An intestinal zinc sensor regulates food intake and developmental growth.

In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis.

Drosophila-associated bacteria differentially shape the nutritional requirements of their host during juvenile growth.

The interplay between nutrition and the microbial communities colonizing the gastrointestinal tract (i.e., gut microbiota) determines juvenile growth trajectory. Nutritional deficiencies trigger developmental delays, and an immature gut microbiota is a hallmark of pathologies related to childhood undernutrition. However, how host-associated bacteria modulate the impact of nutrition on juvenile growth remains elusive. Here, using gnotobiotic Drosophila melanogaster larvae independently associated with Acetobacter pomorumWJL (ApWJL) and Lactobacillus plantarumNC8 (LpNC8), 2 model Drosophila-associated bacteria, we performed a large-scale, systematic nutritional screen based on larval growth in 40 different and precisely controlled nutritional environments. We combined these results with genome-based metabolic network reconstruction to define the biosynthetic capacities of Drosophila germ-free (GF) larvae and its 2 bacterial partners. We first established that ApWJL and LpNC8 differentially fulfill the nutritional requirements of the ex-GF larvae and parsed such difference down to individual amino acids, vitamins, other micronutrients, and trace metals. We found that Drosophila-associated bacteria not only fortify the host's diet with essential nutrients but, in specific instances, functionally compensate for host auxotrophies by either providing a metabolic intermediate or nutrient derivative to the host or by uptaking, concentrating, and delivering contaminant traces of micronutrients. Our systematic work reveals that beyond the molecular dialogue engaged between the host and its bacterial partners, Drosophila and its associated bacteria establish an integrated nutritional network relying on nutrient provision and utilization.

Maintaining immune homeostasis in fly gut.

Like every metazoan species hosting a gut flora, drosophila tolerate commensal microbiota yet remain able to mount an efficient immune response to food-borne pathogens. New findings explain how the quantity of reactive oxygen species in the gut is 'tuned' to microbial burden and how intestinal immune homeostasis is thereby maintained

The importance of being persistent: The first true resident gut symbiont in Drosophila.

In the animal kingdom, nutritional mutualism is a perpetual and intimate dialogue carried out between the host and its associated gut community members. This dialogue affects many aspects of the host's development and physiology. Some constituents of the animal gut microbiota can stably reside within the host for years, and such long-term persistence might be a prerequisite for these microbes to assert their beneficial impact. How long-term persistence is established and maintained is an interesting question, and several classic model organisms associated with cultivable resident strains are used to address this question. However, in Drosophila, this model has long eluded fly geneticists. In this issue of PLOS Biology, Pais and colleagues present the most rigorous and comprehensive demonstration to date that persistence and gut residency do take place in the digestive tract of Drosophila melanogaster. This natural gut isolate of Acetobacter thailandicus stably colonizes the adult fly foregut, accelerates larval maturation, and boosts host fecundity and fertility as efficiently as the known laboratory strains. The discovery of such stable association will be a boon for the Drosophila community interested in host-microbiota interaction, as it not only provides a novel model to unravel the molecular underpinnings of persistence but also opens a new arena for using Drosophila to study the implications of gut persistence in evolution and ecology.

Probiotic from human breast milk, Lactobacillus fermentum, promotes growth in animal model of chronic malnutrition.

BACKGROUND: Chronic undernutrition leads to growth hormone resistance and poor growth in children, which has been shown to be modulated by microbiota. We studied whether Lactobacillus fermentum CECT5716 (Lf CECT5716), isolated from mother's breast milk, could promote juvenile growth through the modulation of lipid absorption in a model of starvation. METHODS: Germ-free (GF) Drosophila melanogaster larvae were inoculated with Lf CECT5716 in conditions of undernutrition with and without infant formula. The impact of Lf CECT5716 on larval growth was assessed 7 days after egg laying (AED) by measuring the larval size and on maturation by measuring the emergence of pupae during 21 days AED. For lipid absorption test, Caco2/TC7 intestinal cells were incubated with Lf CECT5716 and challenged with mixed lipid micelles. RESULTS: The mono-associated larvae with Lf CECT5716 were significantly longer than GF larvae (3.7 vs 2.5 mm; p < 0.0001). The effect was maintained when Lf CECT5716 was added to the infant formula. The maturation time of larvae was accelerated by Lf CECT5716 (12 vs 13.2 days; p = 0.01). Lf CECT5716 did not have significant impact on lipid absorption in Caco2/TC7 cells. CONCLUSIONS: Lf CECT5716 is a growth-promoting strain upon undernutrition in Drosophila, with a maintained effect when added to an infant formula but without effect on lipid absorption in vitro.

