Genetic Screening Revealed the Negative Regulation of miR-310~313 Cluster Members on Imd Pathway during Gram-Negative Bacterial Infection in Drosophila.

Innate immune response is the first line of host defense against pathogenic microorganisms, and its excessive or insufficient activation is detrimental to the organism. Many individual microRNAs (miRNAs) have emerged as crucial post-transcriptional regulators of immune homeostasis in Drosophila melanogaster. However, the synergistical regulation of miRNAs located within a cluster on the Imd-immune pathway remains obscured. In our study, a genetic screening with 52 transgenic UAS-miRNAs was performed to identify ten miRNAs or miRNA clusters, including the miR310~313 cluster, which may function on Imd-dependent immune responses. The miRNA RT-qPCR analysis showed that the expression of miR-310~313 cluster members exhibited an increase at 6-12 h post E. coli infection. Furthermore, the overexpression of the miR-310~313 cluster impaired the Drosophila survival. And the overexpression of miR-310/311/312 reduced Dpt expression, an indication of Imd pathway induced by Gram-negative bacteria. Conversely, the knockdown of miR-310/311/312 led to increases in Dpt expression. The Luciferase reporter expression assays and RT-qPCR analysis confirmed that miR-310~313 cluster members directly co-targeted and inhibited Imd transcription. These findings reveal that the members of the miR-310~313 cluster synergistically inhibit Imd-dependent immune responses by co-targeting the Imd gene in Drosophila.

High-fat diets induce inflammatory IMD/NFκB signaling via gut microbiota remodeling in Drosophila.

High-fat diets (HFDs), a prevailing daily dietary style worldwide, induce chronic low-grade inflammation in the central nervous system and peripheral tissues, promoting a variety of diseases including pathologies associated with neuroinflammation. However, the mechanisms linking HFDs to inflammation are not entirely clear. Here, using a Drosophila HFD model, we explored the mechanism of HFD-induced inflammation in remote tissues. We found that HFDs activated the IMD/NFκB immune pathway in the head through remodeling of the commensal gut bacteria. Removal of gut microbiota abolished such HFD-induced remote inflammatory response. Further experiments revealed that HFDs significantly increased the abundance of Acetobacter malorum in the gut, and the re-association of this bacterium was sufficient to elicit inflammatory response in remote tissues. Mechanistically, Acetobacter malorum produced a greater amount of peptidoglycan (PGN), a well-defined microbial molecular pattern that enters the circulation and remotely activates an inflammatory response. Our results thus show that HFDs trigger inflammation mediated by a bacterial molecular pattern that elicits host immune response.

Microbiome-derived acidity protects against microbial invasion in Drosophila.

Microbial invasions underlie host-microbe interactions resulting in pathogenesis and probiotic colonization. In this study, we explore the effects of the microbiome on microbial invasion in Drosophila melanogaster. We demonstrate that gut microbes Lactiplantibacillus plantarum and Acetobacter tropicalis improve survival and lead to a reduction in microbial burden during infection. Using a microbial interaction assay, we report that L. plantarum inhibits the growth of invasive bacteria, while A. tropicalis reduces this inhibition. We further show that inhibition by L. plantarum is linked to its ability to acidify its environment via lactic acid production by lactate dehydrogenase, while A. tropicalis diminishes the inhibition by quenching acids. We propose that acid from the microbiome is a gatekeeper to microbial invasions, as only microbes capable of tolerating acidic environments can colonize the host. The methods and findings described herein will add to the growing breadth of tools to study microbe-microbe interactions in broad contexts.

The utility of Drosophila melanogaster as a fungal infection model.

Invasive fungal diseases have profound effects upon human health and are on increase globally. The World Health Organization (WHO) in 2022 published the fungal priority list calling for improved public health interventions and advance research. Drosophila melanogaster presents an excellent model system to dissect host-pathogen interactions and has been proved valuable to study immunopathogenesis of fungal diseases. In this review we highlight the recent advances in fungal-Drosophila interplay with an emphasis on the recently published WHO's fungal priority list and we focus on available tools and technologies.

Facultatively ectoparasitic mites as vectors for entomopathogenic bacteria in Drosophila.

Opportunistic bacterial infections are common in insect populations but there is little information on how they are acquired or transmitted. We tested the hypothesis that Macrocheles mites can transmit systemic bacterial infections between Drosophila hosts. We found that 24% of mites acquired detectable levels of bacteria after feeding on infected flies and 87% of infected mites passed bacteria to naïve recipient flies. The probability that a mite could pass Serratia from an infected donor fly to a naïve recipient fly was 27.1%. These data demonstrate that Macrocheles mites are capable of serving as vectors of bacterial infection between insects.

