Biochemical and Biophysical Research Communications
Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract
Introduction
The innate immune system is a conserved host defense program in multicellular organisms. Innate immune signaling in Drosophila depends on two distinct signaling pathways, the Toll and immune deficiency (Imd) pathways [1], [2]. The Imd pathway recognizes Diaminopimelic acid (DAP)-type peptidoglycans derived from Gram-negative bacteria via peptidoglycan recognition protein (PGRP)-LC and PGRP-LE on the plasma membrane or in the cytoplasm [3], [4], [5]. These receptors facilitate downstream signaling that activates nuclear factor-kappa B (NF-κB) and induces the expression of antimicrobial peptides via the adaptor adaptor protein Imd [3], [4]. In contrast, the Toll pathway recognizes Lysine (Lys)-type peptidoglycans derived from Gram-positive bacteria or β-glucans derived from fungi via the PGRP-SA/GNBP1 complex or GNBP3 in the hemolymph, a fluid analogous to blood in insects [5], [6], [7], [8]. The recognition of either activates modular serine protease (ModSP) [9], followed by activation of Spz-processing enzyme (SPE), and cleavage of Spätzle (Spz), which is a protein ligand of the Toll receptor [10], [11]. β-Galactosylation of Spz by the UDP-galactose transporter, Senju, inhibits processing of Spz during steady state and decreases bacterial infection [12]. The active-form of Spz induces conformational changes in the Toll receptor [13], and facilitates the recruitment of an adaptor complex called the Myddosome, which is comprised of Drosophila MyD88 (dMyd88), Tube, and protein kinase Pelle [14], [15], [16], [17]. Myddosome formation at the plasma membrane is maintained by the E3 ligase Sherpa via Lys63-linked ubiquitination [18], and this event induces full activation of intracellular signaling mediated by Pelle and several other protein kinases [18], [19], [20], [21]. Ultimately, these events trigger the phosphorylation and degradation of Cactus, a Drosophila homolog of IκB [19], [20], [21], [22], and nuclear translocation of NF-κB proteins Dif/dorsal, which induces the expression of antimicrobial peptides [23], [24].
The extracellular Toll pathway also recognizes Damage-associated molecular patterns (DAMPs) that have been demonstrated to play roles in host defense and disease progression [25]. Proteases and virulence factors released from Fungi and Gram-positive bacteria activate the serine protease persephone (psh) in the alternative pathway to SPE [9], [26]. Furthermore, improper cell-fate control by mutations in the initiator caspase Dronc or Drosophila Apaf-1 homolog Dark elicits systemic Toll pathway activation in larvae as a result of psh activation [27], [28], and induces dysregulation in energy metabolism via the Drosophila forkhead box protein O (dFoxO)-glycine N-methyltransferase (Gnmt) axis [29]. These reports suggest that damaged and secondary necrotic cells might release uncharacterized intrinsic ligands that extracellularly activate the Toll pathway. However, other than Spz or Drosophila neurotrophins [30], the intrinsic ligands that directly activate the Toll receptor have not yet been identified.
Here, we demonstrate that a Drosophila larva-derived tissue extract, which was partially purified from healthy larvae with a simple procedure, strongly potentiates Toll pathway activation via the Toll receptor in Drosophila cultured cells. Using the activity induced by the extract, we performed a genome-wide RNAi screening for damage-related signaling factors, and found that the Jumonji-like transcription factor Jarid2 is required for host defense via the Toll pathway against bacterial infection in adult flies.
Section snippets
Purification and characterization of the Drosophila larva-derived tissue extract
Third-instar larvae of normal and germ-free wild type (Oregon R), and spzrm7 homozygote mutants were washed in deionized water and immediately homogenized using a beads-based homogenizer (Precellys 24; Bertin Technologies) in acidic conditions supplemented with 0.1% trifluoroacetic acid (TFA; Wako). The lysate was separated into a precipitant (i.e., major proteins, lipids, and nucleotides) and supernatant (i.e., particular peptides and salts). The peptidic fraction in the supernatant was
Purification and characterization of the Drosophila larva-derived tissue extract that potentiates Toll pathway activation
We attempted to purify the active DAMPs from the Drosophila larva-derived tissue extract from whole third instar larvae, since the DAMPs may be released from damaged tissue upon infection of microbes or secondary necrosis. We set up a purification method that primarily salvages peptide compounds (Fig. 1A, and see Materials and Methods), and purified the larva-derived tissue extract from acidic lysates of whole larvae. We examined whether the extract induced Toll pathway activation using a
Discussion
In the current study, we demonstrate that the Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Our analysis suggests that the larva-derived tissue extract may contain direct ligands released from damaged tissue to activate the Toll receptor. Using the activity elicited by the extract, we performed genome-wide RNAi screening for signaling factors that mediate Toll pathway signaling in damage responses. Based on the genome-wide screening
Funding
This work was supported by grants from the Japan Science and Technology Agency (JST); the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT); the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN); a Global COE Research Grant (Tohoku University Ecosystem Adaptability); the Takeda Science Foundation; the Kao Foundation for Arts and Sciences; the Uehara Memorial Foundation; and the Futaba Electronics Memorial Foundation.