Gut Microbiota and Host Juvenile Growth.

Good genes, good food, good friends. That is what parents hope will sustain and nurture the harmonious growth of their children. The impact of the genetic background and nutrition on postnatal growth has been in the spot light for long, but the good friends have come to the scene only recently. Among the good friends perhaps the most crucial ones are those that we are carrying within ourselves. They comprise the trillions of microbes that collectively constitute each individual's intestinal microbiota. Indeed, recent epidemiological and field studies in humans, supported by extensive experimental data on animal models, demonstrate a clear role of the intestinal microbiota on their host's juvenile growth, especially under suboptimal nutrient conditions. Genuinely integrative approaches applicable to invertebrate and vertebrate systems combine tools from genetics, developmental biology, microbiology, nutrition, and physiology to reveal how gut microbiota affects growth both positively and negatively, in healthy and pathological conditions. It appears that certain natural or engineered gut microbiota communities can positively impact insulin/IGF-1 and steroid hormone signaling, thus contributing to the host juvenile development and maturation.

Lactobacillus plantarum favors the early emergence of fit and fertile adult Drosophila upon chronic undernutrition.

Animals are naturally surrounded by a variety of microorganisms with which they constantly interact. Among these microbes, some live in close association with a host and form its microbiota. These communities are being extensively studied, owing to their contributions to shaping various aspects of animal physiology. One of these commensal species, Lactobacillus plantarum, and in particular the L.p.WJL strain, has been shown to promote the growth of Drosophila larvae upon nutrient scarcity, allowing earlier metamorphosis and adult emergence compared with axenic individuals. As for many insects, conditions surrounding the post-embryonic development dictate key adult life history traits in Drosophila, and adjusting developmental timing according to the environment is essential for adult fitness. Thus, we wondered whether the growth acceleration induced by L.p.WJL in a context of poor nutrition could adversely impact the fitness of Drosophila adults. Here, we show that the L.p.WJL -mediated acceleration of growth is not deleterious; adults emerging after an accelerated development are as fit as their axenic siblings. Additionally, the presence of L.p.WJL even leads to a lifespan extension in nutritionally challenged males. These results demonstrate that L.p.WJL is a beneficial partner for Drosophila melanogaster through its entire life cycle. Thus, commensal bacteria allow the earlier emergence and longer survival of fit and fertile individuals and might represent one of the factors contributing to the ecological success of Drosophila.

Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling.

Caspases have been extensively studied as critical initiators and executioners of cell death pathways. However, caspases also take part in non-apoptotic signalling events such as the regulation of innate immunity and activation of nuclear factor-κB (NF-κB). How caspases are activated under these conditions and process a selective set of substrates to allow NF-κB signalling without killing the cell remains largely unknown. Here, we show that stimulation of the Drosophila pattern recognition protein PGRP-LCx induces DIAP2-dependent polyubiquitylation of the initiator caspase DREDD. Signal-dependent ubiquitylation of DREDD is required for full processing of IMD, NF-κB/Relish and expression of antimicrobial peptide genes in response to infection with Gram-negative bacteria. Our results identify a mechanism that positively controls NF-κB signalling via ubiquitin-mediated activation of DREDD. The direct involvement of ubiquitylation in caspase activation represents a novel mechanism for non-apoptotic caspase-mediated signalling.

Nomadic lifestyle of Lactobacillus plantarum revealed by comparative genomics of 54 strains isolated from different habitats.