Prophage proteins alter long noncoding RNA and DNA of developing sperm to induce a paternal-effect lethality.

The extent to which prophage proteins interact with eukaryotic macromolecules is largely unknown. In this work, we show that cytoplasmic incompatibility factor A (CifA) and B (CifB) proteins, encoded by prophage WO of the endosymbiont Wolbachia, alter long noncoding RNA (lncRNA) and DNA during Drosophila sperm development to establish a paternal-effect embryonic lethality known as cytoplasmic incompatibility (CI). CifA is a ribonuclease (RNase) that depletes a spermatocyte lncRNA important for the histone-to-protamine transition of spermiogenesis. Both CifA and CifB are deoxyribonucleases (DNases) that elevate DNA damage in late spermiogenesis. lncRNA knockdown enhances CI, and mutagenesis links lncRNA depletion and subsequent sperm chromatin integrity changes to embryonic DNA damage and CI. Hence, prophage proteins interact with eukaryotic macromolecules during gametogenesis to create a symbiosis that is fundamental to insect evolution and vector control.

Antifungal activity of azoles, allylamines, and 8-hidroxiquinolines, alone and in combination, against Malassezia pachydermatis in vitro and in vivo.

Malassezia pachydermatis is often reported as the causative agent of dermatitis in dogs. This study aims to evaluate the in vitro and in vivo antifungal activity of azoles and terbinafine (TRB), alone and in combination with the 8-hydroxyquinoline derivatives (8-HQs) clioquinol (CQL), 8-hydroxyquinoline-5-(n-4-chlorophenyl)sulfonamide (PH151), and 8-hydroxyquinoline-5-(n-4-methoxyphenyl)sulfonamide (PH153), against 16 M. pachydermatis isolates. Susceptibility to the drugs was evaluated by in vitro broth microdilution and time-kill assays. The Toll-deficient Drosophila melanogaster fly model was used to assess the efficacy of drugs in vivo. In vitro tests showed that ketoconazole (KTZ) was the most active drug, followed by TRB and CQL. The combinations itraconazole (ITZ)+CQL and ITZ+PH151 resulted in the highest percentages of synergism and none of the combinations resulted in antagonism. TRB showed the highest survival rates after seven days of treatment of the flies, followed by CQL and ITZ, whereas the evaluation of fungal burden of dead flies showed a greater fungicidal effect of azoles when compared to the other drugs. Here we showed for the first time that CQL is effective against M. pachydermatis and potentially interesting for the treatment of malasseziosis.

Aedes aegypti microbiome composition covaries with the density of Wolbachia infection.

BACKGROUND: Wolbachia is a widespread bacterial endosymbiont that can inhibit vector competency when stably transinfected into the mosquito, Aedes aegypti, a primary vector of the dengue virus (DENV) and other arboviruses. Although a complete mechanistic understanding of pathogen blocking is lacking, it is likely to involve host immunity induction and resource competition between Wolbachia and DENV, both of which may be impacted by microbiome composition. The potential impact of Wolbachia transinfection on host fitness is also of importance given the widespread release of mosquitos infected with the Drosophila melanogaster strain of Wolbachia (wMel) in wild populations. Here, population-level genomic data from Ae. aegypti was surveyed to establish the relationship between the density of wMel infection and the composition of the host microbiome. RESULTS: Analysis of genomic data from 172 Ae. aegypti females across six populations resulted in an expanded and quantitatively refined, species-level characterization of the bacterial, archaeal, and fungal microbiome. This included 844 species of bacteria across 23 phyla, of which 54 species were found to be ubiquitous microbiome members across these populations. The density of wMel infection was highly variable between individuals and negatively correlated with microbiome diversity. Network analyses revealed wMel as a hub comprised solely of negative interactions with other bacterial species. This contrasted with the large and highly interconnected network of other microbiome species that may represent members of the midgut microbiome community in this population. CONCLUSION: Our bioinformatic survey provided a species-level characterization of Ae. aegypti microbiome composition and variation. wMel load varied substantially across populations and individuals and, importantly, wMel was a major hub of a negative interactions across the microbiome. These interactions may be an inherent consequence of heightened pathogen blocking in densely infected individuals or, alternatively, may result from antagonistic Wolbachia-incompatible bacteria that could impede the efficacy of wMel as a biological control agent in future applications. The relationship between wMel infection variation and the microbiome warrants further investigation in the context of developing wMel as a multivalent control agent against other arboviruses. Video Abstract.

