Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract

doi:10.1016/j.bbrc.2015.09.138

Biochemical and Biophysical Research Communications

Volume 467, Issue 2, 13 November 2015, Pages 400-406

https://doi.org/10.1016/j.bbrc.2015.09.138 Get rights and content

Highlights

•
The Drosophila larva-derived tissue extract activates the Toll pathway.
•
The extract activates Toll without Spz or bacterial components.
•
Genome-wide RNAi screening revealed damage-related signaling factors.

Abstract

Damage-associated molecular patterns (DAMPs), so-called “danger signals,” play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. Here, we demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, we performed ex vivo genome-wide RNAi screening in Drosophila cultured cells, and identified several signaling factors that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that our larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.

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 spz^rm7 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)

B. Lemaitre et al.
The host defense of Drosophila melanogaster
Annu. Rev. Immunol.
(2007)
D. Ferrandon et al.
The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections
Nat. Rev. Immunol.
(2007)
A. Kleino et al.
The Drosophila IMD pathway in the activation of the humoral immune response
Dev. Comp. Immunol.
(2014)
H. Myllymaki et al.
The Drosophila imd signaling pathway
J. Immunol.
(2014)
S. Valanne et al.
The Drosophila Toll signaling pathway
J. Immunol.
(2011)
V. Gobert et al.
Dual activation of the Drosophila toll pathway by two pattern recognition receptors
Science
(2003)
T. Michel et al.
Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein
Nature
(2001)
M. Gottar et al.
Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors
Cell
(2006)
N. Buchon 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)
I.H. Jang et al.
A Spatzle-processing enzyme required for toll signaling activation in Drosophila innate immunity
Dev. Cell
(2006)

A.N. Weber et al.

Binding of the Drosophila cytokine Spatzle to Toll is direct and establishes signaling

Nat. Immunol.

(2003)

M. Yamamoto-Hino et al.

Dynamic regulation of innate immune responses in Drosophila by Senju-mediated glycosylation

Proc. Natl. Acad. Sci. U. S. A.

(2015)

M. Lewisa et al.

Cytokine Spätzle binds to the Drosophila immunoreceptor Toll with a neurotrophin-like specificity and couples receptor activation

Proc. Natl. Acad. Sci. U. S. A.

(2009)

S. Tauszig-Delamasure et al.

Drosophila MyD88 is required for the response to fungal and Gram-positive bacterial infections

Nat. Immunol.

(2002)

H. Sun et al.

Regulated assembly of the Toll signaling complex drives Drosophila dorsoventral patterning

EMBO J.

(2004)

H. Sun et al.

A heterotrimeric death domain complex in Toll signaling

Proc. Natl. Acad. Sci. U. S. A.

(2002)

L.R. Marek et al.

Phosphoinositide Binding by the Toll Adaptor dMyD88 Controls Antibacterial Responses in Drosophila

Immunity

(2012)

H. Kanoh et al.

Genome-wide RNAi screening identifies the E3 ligase Sherpa required for Toll innate immune signaling in Drosophila adults

Sci. Signal

(2015)

Cited by (14)

