The role of Drosophila microbiota in gut homeostasis and immunity

ABSTRACT

The gut epithelia of virtually all animals harbor complex microbial communities that play an important role in maintaining immune and cellular homeostasis. Gut microbiota have evolutionarily adapted to the host gut environment, serving as key regulators of intestinal stem cells to promote a healthy gut barrier and modulate epithelial self-renewal. Disruption of these populations has been associated with inflammatory disorders or cancerous lesions of the intestine. However, the molecular mechanisms controlling gut-microbe interactions are only partially understood due to the high diversity and biologically dynamic nature of these microorganisms. This article reviews the current knowledge on Drosophila gut microbiota and its role in signaling pathways that are crucial for the induction of distinct homeostatic and immune responses. Thanks to the genetic tractability of Drosophila and its cultivable and simple microbiota, this association model offers new efficient tools for investigating the crosstalk between a host and its microbiota while providing a framework for a better understanding of the ecological and evolutionary roles of the microbiome.

KEYWORDS:

• Drosophila
• innate immunity
• gut microbiota
• gut immunity
• gut homeostasis

Introduction

Microorganisms perform a variety of biogeochemical functions ranging from waste decomposition, nutrient cycling, nitrogen fixation, and antibiotic production, which are crucial for robust ecosystems.^1–4 They are a major driving force behind evolution and ecology, forming abundant and diverse microbial communities termed as “microbiome/s” that impact the organismal phenotype of their hosts.^5,6 Microbiomes contribute to nearly every aspect of animal biology including pathogen susceptibility and autoimmunity, adaptation to environmental stresses, range expansion, and metabolism.⁷ For instance, molecular advances show that numerous marine invertebrates require transient microbe-derived chemical cues, often associated with biofilms, to proceed from one developmental stage to the next.^8–10 Additionally, bacteria that shelter in the human gut and intestines assist in digesting endogenous and exogenous carbohydrates, provide vitamins, and maintain immune homeostasis.¹¹ The precise ecological niche of a microbiome is largely dependent on multiple factors including specific interactions among host genotype, diet, and environment.¹² Variations in these factors alter the composition and diversity of the microbiome, which are closely associated with host growth, reproduction, and fitness. Owing to this far-reaching significance, scientists have come to recognize the role of coevolved microbes in the health and disease of eukaryotic hosts, thereby shifting the focus of research to examine the unique complexities of microbiomes and characterize their distributions, abundances, and functional traits.

Recent research has provided new possibilities to investigate microbial communities and address the association between a host and its microbiome. All microorganisms were initially considered harmful external pathogens causing disease to the host and triggering immune responses to eliminate them. However, using advanced molecular tools such as next generation sequencing, previous work has shown that the association between the host and its microbiome can take many forms that may include symbiotic interactions.^13,14 Symbiotic microbiomes, often referred to as “microbiota,” coevolve with their hosts over millennia toward mutualism and homeostasis, establishing intimate relationships that require the proper functioning of host immunity to prevent microbes from over-exploiting host resources while modulating immune tolerance to innocuous stimuli.¹¹ In many animals, the most complex and diverse populations of microbes reside in the gut, a fundamental interface for development, nutrient acquisition, and immune recognition.¹⁵ Gut microorganisms, which include a core microbiota of consistently associated bacteria, are reported to transmit distinct signals that affect functions of both the innate and adaptive immune systems and often result in systemic outcomes that are crucial for various biological processes of their hosts.^16,17 Alterations of these gut microbial communities can cause immune dysregulation, leading to autoimmune disorders and ultimately driving mortality.¹⁵ Hence, characterizing the cellular and molecular interactions of gut microbiota and the host immune system will not only help understand autoimmune diseases but will also provide a new foundation for the design of novel immuno- and microbe-based therapeutics that have tremendous potential to improve human health.