Wolbachia infection at least partially rescues the fertility and ovary defects of several new Drosophila melanogaster bag of marbles protein-coding mutants.

The D. melanogaster protein coding gene bag of marbles (bam) plays a key role in early male and female reproduction by forming complexes with partner proteins to promote differentiation in gametogenesis. Like another germline gene, Sex lethal, bam genetically interacts with the endosymbiont Wolbachia, as Wolbachia rescues the reduced fertility of a bam hypomorphic mutant. Here, we explored the specificity of the bam-Wolbachia interaction by generating 22 new bam mutants, with ten mutants displaying fertility defects. Nine of these mutants trend towards rescue by the wMel Wolbachia variant, with eight statistically significant at the fertility and/or cytological level. In some cases, fertility was increased a striking 20-fold. There is no specificity between the rescue and the known binding regions of bam, suggesting wMel does not interact with one singular bam partner to rescue the reproductive phenotype. We further tested if wMel interacts with bam in a non-specific way, by increasing bam transcript levels or acting upstream in germline stem cells. A fertility assessment of a bam RNAi knockdown mutant reveals that wMel rescue is specific to functionally mutant bam alleles and we find no obvious evidence of wMel interaction with germline stem cells in bam mutants.

Acetobacter pomorum in the Drosophila gut microbiota buffers against host metabolic impacts of dietary preservative formula and batch variation in dietary yeast.

Gut microbiota are fundamentally important for healthy function in animal hosts. Drosophila melanogaster is a powerful system for understanding host-microbiota interactions, with modulation of the microbiota inducing phenotypic changes that are conserved across animal taxa. Qualitative differences in diet, such as preservatives and dietary yeast batch variation, may affect fly health indirectly via microbiota, and may potentially have hitherto uncharacterized effects directly on the fly. These factors are rarely considered, controlled, and are not standardized among laboratories. Here, we show that the microbiota's impact on fly triacylglyceride (TAG) levels-a commonly-measured metabolic index-depends on both preservatives and yeast, and combinatorial interactions among the three variables. In studies of conventional, axenic, and gnotobiotic flies, we found that microbial impacts were apparent only on specific yeast-by-preservative conditions, with TAG levels determined by a tripartite interaction of the three experimental factors. When comparing axenic and conventional flies, we found that preservatives caused more variance in host TAG than microbiota status, and certain yeast-preservative combinations even reversed effects of microbiota on TAG. Preservatives had major effects in axenic flies, suggesting either direct effects on the fly or indirect effects via media. However, Acetobacter pomorum buffers the fly against this effect, despite the preservatives inhibiting growth, indicating that this bacterium benefits the host in the face of mutual environmental toxicity. Our results suggest that antimicrobial preservatives have major impacts on host TAG, and that microbiota modulates host TAG dependent on the combination of the dietary factors of preservative formula and yeast batch. IMPORTANCE Drosophila melanogaster is a premier model for microbiome science, which has greatly enhanced our understanding of the basic biology of host-microbe interactions. However, often overlooked factors such as dietary composition, including yeast batch variability and preservative formula, may confound data interpretation of experiments within the same lab and lead to different findings when comparing between labs. Our study supports this notion; we find that the microbiota does not alter host TAG levels independently. Rather, TAG is modulated by combinatorial effects of microbiota, yeast batch, and preservative formula. Specific preservatives increase TAG even in germ-free flies, showing that a commonplace procedure in fly husbandry alters metabolic physiology. This work serves as a cautionary tale that fly rearing methodology can mask or drive microbiota-dependent metabolic changes and also cause microbiota-independent changes.

Delayed host mortality and immune response upon infection with P. aeruginosa persister cells.

Chronic infections are a heavy burden on healthcare systems worldwide. Persister cells are thought to be largely responsible for chronic infection due to their tolerance to antimicrobials and recalcitrance to innate immunity factors. Pseudomonas aeruginosa is a common and clinically relevant pathogen that contains stereotypical persister cells. Despite their importance in chronic infection, there have been limited efforts to study persister cell infections in vivo. Drosophila melanogaster has a well-described innate immune response similar to that of vertebrates and is a good candidate for the development of an in vivo model of infection for persister cells. Similar to what is observed in other bacterial strains, in this work we found that infection with P. aeruginosa persister cells resulted in a delayed mortality phenotype in Caenorhabditis elegans, Arabidopsis thaliana, and D. melanogaster compared to infection with regular cells. An in-depth characterization of infected D. melanogaster found that bacterial loads differed between persister and regular cells' infections during the early stages. Furthermore, hemocyte activation and antimicrobial peptide expression were delayed/reduced in persister infections over the same time course, indicating an initial suppression of, or inability to elicit, the fly immune response. Overall, our findings support the use of D. melanogaster as a model in which to study persister cells in vivo, where this bacterial subpopulation exhibits delayed virulence and an attenuated immune response.