The ability of bacteria to adapt to diverse environmental conditions is well-known. The process of bacterial adaptation to a niche has been linked to large changes in the genome content, showing that many bacterial genomes reflect the constraints imposed by their habitat. However, some highly versatile bacteria are found in diverse habitats that almost share nothing in common. Lactobacillus plantarum is a lactic acid bacterium that is found in a large variety of habitat. With the aim of unravelling the link between evolution and ecological versatility of L. plantarum, we analysed the genomes of 54 L. plantarum strains isolated from different environments. Comparative genome analysis identified a high level of genomic diversity and plasticity among the strains analysed. Phylogenomic and functional divergence studies coupled with gene-trait matching analyses revealed a mixed distribution of the strains, which was uncoupled from their environmental origin. Our findings revealed the absence of specific genomic signatures marking adaptations of L. plantarum towards the diverse habitats it is associated with. This suggests fundamentally similar trends of genome evolution in L. plantarum, which occur in a manner that is apparently uncoupled from ecological constraint and reflects the nomadic lifestyle of this species.

Lactobacilli-Host mutualism: "learning on the fly".

Metazoans establish with microorganisms complex interactions for their mutual benefits. Drosophila, which has already proven useful host model to study several aspects of innate immunity and host-bacteria pathogenic associations has become a powerful model to dissect the mechanisms behind mutualistic host-microbe interactions. Drosophila microbiota is composed of simple and aerotolerant bacterial communities mostly composed of Lactobacillaceae and Acetobactereaceae. Drosophila mono- or poly-associated with lactobacilli strains constitutes a powerful model to dissect the complex interplay between lactobacilli and host biologic traits. Thanks to the genetic tractability of both Drosophila and lactobacilli this association model offers a great opportunity to reveal the underlying molecular mechanisms. Here, we review our current knowledge about how the Drosophila model is helping our understanding of how lactobacilli shapes host biology.

Toll-like receptors--taking an evolutionary approach.

The Toll receptor was initially identified in Drosophila melanogaster for its role in embryonic development. Subsequently, D. melanogaster Toll and mammalian Toll-like receptors (TLRs) have been recognized as key regulators of immune responses. After ten years of intense research on TLRs and the recent accumulation of genomic and functional data in diverse organisms, we review the distribution and functions of TLRs in the animal kingdom. We provide an evolutionary perspective on TLRs, which sheds light on their origin at the dawn of animal evolution and suggests that different TLRs might have been co-opted independently during animal evolution to mediate analogous immune functions.

Structure-function analysis of Lactiplantibacillus plantarum DltE reveals D-alanylated lipoteichoic acids as direct cues supporting Drosophila juvenile growth.

Metazoans establish mutually beneficial interactions with their resident microorganisms. However, our understanding of the microbial cues contributing to host physiology remains elusive. Previously, we identified a bacterial machinery encoded by the dlt operon involved in Drosophila melanogaster's juvenile growth promotion by Lactiplantibacillus plantarum. Here, using crystallography combined with biochemical and cellular approaches, we investigate the physiological role of an uncharacterized protein (DltE) encoded by this operon. We show that lipoteichoic acids (LTAs) but not wall teichoic acids are D-alanylated in Lactiplantibacillus plantarumNC8 cell envelope and demonstrate that DltE is a D-Ala carboxyesterase removing D-Ala from LTA. Using the mutualistic association of L. plantarumNC8 and Drosophila melanogaster as a symbiosis model, we establish that D-alanylated LTAs (D-Ala-LTAs) are direct cues supporting intestinal peptidase expression and juvenile growth in Drosophila. Our results pave the way to probing the contribution of D-Ala-LTAs to host physiology in other symbiotic models.

Intestinal GCN2 controls Drosophila systemic growth in response to Lactiplantibacillus plantarum symbiotic cues encoded by r/tRNA operons.