A major challenge in mechanistic studies addressing the role of the microbiota in host immunity is to identify the contribution of individual bacterial microbes and interactions among species in shaping microbe-dependent traits and host fitness. The mammalian gut microbiome, for example, can vary greatly between individuals and is composed of approximately 1,000 species of microorganisms.¹⁸

Previous work has demonstrated that the composition and function of gut microbiota depend mainly on the degree of functional modularity of the microbiota.¹⁹ Some gut microbiota-dependent traits are mediated by bacterial species acting independently, such that the microbiota is functionally modular, while other functions are the product of interactions among several bacterial species.¹⁹ A powerful route to decipher these relationships and investigate the degree of functional modularity of gut microbiota is provided by gnotobiotic animals, especially insect models such as the fruit fly Drosophila melanogaster.¹³ These are animals that are experimentally colonized by known sets of microorganisms to allow studies of single-species, dual-species, and multispecies microbiota to unambiguously parse their specific contributions to host immunity.^20,21 Unlike gnotobiotic mammals, Drosophila model systems lack the complexity and diversity of associations with microorganisms, making it possible to examine the contribution of specific bacteria and the entire microbiome toward host physiological and immunological processes. This review summarizes the current knowledge of (1) Drosophila gut anatomy and compartmentalization, (2) the adult microbiota composition and its transient nature, and (3) the role of gut microbiota in immunity and intestinal tissue homeostasis which modulates host fitness and lifespan.

Drosophila gut anatomy

Drosophila is a valuable experimental model that is amenable to different types of in-depth studies ranging from classic genetics to large-scale systems approaches.^22–24 The short generation times of fruit flies, their relative ease of manipulation, and advanced molecular tools allow researchers to unravel the specific mechanisms by which gut microorganisms influence the fitness, health, and behavior of their hosts, thereby making host-gut microbiota interactions a more navigable field. In addition, the Drosophila gut has striking similarities to the human gastrointestinal tract in both structure and function, with studies showing a high degree of conserved signaling pathways for gut damage repairs and protection against diseases.^25,26 The Drosophila gut, which consists of a long tube lined by an epithelial monolayer, is divided into three discrete domains of different developmental origins, the foregut, midgut, and hindgut (Figures 1).²⁷ Both anatomical specializations and regional compartmentalization of the gut enable sequential ingestion, storage, digestion, absorption, and defecation. The pharynx, esophagus, and crop, a structure used for food storage prior to digestion, are all parts of the foregut, which is of ectodermal origin. At the foregut/midgut junction, a complex bulb-shaped organ termed the cardia (proventriculus) functions as a gastric valve to control food entry into the midgut and produces the peritrophic matrix, a major site of antimicrobial peptide (AMP) synthesis.²⁸ The midgut is derived from the endoderm and acts as the entry site not only for nutrients like food and water, but also for pathogens, such as harmful bacteria, viruses, and toxins. The hindgut and Malpighian tubules, which form close to the junction between the hindgut and posterior midgut, are ectodermal in origin. Together, they form the functional analogs of the mammalian kidney, regulating water and ion homeostasis over a wide range of external or dietary osmolarities.²⁹

Figure 1. Drosophila gut structure. (A) Adult Drosophila digestive tract (lateral). (B) Anatomical structure of adult Drosophila digestive tract. (C) General composition and cell types of adult Drosophila midgut.