Environmental microbes promote phenotypic plasticity in reproduction and sleep behaviour.

The microbiome has been hypothesized as a driving force of phenotypic variation in host organisms that is capable of extending metabolic processes, altering development and in some cases, conferring novel functions that are critical for survival. Only a few studies have directly shown a causal role for the environmental microbiome in altering host phenotypic features. To assess the extent to which environmental microbes induce variation in host life-history traits and behaviour, we inoculated axenic Drosophila melanogaster with microbes isolated from drosophilid populations collected from two different field sites and generated two populations with distinct bacterial and fungal profiles. We show that microbes isolated from environmental sites with modest abiotic differences induce large variation in host reproduction, fatty acid levels, stress tolerance and sleep behaviour. Importantly, clearing microbes from each experimental population removed the phenotypic differences. The results support the causal role of environmental microbes as drivers of host phenotypic plasticity and potentially, rapid adaptation and evolution.

Gut microbes predominantly act as living beneficial partners rather than raw nutrients.

Animals and their gut microbes mutually benefit their health. Nutrition plays a central role in this, directly influencing both host and microbial fitness and the nature of their interactions. This makes nutritional symbioses a complex and dynamic tri-system of diet-microbiota-host. Despite recent discoveries on this field, full control over the interplay among these partners is challenging and hinders the resolution of fundamental questions, such as how to parse the gut microbes' effect as raw nutrition or as symbiotic partners? To tackle this, we made use of the well-characterized Drosophila melanogaster/Lactiplantibacillus plantarum experimental model of nutritional symbiosis to generate a quantitative framework of gut microbes' effect on the host. By coupling experimental assays and Random Forest analysis, we show that the beneficial effect of L. plantarum strains primarily results from the active relationship as symbionts rather than raw nutrients, regardless of the bacterial strain. Metabolomic analysis of both active and inactive bacterial cells further demonstrated the crucial role of the production of beneficial bacterial metabolites, such as N-acetylated-amino-acids, as result of active bacterial growth and function. Altogether, our results provide a ranking and quantification of the main bacterial features contributing to sustain animal growth. We demonstrate that bacterial activity is the predominant and necessary variable involved in bacteria-mediated benefit, followed by strain-specific properties and the nutritional potential of the bacterial cells. This contributes to elucidate the role of beneficial bacteria and probiotics, creating a broad quantitative framework for host-gut microbiome that can be expanded to other model systems.

Ecology-relevant bacteria drive the evolution of host antimicrobial peptides in Drosophila.

Antimicrobial peptides are host-encoded immune effectors that combat pathogens and shape the microbiome in plants and animals. However, little is known about how the host antimicrobial peptide repertoire is adapted to its microbiome. Here, we characterized the function and evolution of the Diptericin antimicrobial peptide family of Diptera. Using mutations affecting the two Diptericins (Dpt) of Drosophila melanogaster, we reveal the specific role of DptA for the pathogen Providencia rettgeri and DptB for the gut mutualist Acetobacter. The presence of DptA- or DptB-like genes across Diptera correlates with the presence of Providencia and Acetobacter in their environment. Moreover, DptA- and DptB-like sequences predict host resistance against infection by these bacteria across the genus Drosophila. Our study explains the evolutionary logic behind the bursts of rapid evolution of an antimicrobial peptide family and reveals how the host immune repertoire adapts to changing microbial environments.

Inside help from the microbiome.

Elucidating the role of one of the proteins produced by Lactiplantibacillus plantarum reveals a new molecule that allows this gut bacterium to support the development of fruit fly larvae.

Intake of caffeine containing sugar diet remodels gut microbiota and perturbs Drosophila melanogaster immunity and lifespan.