Author contributions
H.K. and T.K. conceived this study. H.K., T.K., R.W., and S.N. designed the experiments. S.N. provided the procedure for the larva-derived tissue extract. T.K. found the Drosomycin-inducing activity in larva-derived tissue extract. H.K., T.K., and R.W. performed the experiments for Fig. 1, and the data were analyzed by H.K., T.K., and R.W. T.K. performed the experiments for Fig. 2, and the data were analyzed by H.K. L.L.T. performed the experiments for Fig. 3, and the data were analyzed by H.K,
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgments
We are grateful to the Bloomington Stock Center, the Drosophila Genomics Resource Center at Indiana University, the Drosophila Genetic Resource Center at the Kyoto Institute of Technology, the Drosophila RNAi Screening Center, the Genetic Strain Research Center of National Institute of Genetics, and the Vienna Drosophila RNAi Center for fly stocks.
References (37)
- et al.
The host defense of Drosophila melanogaster
Annu. Rev. Immunol.
(2007) - et al.
The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections
Nat. Rev. Immunol.
(2007) - et al.
The Drosophila IMD pathway in the activation of the humoral immune response
Dev. Comp. Immunol.
(2014) - et al.
The Drosophila imd signaling pathway
J. Immunol.
(2014) - et al.
The Drosophila Toll signaling pathway
J. Immunol.
(2011) - et al.
Dual activation of the Drosophila toll pathway by two pattern recognition receptors
Science
(2003) - et al.
Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein
Nature
(2001) - et al.
Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors
Cell
(2006) - et al.
A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway
Proc. Natl. Acad. Sci. U. S. A.
(2009) - et al.
A Spatzle-processing enzyme required for toll signaling activation in Drosophila innate immunity
Dev. Cell
(2006)
Binding of the Drosophila cytokine Spatzle to Toll is direct and establishes signaling
Nat. Immunol.
Dynamic regulation of innate immune responses in Drosophila by Senju-mediated glycosylation
Proc. Natl. Acad. Sci. U. S. A.
Cytokine Spätzle binds to the Drosophila immunoreceptor Toll with a neurotrophin-like specificity and couples receptor activation
Proc. Natl. Acad. Sci. U. S. A.
Drosophila MyD88 is required for the response to fungal and Gram-positive bacterial infections
Nat. Immunol.
Regulated assembly of the Toll signaling complex drives Drosophila dorsoventral patterning
EMBO J.
A heterotrimeric death domain complex in Toll signaling
Proc. Natl. Acad. Sci. U. S. A.
Phosphoinositide Binding by the Toll Adaptor dMyD88 Controls Antibacterial Responses in Drosophila
Immunity
Genome-wide RNAi screening identifies the E3 ligase Sherpa required for Toll innate immune signaling in Drosophila adults
Sci. Signal
Cited by (14)
Regulating metabolism to shape immune function: Lessons from Drosophila
2023, Seminars in Cell and Developmental BiologyCitation Excerpt :In Drosophila, mechanical damage to larval tissues without cuticle puncture is sufficient to activate Drosomycin expression, in a manner partially dependent on Toll signaling pathway components [85]. Work is ongoing to identify molecules that serve as damage associated molecular patterns in the fruit fly [86,87]. Intracellular pathogens that invade host cells through phagocytosis and establish residence in the cytosol or vacuoles are protected from the humoral arm of the immune response [88].
Dual comprehensive approach to decipher the Drosophila Toll pathway, ex vivo RNAi screenings and immunoprecipitation-mass spectrometry
2019, Biochemical and Biophysical Research CommunicationsCharacterization of Spz5 as a novel ligand for Drosophila Toll-1 receptor
2018, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Statistical analyses were performed by the Student's t-test, and P values < 0.05 were considered significant. We previously reported that Drosophila larva-derived tissue extracts exhibit ligand activity for the Toll-1 receptor, and the activity is protease liable but different from Spz [20]. Further, we presented in that study that RNAi-mediated knockdown of Spz4 in adult flies moderately disrupted Toll pathway activation following infections caused by Gram-positive bacteria.
Dynamic miRNA-mRNA regulations are essential for maintaining Drosophila immune homeostasis during Micrococcus luteus infection
2018, Developmental and Comparative ImmunologyCitation Excerpt :The JAK/STAT pathway is also involved in Drosophila innate immune response (Agaisse and Perrimon, 2004; Myllymäki and Rämet, 2014), but it remains poorly understood by compared to Toll and Imd pathways. In recent years, many studies have identified new components and regulators involved in Drosophila immune responses, such as Sherpa, Pitslre, Doa, and Jarid2 are new members of the Toll pathway (Kanoh et al., 2015a, 2015b), whilst Iap2, Akirin, CYLD, Caspar, Dnr1, Zfh1 and Pirk can activete or inhibit the immune response of Imd pathway (Tsichritzis et al., 2007; Valanne et al., 2007; Goto and Geijn van de, 2008; Kleino et al., 2008). In fact, the Drosophila innate immune response is controlled and fine-tuned at multiple levels, in which may Integrate a complex network of gene regulation to prevent unwanted overactivation of immune response.
Unexpected role of the IMD pathway in Drosophila gut defense against Staphylococcus aureus
2018, Biochemical and Biophysical Research CommunicationsIdentification and expression analysis of IκB and NF-κB genes from Cyclina sinensis
2016, Fish and Shellfish ImmunologyCitation Excerpt :The results illustrated that different pathogenic infection activated degradation of IκB and NF-κB transcription that involved in innate immune response. In Drosophila, the Toll pathway was involved in mediating damage responses during bacterial infection and improper cell-fate control [31,32]. Two pathways that initiated downstream TLR signaling are known, namely the MyD88 and TIR domain-containing adapter inducing IFN-β(TRIF)-dependent pathways.
- 1
Present address: Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, Japan.
- 2
Present address: Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan.
- 3
These authors contributed equally to this work.