Symbiotic bacteria interact with their host through symbiotic cues. Here, we took advantage of the mutualism between Drosophila and Lactiplantibacillus plantarum (Lp) to investigate a novel mechanism of host-symbiont interaction. Using chemically defined diets, we found that association with Lp improves the growth of larvae-fed amino acid-imbalanced diets, even though Lp cannot produce the limiting amino acid. We show that in this context Lp supports its host's growth through a molecular dialogue that requires functional operons encoding ribosomal and transfer RNAs (r/tRNAs) in Lp and the general control nonderepressible 2 (GCN2) kinase in Drosophila's enterocytes. Our data indicate that Lp's r/tRNAs are packaged in extracellular vesicles and activate GCN2 in a subset of larval enterocytes, a mechanism necessary to remodel the intestinal transcriptome and ultimately to support anabolic growth. Based on our findings, we propose a novel beneficial molecular dialogue between host and microbes, which relies on a non-canonical role of GCN2 as a mediator of non-nutritional symbiotic cues encoded by r/tRNA operons.

Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice.

The intestinal microbiota is known to influence postnatal growth. We previously found that a strain of Lactiplantibacillus plantarum (strain LpWJL) buffers the adverse effects of chronic undernutrition on the growth of juvenile germ-free mice. Here, we report that LpWJL sustains the postnatal growth of malnourished conventional animals and supports both insulin-like growth factor-1 (IGF-1) and insulin production and activity. We have identified cell walls isolated from LpWJL, as well as muramyl dipeptide and mifamurtide, as sufficient cues to stimulate animal growth despite undernutrition. Further, we found that NOD2 is necessary in intestinal epithelial cells for LpWJL-mediated IGF-1 production and for postnatal growth promotion in malnourished conventional animals. These findings indicate that, coupled with renutrition, bacteria cell walls or purified NOD2 ligands have the potential to alleviate stunting.

Two roles for the Drosophila IKK complex in the activation of Relish and the induction of antimicrobial peptide genes.

The Drosophila NF-kappaB transcription factor Relish is an essential regulator of antimicrobial peptide gene induction after gram-negative bacterial infection. Relish is a bipartite NF-kappaB precursor protein, with an N-terminal Rel homology domain and a C-terminal IkappaB-like domain, similar to mammalian p100 and p105. Unlike these mammalian homologs, Relish is endoproteolytically cleaved after infection, allowing the N-terminal NF-kappaB module to translocate to the nucleus. Signal-dependent activation of Relish, including cleavage, requires both the Drosophila IkappaB kinase (IKK) and death-related ced-3/Nedd2-like protein (DREDD), the Drosophila caspase-8 like protease. In this report, we show that the IKK complex controls Relish by direct phosphorylation on serines 528 and 529. Surprisingly, these phosphorylation sites are not required for Relish cleavage, nuclear translocation, or DNA binding. Instead they are critical for recruitment of RNA polymerase II and antimicrobial peptide gene induction, whereas IKK functions noncatalytically to support Dredd-mediated cleavage of Relish.

DltC acts as an interaction hub for AcpS, DltA and DltB in the teichoic acid D-alanylation pathway of Lactiplantibacillus plantarum.

Teichoic acids (TA) are crucial for the homeostasis of the bacterial cell wall as well as their developmental behavior and interplay with the environment. TA can be decorated by different modifications, modulating thus their biochemical properties. One major modification consists in the esterification of TA by D-alanine, a process known as D-alanylation. TA D-alanylation is performed by the Dlt pathway, which starts in the cytoplasm and continues extracellularly after D-Ala transportation through the membrane. In this study, we combined structural biology and in vivo approaches to dissect the cytoplasmic steps of this pathway in Lactiplantibacillus plantarum, a bacterial species conferring health benefits to its animal host. After establishing that AcpS, DltB, DltC1 and DltA are required for the promotion of Drosophila juvenile growth under chronic undernutrition, we solved their crystal structure and/or used NMR and molecular modeling to study their interactions. Our work demonstrates that the suite of interactions between these proteins is ordered with a conserved surface of DltC1 docking sequentially AcpS, DltA and eventually DltB. Altogether, we conclude that DltC1 acts as an interaction hub for all the successive cytoplasmic steps of the TA D-alanylation pathway.

Approaches to discern if microbiome associations reflect causation in metabolic and immune disorders.