The diet-microbiome-immunity axis is one among the many arms that draw up the "we are what we intake" proclamation. As such, studies on the effect of food and beverage intake on the gut environment and microbiome and on modulating immunological responses and the host's susceptibility to pathogens are on the rise. A typical accompaniment in different sustenance we consume on daily basis is the trimethylxanthine alkaloid caffeine. Being a chief component in our regular aliment, a better understanding of the effect of caffeine containing food and beverages on our gut-microbiome-immunity axis and henceforth on our health is much needed. In this study, we shed more light on the effect of oral consumption of caffeine supplemented sugar diet on the gut environment, specifically on the gut microbiota, innate immunity and host susceptibility to pathogens using the Drosophila melanogaster model organism. Our findings reveal that the oral intake of a dose-specific caffeine containing sucrose/agarose sugar diet causes a significant alteration within the fly gut milieu demarcated by microbial dysbiosis and an elevation in the production of reactive oxygen species and expression of immune-deficiency (Imd) pathway-dependent antimicrobial peptide genes. The oral intake of caffeine containing sucrose/agarose sugar diet also renders the flies more susceptible to bacterial infection and shortens their lifespan in both infection and non-infection settings. Our findings set forth additional insight into the potentiality of diet to alter the gut milieu and highlight the importance of dietary control on health.

Rapid but narrow - Evolutionary adaptation and transcriptional response of Drosophila melanogaster to toxic mould.

Insects have adapted to a multitude of environmental conditions, including the presence of xenobiotic noxious substances. Environmental microorganisms, particularly rich on ephemeral resources, employ these noxious chemicals in a chemical warfare against predators and competitors, driving co-evolutionary adaptations. In order to analyse how environmental microbes may be driving such evolutionary adaptations, we experimentally evolved Drosophila melanogaster populations by exposing larvae to the toxin-producing mould Aspergillus nidulans that infests the flies' breeding substrate. To disentangle the effects of the mycotoxin Sterigmatocystin from other substrate modifications inflicted by the mould, we used the following four selection regimes: (i) control without fungus, (ii) A. nidulans wild type, (iii) a mutant of A. nidulans ΔlaeA with impaired toxin production, (iv) synthetic Sterigmatocystin. Experimental evolution was carried out in five independent D. melanogaster populations each, for a total of 11 generations. We further combined our evolution experiment with transcriptome analysis to identify evolutionary shifts in gene expression due to the selection regimes and mould confrontation. Populations that evolved in presence of the toxin-producing mould or the pure mycotoxin rapidly adapted to the respective conditions and showed higher viability in subsequent confrontations. Yet, mycotoxin-selected populations had no advantage in A. nidulans wild type confrontation. Moreover, distinctive changes in gene expression related to the selection-regime contrast were only associated with the toxin-producing-fungus regime and comprised a narrow set of genes. Thus, it needs the specific conditions of the selection agent to enable adaptation to the fungus.

Flagellar Genes Are Associated with the Colonization Persistence Phenotype of the Drosophila melanogaster Microbiota.

In this work, we use Drosophila melanogaster as a model to identify bacterial genes necessary for bacteria to colonize their hosts independent of the bulk flow of diet. Early work on this model system established that dietary replenishment drives the composition of the D. melanogaster gut microbiota, and subsequent research has shown that some bacterial strains can stably colonize, or persist within, the fly independent of dietary replenishment. Here, we reveal transposon insertions in specific bacterial genes that influence the bacterial colonization persistence phenotype by using a gene association approach. We initially established that different bacterial strains persist at various levels, independent of dietary replenishment. We then repeated the analysis with an expanded panel of bacterial strains and performed a metagenome-wide association (MGWA) study to identify distinct bacterial genes that are significantly correlated with the level of colonization by persistent bacterial strains. Based on the MGWA study, we tested if 44 bacterial transposon insertion mutants from 6 gene categories affect bacterial persistence within the flies. We identified that transposon insertions in four flagellar genes, one urea carboxylase gene, one phosphatidylinositol gene, one bacterial secretion gene, and one antimicrobial peptide (AMP) resistance gene each significantly influenced the colonization of D. melanogaster by an Acetobacter fabarum strain. Follow-up experiments revealed that each flagellar mutant was nonmotile, even though the wild-type strain was motile. Taken together, these results reveal that transposon insertions in specific bacterial genes, including motility genes, are necessary for at least one member of the fly microbiota to persistently colonize the fly. IMPORTANCE Despite the growing body of research on the microbiota, the mechanisms by which the microbiota colonizes a host can still be further elucidated. This study identifies bacterial genes that are associated with the colonization persistence phenotype of the microbiota in Drosophila melanogaster, which reveals specific bacterial factors that influence the establishment of the microbiota within its host. The identification of specific genes that affect persistence can help inform how the microbiota colonizes a host. Furthermore, a deeper understanding of the genetic mechanisms of the establishment of the microbiota could aid in the further development of the Drosophila microbiota as a model for microbiome research.