In contrast to the ectodermally-derived regions of the intestinal epithelium, which remain relatively poorly understood, the midgut has been characterized in extensive detail in recent years. The adult Drosophila midgut, one of the largest fly organs, consists of a simple epithelium, surrounded by visceral muscles, nerves, and trachea (Figure 1c). The epithelium is maintained by the proliferative activity of pluripotent intestinal stem cells (ISCs) that ensure the control of remarkably rapid cell renewal to replace dead or damaged cells.³⁰ ISCs divide and generate enteroblasts (EBs), which are postmitotic, immature cells that gradually differentiate into either absorptive enterocytes (ECs) or secretory enteroendocrine (EE) cells (Figure 1c).^28,31 Anatomical and patterning studies have long distinguished the adult midgut into three segments, the anterior, middle, and posterior, based on specific histological and cellular features, stem cell proliferation rates, physical properties (as inferred from longitudinal gradients of pH dye-indicators in the lumen), and gene expression profiles. The anterior portion releases digestive enzymes to break down food entering from the cardia.³² The middle midgut has an acidic pH (<4.0) and is itself tripartite in organization. It holds specialized enterocytes known as interstitial cells, interspersed with morphologically distinctive “copper cells” that act similarly to the vertebrate gastric parietal cells in that they both secrete acid.³³ The posterior midgut epithelium has been best studied to investigate self-renewing and multipotent stem cell functions due to its physiological equivalence to the human small intestine.^31,32 Although this rudimentary compartmentalization of the Drosophila midgut is widely used, other studies have suggested more complex regionalization based on the multifaceted molecular mechanisms underlying the regenerative and functional activities of these subdivisions.^27,28,30,32 This calls for further studies to better understand the maintenance and plasticity of midgut compartmentalization in the adult. Nevertheless, the simple cell lineage of Drosophila gut cells and availability of abundant mutants make it an ideal model system for studying gut infections and disorders, as well as gut microbiota-mediated homeostasis and immunity.

Drosophila gut microbiota

Drosophila has recently come to the forefront of gut microbiome function and immunity research fields due to the simplicity of its microbiota, resemblance to mammal systems, and resourceful methods to rear germ-free flies.³⁴ In addition to endosymbionts, the fruit fly harbors a core gut microbiota that is promoted by positive interactions (commensalism (+/0), mutualism (+/+)) among microorganisms, favoring shared persistence. The key difference between endosymbionts and microbiomes is in their mode of transmission. Endosymbionts are transferred via a maternal transmission with high fidelity whereas the broader microbiome does not rely much on maternal transmission and is transferred via environmental factors.^35–37 Drosophila microbiota can be passed to offspring through contamination of eggshells, which are ingested by larvae after hatching.³⁸ The microbiota counts increase until the larvae reach the third-instar developmental stage, after which they diminish because of the increased expression of several AMP genes at the pupal stage. Newly eclosed adult flies thereby have very few living microbes in the gut. However, the gut microbiota shifts as flies age toward increased abundance and variability in composition.³⁸ Such changes are promoted with ingested microorganisms that are routinely released back to the external environment via feces, sloughed skin, and fluid secretions, which serve as a reservoir for the microbiota and provide a mechanism of oral transmission to adults.³⁹ The capacity to persist in the Drosophila gut varies among microbial isolates depending on their rates of proliferation and emigration out of the host.³⁰ For these reasons, it is essential to monitor the taxonomic and functional composition of the Drosophila gut microbiota in the context of genetic and environmental factors that influence the relationship between a host and its microbiota.

The microbiota composition varies within and among Drosophila populations and species, with up to 20 species comprising more than 90% of all microbes in the gut.⁴⁰ Natural Drosophila populations consume microbes associated with rotting fruit, including various yeasts and bacteria. Ascomycete yeasts, predominantly Saccharomycetales (Hanseniaspora and Pichia), are prevalent in wild Drosophila but can be eliminated with the use of antifungal agents in laboratory diets.^41,42 Most importantly, the Drosophila gut microbiota is dominated by bacteria, especially Proteobacteria (Acetobacteraceae and Enterobacteriaceae) and Firmicutes (Lactobacillus and Enterococcus), which are present in the crop, midgut, and hindgut with densities up to 10⁶ cells per fly.²⁵ Interestingly, female and male flies do not exhibit consistent differences in microbial composition or abundance.⁴³ However, microbiota changes are evident throughout various developmental stages as flies age, showing that Acetobacter persici and Lactobacillus brevis are dominant species in young flies while Acetobacter malorum and Lactobacillus plantarum are dominant in older individuals.⁴⁴ Axenic (microbiota-free) or derivative gnotobiotic flies can be generated via embryo bleaching and culture on sterile food, followed by association with defined species for the latter, which allows precise quantification and manipulation of the microorganisms that cycle between food and Drosophila hosts.^19,45,46 Such experimental screens, coupled with genomic tools aimed to capture the full extent of microbial diversity and function within the gut, have become integral to understanding host health and conditioning of host immune defenses against pathogens.