Our understanding of microorganisms residing within our gut and their roles in the host metabolism and immunity advanced greatly over the past 20 years. Currently, microbiome studies are shifting from association and correlation studies to studies demonstrating causality of identified microbiome signatures and identification of molecular mechanisms underlying these interactions. This transformation is crucial for the efficient translation into clinical application and development of targeted strategies to beneficially modulate the intestinal microbiota. As mechanistic studies are still quite challenging to perform in humans, the causal role of microbiota is frequently evaluated in animal models that need to be appropriately selected. Here, we provide a comprehensive overview on approaches that can be applied in addressing causality of host-microbe interactions in five major animal model organisms (Caenorhabditis elegans, Drosophila melanogaster, zebrafish, rodents, and pigs). We particularly focused on discussing methods available for studying the causality ranging from the usage of gut microbiota transfer, diverse models of metabolic and immune perturbations involving nutritional and chemical factors, gene modifications and surgically induced models, metabolite profiling up to culture-based approached. Furthermore, we addressed the impact of the gut morphology, physiology as well as diet on the microbiota composition in various models and resulting species specificities. Finally, we conclude this review with the discussion on models that can be applied to study the causal role of the gut microbiota in the context of metabolic syndrome and host immunity. We hope this review will facilitate important considerations for appropriate animal model selection.

Microbial and nutritional influence on endocrine control of growth.

The worrying number of children suffering from undernutrition and consequent stunting worldwide makes the understanding of the relationship between nutritional status and postnatal growth crucial. Moreover, it is now well established that undernourished children harbor an altered microbiota, correlating with impaired growth. In this review, we describe how murine models have been used to explore the functional relationships between endocrine regulation of growth, nutrition and gut microbiota. In numerous Mammalian species, postnatal growth is mainly regulated by the conserved GH/IGF1 somatotropic axis that acts through endocrine and paracrine pathways, notably enabling longitudinal bone growth. Recent studies have demonstrated that the microbiota effects on growth could involve a modulation of GH and IGF1 circulating levels. Besides, the GH/IGF1 somatotropic axis may regulate the gut microbiota composition and diversity. Studying the bidirectional relationship between growth hormones and the gut microbiome could therefore help developing microbiota-targeting therapies in order to reduce the long-term consequences of stunting.

Microbial Modulation of the Development and Physiology of the Enteric Nervous System.

The gastrointestinal tract harbors an intrinsic neuronal network, the enteric nervous system (ENS). The ENS controls motility, fluid homeostasis, and blood flow, but also interacts with other components of the intestine such as epithelial and immune cells. Recent studies indicate that gut microbiota diversification, which occurs alongside postnatal ENS maturation, could be critical for the development and function of the ENS. Here we discuss the possibility that this functional relationship starts in utero, whereby the maternal microbiota would prime the developing ENS and shape its physiology. We review ENS/microbiota interactions and their modulation in physiological and pathophysiological contexts. While microbial modulation of the ENS physiology is now well established, further studies are required to understand the contribution of the gut microbiota to the development and pathology of the ENS and to reveal the precise mechanisms underlying microbiota-to-ENS communications.

A standardized gnotobiotic mouse model harboring a minimal 15-member mouse gut microbiota recapitulates SOPF/SPF phenotypes.

Mus musculus is the classic mammalian model for biomedical research. Despite global efforts to standardize breeding and experimental procedures, the undefined composition and interindividual diversity of the microbiota of laboratory mice remains a limitation. In an attempt to standardize the gut microbiome in preclinical mouse studies, here we report the development of a simplified mouse microbiota composed of 15 strains from 7 of the 20 most prevalent bacterial families representative of the fecal microbiota of C57BL/6J Specific (and Opportunistic) Pathogen-Free (SPF/SOPF) animals and the derivation of a standardized gnotobiotic mouse model called GM15. GM15 recapitulates extensively the functionalities found in the C57BL/6J SOPF microbiota metagenome, and GM15 animals are phenotypically similar to SOPF or SPF animals in two different facilities. They are also less sensitive to the deleterious effects of post-weaning malnutrition. In this work, we show that the GM15 model provides increased reproducibility and robustness of preclinical studies by limiting the confounding effect of fluctuation in microbiota composition, and offers opportunities for research focused on how the microbiota shapes host physiology in health and disease.