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In Drosophila , the endoplasmic reticulum-associated protein degradation (ERAD) is engaged in regulating pleiotropic biological processes, with regard to retinal degeneration, intestinal homeostasis, and organismal development. The extent to which it functions in controlling the fly innate immune defense, however, remains largely unknown. Here, we show that blockade of the ERAD in fat bodies antagonizes the Toll but not the IMD innate immune defense in Drosophila . Genetic approaches further suggest a functional role of Me31B in the ERAD-mediated fly innate immunity. Moreover, we provide evidence that silence of Xbp1 other than PERK or Atf6 partially rescues the immune defects by the dysregulated ERAD in fat bodies. Collectively, our study uncovers an essential function of the ERAD in mediating the Toll innate immune reaction in Drosophila ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhu, Liu, Zhang, Zhang, Han, Duan, Jin, Guo, She, Xiao, Goto, Cai and Ji.)

Drosophila immune deficiency (IMD) pathway is similar to the human tumor necrosis factor receptor (TNFR) signaling pathway and is preferentially activated by Gram-negative bacterial infection. Recent studies highlighted the importance of IMD pathway regulation as it is tightly controlled by numbers of negative regulators at multiple levels. Here, we report a new negative regulator of the IMD pathway, Verloren (Velo). Silencing of Velo led to constitutive expression of the IMD pathway dependent antimicrobial peptides (AMPs), and Escherichia coli stimulation further enhanced the AMP expression. Epistatic analysis indicated that Velo knock-down mediated AMP upregulation is dependent on the canonical members of the IMD pathway. The immune fluorescent study using overexpression constructs revealed that Velo resides both in the nucleus and cytoplasm, but the majority (~ 75%) is localized in the nucleus. We also observed from in vivo analysis that Velo knock-down flies exhibit significant upregulation of the AMP expression and reduced bacterial load. Survival experiments showed that Velo knock-down flies have a short lifespan and are susceptible to the infection of pathogenic Gram-negative bacteria, P. aeruginosa. Taken together, these data suggest that Velo is an additional new negative regulator of the IMD pathway, possibly acting in both the nucleus and cytoplasm., (© 2021. The Author(s).)

The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor κB (NF-κB) signaling pathway, we undertook an in vitro RNAi screen targeting E3 ubiquitin ligases specifically and identified the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) as a new actor in the IMD pathway. Hyd mediated Lys63 (K63)-linked polyubiquitination of the NF-κB cofactor Akirin was required for efficient binding of Akirin to the NF-κB transcription factor Relish. We showed that this Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-κB transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. We showed further that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-κB pathway for the activation of Interleukin 6 (IL6) transcription by LPS or IL-1β in cultured human cells. Our findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases., Competing Interests: The authors have declared that no competing interests exist.

Innate immunity is an evolutionarily conserved host defense system against infections. The fruit fly Drosophila relies solely on innate immunity for infection defense, and the conservation of innate immunity makes Drosophila an ideal model for understanding the principles of innate immunity, which comprises both humoral and cellular responses. The mechanisms underlying the coordination of humoral and cellular responses, however, has remained unclear. Previously, we identified Gyc76C, a receptor-type guanylate cyclase that produces cyclic guanosine monophosphate (cGMP), as an immune receptor in Drosophila . Gyc76C mediates the induction of antimicrobial peptides for humoral responses by a novel cGMP pathway including a membrane-localized cGMP-dependent protein kinase, DG2, through downstream components of the Toll receptor such as dMyD88. Here we show that Gyc76C is also required for the proliferation of blood cells (hemocytes) for cellular responses to bacterial infections. In contrast to Gyc76C-dependent antimicrobial peptide induction, Gyc76C-dependent hemocyte proliferation is meditated by a small GTPase, Ras85D, and not by DG2 or dMyD88, indicating that Gyc76C mediates the cellular and humoral immune responses in distinct ways., (Copyright © 2020 Iwashita, Suzuki, Goto, Oyama, Kanoh, Kuraishi, Fuse, Yano, Oshima, Dow, Davies and Kurata.)

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKβ and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKβ and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKβ to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.

The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-B family, the Toll and the immune deficiency (IMD) pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study from this laboratory reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. Here, we focus on DNA methyltransferase-associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-B protein. We show that silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of Escherichia coli-induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma-associated protein 55 kDa (BAP55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling.