Drosophila gut microbiota in immune responses

The gut, which represents one of the largest interfaces between the host internal and external environments, is perpetually exposed to various inputs including nutrients and infectious agents such as bacteria, viruses, fungi, and parasites. Resident gut microbiota in turn offers numerous benefits to the host through a range of physiological functions including strengthening gut integrity or shaping the intestinal epithelium, harvesting energy, protecting against pathogens, and regulating host immunity.^47–51 The intersection between gut microbiota and the host immune system during infections has been particularly intriguing, suggesting unappreciated interrelationships between pathogens and microbiota-dependent responses.⁴⁵ Several Drosophila studies addressing this topic confirm that gut microbiota improves host survival in the presence of well-established bacterial pathogens such as Pseudomonas aeruginosa or Serratia marcescens.^52–54 The question of how certain microbiota species mediate the observed salubrious effects is an interesting area of research, with evidence showing that the Drosophila gut microbiota influences both host resistance and resilience during microbial infections.⁵⁵ Resistance is characterized by the local production of antimicrobial compounds such as reactive oxygen species (ROS) and AMPs whereas resilience primarily depends on the ability of the intestinal epithelium to regenerate during and after the infectious episode.

Activation of immune signaling pathways in the gut

Previous work shows that Drosophila gut microbiota increases the basal levels of the tightly regulated Janus kinase/Signal transducers and activators of transcription (JAK/STAT), c-Jun NH₂-terminal kinase (JNK), and Immune deficiency (Imd) signaling pathways, which are not only involved in ISC proliferation and differentiation in infected guts, but also critical for the maintenance of gut homeostasis in uninfected guts (Figure 2).⁵⁶ The JAK/STAT signaling is mainly activated in the ISCs while the JNK and Imd pathways are primarily induced in the ECs.^57,58 Data suggest that flies raised in axenic conditions express lower levels of Unpaired 3 (upd3), puckered, and Diptericin genes, which are reporters of the JAK/STAT, JNK, and Imd pathway activities, respectively, compared to conventionally reared flies. The microbe-induced JAK/STAT pathway is linked to stem cell division, as demonstrated by the presence of cells double-positive for phospho-histone 3 (PH3), a marker for dividing cells, and escargot, a marker for ISCs and EBs, in flies.^59,60 Similarly, direct genetic induction of JNK signaling, a mitogen activated protein kinase- (MAPK-) type kinase cascade that branches out from the

Figure 2. Function of Drosophila gut microbiota in homeostasis and immunity. Drosophila gut microbiota modulates several signaling pathways that have profound effects on its gut homeostasis and immunity. Bacterial-derived metabolites such as uracil induce the generation of reactive oxygen species (ROS) through the NADPH oxidase (Nox) and dual-oxidase (Duox), which are involved in intestinal stem cell (ISC) regulation. Gut microbiota also increases the expression of the immune deficiency (Imd) pathway that is responsible for the production of Relish/NF-κB-dependent antimicrobial peptides (AMPs), the Janus kinase/Signal transducers and activators of transcription (JAK/STAT) pathway that controls multiple biological processes during ISC proliferation and homeostasis, and the c-Jun NH₂-terminal kinase (JNK) pathway that promotes epithelium renewal during injury and repair states.

