chronic inflammation. These results reveal that bacteria with distinct abilities to activate uracil-induced gut inflammation, in terms of intensity and duration, act as critical factors that determine homeostasis or pathogenesis in gut-microbe interactions.

Abstract IA4: Synthetic lethal screens in Drosophila

Abstract During development and homeostasis, cells integrate numerous signals that are either systemic or produced by neighboring cells, informing them of the state of the organism, tissues, and neighboring cells. How cells integrate and interpret these various signals is one of the major focuses of our laboratory. We are particularly interested in identifying nodes of signaling networks that allow the crosstalk between canonical pathways and to study the dynamics of signal integration. I will discuss our current studies on the crosstalk between Insulin and Hippo networks and present our approach using mutant cell lines generated with TALENs to perform synthetic lethal screens. Further, I will present both cell lines and in vivo studies in Drosophila gut stem cells aimed to study the dynamics of signaling pathways activation. Citation Format: Norbert Perrimon. Synthetic lethal screens in Drosophila. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr IA4.

Mechanisms and consequence of bacteria detection by the Drosophila gut epithelium

Since insect mostly developed on decaying matter and contaminated fruits, they are constantly ingesting bacteria. The insect model, Drosophila, is therefore well adapted to study the interactions that take place between the gut epithelia and either resident or infectious bacteria. In order to provide an ad hoc immune response, gut epithelial cells must detect the presence of bacteria. In a recent report, Bosco-Drayon et al. identify the main receptors by which Drosophila sense gut associated bacteria and analyze how this bacteria-receptor interaction translate into gene activation.

Big bang gene modulates gut immune tolerance in Drosophila

Chronic inflammation of the intestine is detrimental to mammals. Similarly, constant activation of the immune response in the gut by the endogenous flora is suspected to be harmful to Drosophila. Therefore, the innate immune response in the gut of Drosophila melanogaster is tightly balanced to simultaneously prevent infections by pathogenic microorganisms and tolerate the endogenous flora. Here we describe the role of the big bang (bbg) gene, encoding multiple membrane-associated PDZ (PSD-95, Discs-large, ZO-1) domain-containing protein isoforms, in the modulation of the gut immune response. We show that in the adult Drosophila midgut, BBG is present at the level of the septate junctions, on the apical side of the enterocytes. In the absence of BBG, these junctions become loose, enabling the intestinal flora to trigger a constitutive activation of the anterior midgut immune response. This chronic epithelial inflammation leads to a reduced lifespan of bbg mutant flies. Clearing the commensal flora by antibiotics prevents the abnormal activation of the gut immune response and restores a normal lifespan. We now provide genetic evidence that Drosophila septate junctions are part of the gut immune barrier, a function that is evolutionarily conserved in mammals. Collectively, our data suggest that septate junctions are required to maintain the subtle balance between immune tolerance and immune response in the Drosophila gut, which represents a powerful model to study inflammatory bowel diseases.

Acanthopanax senticosus extracts have a protective effect on Drosophila gut immunity

Ethnopharmacological relevanceAanthopanax senticosus (A. senticosus) Harms is a classical adaptogenic agent used in China. It has been applied as an analeptic aid to improve weakened physical status. However, little is known about the effects of A. senticosus on inflammatory disease processes. Materials and methodsFlies fed with standard cornmeal–yeast medium were used as controls, and the treatment groups contained 10% of A. senticosus aqueous extracts (root or fruit) in standard medium. Survival rate was performed by feeding a vial containing five layers of filter paper hydrated with 5% sucrose solution contaminated with pathogenic or toxic compounds. Imaging of the guts was viewed under the microscope. Death cells were detected by 7-AAD staining. ResultsThe A. senticosus extract improved the survival rate, attenuated the death of intestinal epithelial cells, promoted the expression of antimicrobial peptide genes, and decreased the formation of melanotic masses. Moreover, our results indicated that the protective effect of fruit is much higher than that of root extracts. ConclusionsA. senticosus extracts have a protective effect on Drosophila gut immunity and stress response, and may contribute to the prevention of inflammatory diseases induced by pathogenic and toxic compounds.

Defining the interorgan communication network: systemic coordination of organismal cellular processes under homeostasis and localized stress

Following the acquisition of multicellularity, organisms with increasing levels of specialized cells, tissues, and organs emerged during evolution. To coordinate specialized organs, long-distance interorgan communication systems appeared. The central nervous system evolved to regulate many organ behaviors, using hormones or neurons. In addition, organs developed systems to directly communicate their states to one another. This is illustrated by the lack of nervous systems in plants and simple animals like sponges, which can perform complex systemic functions (Lough and Lucas, 2006; Srivastava et al., 2010). Developmental or homeostatic events within cells or tissues have been extensively studied. For example, maintenance of the integrity of the Drosophila gut involves stem cell proliferation and differentiation, partially driven by local JAK/STAT, EGF, MAPK, and Wnt signaling (Panayidou and Apidianakis, 2013). Recently, it has become clear that individual organs themselves are also able to communicate their states. However, the nature of the interorgan signaling mechanisms remains largely a mystery. Here, we review the emerging data supporting the existence of a vast interorgan communication network (ICN). The ICN is the network of peptides, proteins, and metabolites that act between organs to coordinate essential and specialized cellular processes under homeostasis and stress (Figure ​(Figure1).1). We propose that studies in Drosophila, where, unlike in mammals, biochemical studies can be combined with genome-wide in vivo tissue-specific genetic screens, are poised to identify many ICN components. Characterization of the ICN will further understanding of systemic diseases such as cancer-associated muscle cachexia. Figure 1 Overview of the interorgan communication network (ICN). The ICN is the network of peptides, proteins, and metabolites that act between organs to coordinate organismal cellular processes under homeostasis and stress. Organs in the body secrete factors ...