Intracellular bacteria cause serious infectious diseases such as tuberculosis, shigellosis, and listeriosis. The Drosophila peptidoglycan recognition protein (PGRP)-LE functions as an important host pattern recognition receptor against intracellular bacteria such as Listeria monocytogenes. One PGRP-LE-mediated intracellular response against L. monocytogenes infection is the induction of autophagy, a conserved intracellular degradation system. Here, to further elucidate PGRP-LE-mediated intracellular innate immune responses, we performed a strategic microarray analysis and identified the Listericin gene, whose expression is induced in response to L. monocytogenes infection in a PGRP-LE-dependent manner. RNA interference and overexpression experiments demonstrated that Listericin gene induction is cooperatively regulated by PGRP-LE and the JAK-STAT (Janus kinase-signal transducers and activators of transcription) pathway. An in vitro cell culture assay showed that Listericin is secreted as processed forms and suppresses the growth of L. monocytogenes and Gram-negative bacteria. A colony formation unit assay clearly demonstrated that induction of the Listericin gene suppresses not only the growth of L. monocytogenes but also the growth of Gram-negative bacteria in vivo. Based on these findings, we propose that the Listericin gene encodes a novel antibacterial peptide-like protein whose induction is cooperatively regulated by PGRP-LE and the JAK-STAT pathway.

During a genome-wide screen with RNA-mediated interference, we isolated CG8580 as a gene involved in the innate immune response of Drosophila melanogaster. CG8580, which we called Akirin, encoded a protein that acted in parallel with the NF-kappaB transcription factor downstream of the Imd pathway and was required for defense against Gram-negative bacteria. Akirin is highly conserved, and the human genome contains two homologs, one of which was able to rescue the loss-of-function phenotype in drosophila cells. Akirins were strictly localized to the nucleus. Knockout of both Akirin homologs in mice showed that one had an essential function downstream of the Toll-like receptor, tumor necrosis factor and interleukin (IL)-1beta signaling pathways leading to the production of IL-6. Thus, Akirin is a conserved nuclear factor required for innate immune responses.

Hemolectin has been identified as a candidate clotting factor in Drosophila. We reassessed the domain structure of Hemolectin (Hml) and propose that instead of C-type lectin domains, the two discoidin domains are most likely responsible for the protein's lectin activity. We also tested Hml's role in coagulation and immunity in Drosophila. Here we describe the isolation of a new hml allele in a forward screen for coagulation mutants, and our characterization of this and two other hml alleles, one of which is a functional null. While loss of Hml had strong effects on larval hemolymph coagulation ex vivo, mutant larvae survived wounding. Drosophila thus possesses redundant hemostatic mechanisms. We also found that loss of Hml in immune-handicapped adults rendered them more sensitive to Gram(-) bacterial infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.

We have previously identified and characterized Drosophila hemolectin (Hml) in the conditioned medium of a Drosophila cell line. The deduced amino acids sequence contains a number of domains conserved in human von Willebrand factor, coagulation factor V/VIII, and complement factors. To characterize Hml localization and function, we have established two transgenic lines (hml-GAL4 and UAS-hmlRNAi). By crossing hml-GAL4 with UAS-eGFP, we observed that Hml is specifically expressed in embryonic and in larval hemocytes. We determined that Hml is expressed in a subpopulation of plasmatocytes and crystal cells but not in lamellocytes. hml RNAi larvae are viable and do not display obvious developmental defects under normal conditions. However, they show a bleeding defect upon injury. We confirmed that the failure to seal a wound is not due to a defect in melanin production or in phenoloxidase activity. The expression of antimicrobial peptides was not significantly affected on hml RNAi adults. Altogether, our data strongly suggest that Hml is involved in hemostasis and/or coagulation in Drosophila larvae.

Double-stranded RNA (dsRNA) gene interference is an efficient method to silence gene expression in a sequence-specific manner. Here we show that the direct injection of dsRNA can be used in adult Drosophila flies to disrupt function of endogenous genes in vivo. As a proof of principle, we have used this method to silence components of a major signaling cascade, the Toll pathway, which controls fruit fly resistance to fungal and Gram-positive bacterial infections. We demonstrate that the knockout is efficient only if dsRNA is injected in 4- or more day-old flies and that it lasts for at least 1 week. Furthermore, we report dsRNA-based epistatic gene analysis via injection of a mixture of two dsRNAs and propose that injection of dsRNA represents a powerful method for rapid functional analysis of genes in Drosophila melanogaster adults, particularly of those whose mutations are lethal during development.