Imd pathway, promotes large increases not only in midgut mitoses, but also in a pool of cells expressing the stem cell markers.⁶⁰ Therefore, JNK is activated to maintain gut regeneration during homeostasis, cytoprotection during infections and chronic stress, as well as pathological responses during aging.⁶¹ The Imd pathway, which parallels the mammalian tumor necrosis factor α receptor (TNFR) signaling pathway, is triggered upon recognition of Gram-negative bacteria with DAP-type peptidoglycan (DAP-PGN) by the transmembrane receptor PGRP-LC and, specifically in the gut, by the cytoplasmic PGRP-LE receptor.⁶² Negative regulators such as PGRP-SC and PGRP-LB amidases participate in the process of ubiquitination/de-ubiquitination in the Imd pathway to ensure an appropriate level of immune responses and establish immune tolerance to gut microbiota (Figure 2).⁶³ Evidently, axenic flies carrying mutations in Relish or PGRP-LC of the Imd pathway exhibit reduced ISC division and intestinal epithelium renewal in comparison to conventionally raised flies, thus further confirming that gut microbiota conditions the basal level of epithelium renewal by stimulating ISC proliferation.⁵⁶ Collectively, these results propose that infectious microbes reactivate rather than initiate some of the molecular and cellular responses that naturally occur in uninfected flies including NF-κB responses. Of note, microbiota-mediated priming of the immune responses has also been observed in Drosophila viral infection models and human systems.^64,65 The activated NF-κB transcription factors such as Relish promote the expression of AMPs to limit both pathogen and commensal bacterial growth in a feedback loop, therefore preventing abnormalities (dysbiosis) in the intestinal microbiota and extending the host lifespan.⁶⁶

Reactive oxygen species (ROS) production in the gut

Gut microbiota species are capable of biotransforming both host-derived components such as bile acids and dietary substances to generate various metabolites that affect the host’s gut environment and immunological balance.⁶⁷ Although there is no evidence for a role of the Toll pathway in the midgut, Gram-positive bacteria with lysine-type peptidoglycan, commonly identified as Drosophila gut microbiota members, can induce a basal immune reaction in the gut through the release of metabolites that activate ROS production by the NADPH oxidase (Nox) and dual-oxidase (Duox) in the plasma membrane.^67–71 These bacteria include Leuconostoc pseudomesenteroides, Lactobacillus brevis, Lactobacillus plantarum, and Enterococcus faecalis. While it had long been believed that ROS are inevitable pro-inflammatory toxins that accelerate tissue injury when the amounts exceed certain thresholds, research also implicates basal, low-level ROS signaling in normal gut homeostatic maintenance processes involving cell proliferation, migration, differentiation, and gene expression.⁷² Lactic-acid producing bacteria such as Lactobacilli are found to be especially potent inducers of Nox-dependent ROS generation and consequent cellular proliferation in the Drosophila ECs, which is vital during midgut development and homeostasis.^71,73 Studies show that ROS produced by Nox are required for p38 activation in enterocytes following infection or wounding, and for ISC activation upon infection or detergent exposure.⁷⁴ However, the exact mechanisms underlying Nox activation and function have not yet been determined (Figure 2). Further examination of signaling events in the ISC microenvironments will provide critical insights into how microbiota-elicited Nox-ROS responses alter homeostatic immune cell signaling in the gut.

Bacterial-derived uracil acts as a ligand for Duox-mediated ROS production in the Drosophila gut epithelia, which also plays an instructive role in the activation of stem cell signaling during injury and repair states.^56,75,76 Duox is regulated transcriptionally by a p38 MAPK pathway that triggers Activation Transcription Factor 2 (ATF2). The release of uracil by microbiota species such as Commensalibacter intestini A911^T, Acetobacter pomorum, and Lactobacillus plantarum activates an unknown G protein-coupled receptor, leading to the enzymatic induction of Duox at a basal level through the Gαq-phospholipase Cβ-Ca²⁺ signaling pathway (Figure 2).^77–80 Axenic flies lacking the ability to produce ROS generation (Gαq mutant flies or Duox knockdown flies) exhibit a reduced level of ISC proliferation and are susceptible to the ingestion of dietary yeast and several bacterial species.^56,75,76 Further analyses show that Duox-catalyzed ROS generation is absent in distinct bacterial species that make up the gut microbiota in Drosophila, indicating that distinctions among commensal bacteria in Drosophila can be made based on the in vivo Duox-activating ability of each bacterium.⁷⁵ Together, these observations indicate that Duox is dynamically modulated by gut microbiota to regulate host redox biochemistry, limit dysbiosis, and directly intervene in stem cell signaling pathways.

