Antimicrobial peptides extend lifespan in Drosophila.

Antimicrobial peptides (AMPs) are important defense molecules of the innate immune system. High levels of AMPs are induced in response to infections to fight pathogens, whereas moderate levels induced by metabolic stress are thought to shape commensal microbial communities at barrier tissues. We expressed single AMPs in adult flies either ubiquitously or in the gut by using the inducible GeneSwitch system to tightly regulate AMP expression. We found that activation of single AMPs, including Drosocin, resulted in a significant extension of Drosophila lifespan. These animals showed reduced activity of immune pathways over lifetime, less intestinal regenerative processes, reduced stress response and a delayed loss of gut barrier integrity. Furthermore, intestinal Drosocin induction protected the animals against infections with the natural Drosophila pathogen Pseudomonas entomophila, whereas a germ-reduced environment prevented the lifespan extending effect of Drosocin. Our study provides new insights into the crosstalk of innate immunity, intestinal homeostasis and ageing.

Chitinases and Imaginal disc growth factors organize the extracellular matrix formation at barrier tissues in insects.

The cuticle forms an apical extracellular-matrix (ECM) that covers exposed organs, such as epidermis, trachea and gut, for organizing morphogenesis and protection of insects. Recently, we reported that cuticle proteins and chitin are involved in ECM formation. However, molecular mechanisms that control assembly, maturation and replacement of the ECM and its components are not well known. Here we investigated the poorly described glyco-18-domain hydrolase family in Drosophila and identified the Chitinases (Chts) and imaginal-disc-growth-factors (Idgfs) that are essential for larval and adult molting. We demonstrate that Cht and idgf depletion results in deformed cuticles, larval and adult molting defects, and insufficient protection against wounding and bacterial infection, which altogether leads to early lethality. We show that Cht2/Cht5/Cht7/Cht9/Cht12 and idgf1/idgf3/idgf4/idgf5/idgf6 are needed for organizing proteins and chitin-matrix at the apical cell surface. Our data indicate that normal ECM formation requires Chts, which potentially hydrolyze chitin-polymers. We further suggest that the non-enzymatic idgfs act as structural proteins to maintain the ECM scaffold against chitinolytic degradation. Conservation of Chts and Idgfs proposes analogous roles in ECM dynamics across the insect taxa, indicating that Chts/Idgfs are new targets for species specific pest control.

Forkhead, a new cross regulator of metabolism and innate immunity downstream of TOR in Drosophila.

Antimicrobial peptides (AMPs) are conserved cationic peptides which act both as defense molecules of the host immune system and as regulators of the commensal microbiome. Expression of AMPs is induced in response to infection by the Toll and Imd pathway. Under non-infected conditions, the transcription factor dFOXO directly regulates a set of AMP expression at low levels when nutrients are limited. Here we have analyzed whether target of rapamycin (TOR), another major regulator of growth and metabolism, also modulates AMP responses in Drosophila. We found that downregulation of TOR by feeding the drug rapamycin or by overexpressing the negative TOR regulators TSC1/TSC2, resulted in a specific induction of the AMPs Diptericin (Dpt) and Metchnikowin (Mtk). In contrast, overexpression of Rheb, which positively regulates TOR led to a repression of the two AMPs. Genetic and pharmacological experiments indicate that Dpt and Mtk activation is controlled by the transcription factor Forkhead (FKH), the founding member of the FoxO family. Shuttling of FKH from the cytoplasm to the nucleus is induced in the fat body and in the posterior midgut in response to TOR downregulation. The FKH-dependent induction of Dpt and Mtk can be triggered in dFOXO null mutants and in immune-compromised Toll and IMD pathway mutants indicating that FKH acts in parallel to these regulators. Together, we have discovered that FKH is the second conserved member of the FoxO family cross-regulating metabolism and innate immunity. dFOXO and FKH, which are activated upon downregulation of insulin or TOR activities, respectively, act in parallel to induce different sets of AMPs, thereby modulating the immune status of metabolic tissues such as the fat body or the gut in response to the oscillating energy status of the organism.

Targeting multifunctional proteins by virtual screening: structurally diverse cytohesin inhibitors with differentiated biological functions.

Virtual screening (VS) of chemical libraries formatted in silico provides an alternative to experimental high-throughput screening (HTS) for the identification of small molecule modulators of protein function. We have tailored a VS approach combining fingerprint similarity searching and support vector machine modeling toward the identification of small molecular probes for the study of cytohesins, a family of cytoplasmic regulator proteins with multiple cellular functions. A total of 40 new structurally diverse inhibitors were identified, and 26 of these compounds were more active than the primary VS template, a single known inhibitory chemotype, in at least one of three different assays (guanine nucleotide exchange, Drosophila insulin signaling, and human leukocyte cell adhesion). Moreover, these inhibitors displayed differential inhibitory profiles. Our findings demonstrate that, at least for the cytohesins, computational extrapolation from known active compounds was capable of identifying small molecular probes with highly diversified functional profiles.

FOXO-dependent regulation of innate immune homeostasis.

The innate immune system represents an ancient host defence mechanism that protects against invading microorganisms. An important class of immune effector molecules to fight pathogen infections are antimicrobial peptides (AMPs) that are produced in plants and animals. In Drosophila, the induction of AMPs in response to infection is regulated through the activation of the evolutionarily conserved Toll and immune deficiency (IMD) pathways. Here we show that AMP activation can be achieved independently of these immunoregulatory pathways by the transcription factor FOXO, a key regulator of stress resistance, metabolism and ageing. In non-infected animals, AMP genes are activated in response to nuclear FOXO activity when induced by starvation, using insulin signalling mutants, or by applying small molecule inhibitors. AMP induction is lost in foxo null mutants but enhanced when FOXO is overexpressed. Expression of AMP genes in response to FOXO activity can also be triggered in animals unable to respond to immune challenges due to defects in both the Toll and IMD pathways. Molecular experiments at the Drosomycin promoter indicate that FOXO directly binds to its regulatory region, thereby inducing its transcription. In vivo studies in Drosophila, but also studies in human lung, gut, kidney and skin cells indicate that a FOXO-dependent regulation of AMPs is evolutionarily conserved. Our results indicate a new mechanism of cross-regulation of metabolism and innate immunity by which AMP genes can be activated under normal physiological conditions in response to the oscillating energy status of cells and tissues. This regulation seems to be independent of the pathogen-responsive innate immunity pathways whose activation is often associated with tissue damage and repair. The sparse production of AMPs in epithelial tissues in response to FOXO may help modulating the defence reaction without harming the host tissues, in particular when animals are suffering from energy shortage or stress.