Conclusions and perspectives

The digestive tract has an exceptional capacity for homeostasis, as demonstrated by the efficient responses to variations in cell loss with corresponding changes in rates of self-renewal and differentiation. Gut tissue maintenance is extremely important to help preserve physical barrier integrity and proper immune function, thereby preventing the invasion of pathogenic microbes, excessive renewal of epithelial cells, and tumor overgrowth, while generating immune effectors such as ROS and AMPs. In both mammals and insects, the gut microbiota modulates host metabolism, epithelium physiology, and immune responses. Resident microbes particularly enhance host fitness and lifespan by eliciting immune responses that shape the commensal community while eliminating unwanted pathogens. The exact composition and function of these microbial communities is determined by an intricate interplay of genetic and environmental factors. Changes in healthy microbiota composition have been associated with pathologies like inflammatory bowel disease, diabetes, neurological disorders, chronic inflammation, and cancer, making scientists from across disciplines converge on the pressing need to better understand how the microbiome is formed and its role in shaping host phenotype, ecology, and evolution. However, it is extremely difficult to demonstrate the mechanistic connections between host immune genotype, microbiota structure, and disease phenotype due to the enormous diversity of mammalian microbiota and genetic complexity of the immune system.

The gastrointestinal tract in the adult Drosophila has been used extensively as a model system for exploring the mechanisms underlying digestion, absorption and excretion, and stem cell plasticity. The striking structural similarity of the Drosophila and human guts, in addition to major conserved signaling pathways involved in immunity and tissue regeneration, have especially made the Drosophila midgut an ideal model for revelatory studies of host-microbiota interactions and innate immunity. Research shows that Drosophila microbiota actively participates in the homeostasis and immunity of gut epithelia, including AMP- and ROS-based responses, therefore immunologically conditions the gut with efficient antimicrobial signaling to eliminate pathogens while tolerating commensal species. Moreover, gut microbiota is also involved in epithelium renewal, which is an essential component of Drosophila defense against infections. This demonstrates that gut homeostasis is achieved by a complex crosstalk of the immune response, gut microbiota, and stem cell activity. Elucidating the regulatory mechanisms underlying these interactions remains largely at the forefront of studies in disease modeling and drug development, and has potential for regenerative medicine research, thus providing novel perspectives of gut-microbiota associations, their role in health and disease, as well as therapeutic exploitation of beneficial bacteria.

Despite the tractability of the fly-microbiome model, identifying the underlying mechanisms driving organismal phenotypes will be difficult to decouple from nutritional influences. Changes in microbial growth rates, in the gut or in the habitat, dramatically shift nutritional contributions to the host, which is not comparable between flies and humans. Other issues include the lack of an adaptive immune system, methods to measure behavioral tendencies, and drastically different drug effects when compared to human studies. Nonetheless, several genes and signaling pathways that play a role in host immune response in humans have been identified in flies and are being used to study a plethora of human diseases. Such studies demonstrate the importance of Drosophila in uncovering mechanisms of host – microbe interactions and human diseases even if the exact disease does not occur in flies, thus providing a powerful model to understand how, and whether, hosts differentiate between the microbes they encounter across this spectrum of associations.

Author contributions

Ghada Tafesh-Edwards designed and conceptualized the review. Ghada Tafesh-Edwards prepared the original draft and graphical designs of the review. Ghada Tafesh-Edwards and Ioannis Eleftherianos edited and reviewed the final manuscript. Both authors approved the final version of the manuscript.

Acknowledgments

We thank members of the Department of Biological Sciences at George Washington University for discussions and critical reading of this Review.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

No new data were generated or analyzed in support of this research.

Additional information

Funding

This article did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.