Regulating metabolism to shape immune function: Lessons from Drosophila
2023, Seminars in Cell and Developmental Biology
Citation 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].
Infection with pathogenic microbes is a severe threat that hosts manage by activating the innate immune response. In Drosophila melanogaster, the Toll and Imd signaling pathways are activated by pathogen-associated molecular patterns to initiate cellular and humoral immune processes that neutralize and kill invaders. The Toll and Imd signaling pathways operate in organs such as fat body and gut that control host nutrient metabolism, and infections or genetic activation of Toll and Imd signaling also induce wide-ranging changes in host lipid, carbohydrate and protein metabolism. Metabolic regulation by immune signaling can confer resistance to or tolerance of infection, but it can also lead to pathology and susceptibility to infection. These immunometabolic phenotypes are described in this review, as are changes in endocrine signaling and gene regulation that mediate survival during infection. Future work in the field is anticipated to determine key variables such as sex, dietary nutrients, life stage, and pathogen characteristics that modify immunometabolic phenotypes and, importantly, to uncover the mechanisms used by the immune system to regulate metabolism.
Dual comprehensive approach to decipher the Drosophila Toll pathway, ex vivo RNAi screenings and immunoprecipitation-mass spectrometry
2019, Biochemical and Biophysical Research Communications
The Drosophila Toll pathway is involved in embryonic development, innate immunity, and cell-cell interactions. However, compared to the mammalian Toll-like receptor innate immune pathway, its intracellular signaling mechanisms are not fully understood. We have previously performed a series of ex vivo genome-wide RNAi screenings to identify genes required for the activation of the Toll pathway. In this study, we have conducted an additional genome-wide RNAi screening using the overexpression of Tube, an adapter molecule in the Toll pathway, and have performed a co-immunoprecipitation assay to identify components present in the dMyd88-Tube complex. Based on the results of these assays, we have performed a bioinformatic analysis, and describe candidate molecules and post-translational modifications that could be involved in Drosophila Toll signaling.
Characterization of Spz5 as a novel ligand for Drosophila Toll-1 receptor
2018, Biochemical and Biophysical Research Communications
Citation 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.
The Drosophila Toll-1 receptor is involved in embryonic development, innate immunity, and tissue homeostasis. Currently, as a ligand for the Toll-1 receptor, only Spätzle (Spz) has been identified and characterized. We previously reported that Drosophila larva-derived tissue extract contains ligand activity for the Toll-1 receptor, which differs from Spz based on the observation that larval extract prepared from spz mutants possessed full ligand activity. Here, we demonstrate that Spz5, a member of the Spz family of proteins, functions as a ligand for the Toll-1 receptor. Processing of Spz5 by Furin protease, which is known to be important for ligand activity of Spz5 to Toll-6, is not required for its function to the Toll-1 receptor. By generating a spz5 null mutant, we further showed that the Toll-1 ligand activity of larva-derived extract is mainly derived from Spz5. Finally, we found a genetic interaction between spz and spz5 in terms of developmental processes. This study identified a novel ligand for the Drosophila Toll-1 receptor, providing evidence that Toll-1 is a multi-ligand receptor, similar to the mammalian Toll-like receptor.
Dynamic miRNA-mRNA regulations are essential for maintaining Drosophila immune homeostasis during Micrococcus luteus infection
2018, Developmental and Comparative Immunology
Citation 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.
Pathogen bacteria infections can lead to dynamic changes of microRNA (miRNA) and mRNA expression profiles, which may control synergistically the outcome of immune responses. To reveal the role of dynamic miRNA-mRNA regulation in Drosophila innate immune responses, we have detailedly analyzed the paired miRNA and mRNA expression profiles at three time points during Drosophila adult males with Micrococcus luteus (M. luteus) infection using RNA- and small RNA-seq data. Our results demonstrate that differentially expressed miRNAs and mRNAs represent extensively dynamic changes over three time points during Drosophila with M. luteus infection. The pathway enrichment analysis indicates that differentially expressed genes are involved in diverse signaling pathways, including Toll and Imd as well as orther signaling pathways at three time points during Drosophila with M. luteus infection. Remarkably, the dynamic change of miRNA expression is delayed by compared to mRNA expression change over three time points, implying that the “time” parameter should be considered when the function of miRNA/mRNA is further studied. In particular, the dynamic miRNA-mRNA regulatory networks have shown that miRNAs may synergistically regulate gene expressions of different signaling pathways to promote or inhibit innate immune responses and maintain homeostasis in Drosophila, and some new regulators involved in Drosophila innate immune response have been identified. Our findings strongly suggest that miRNA regulation is a key mechanism involved in fine-tuning cooperatively gene expressions of diverse signaling pathways to maintain innate immune response and homeostasis in Drosophila. Taken together, the present study reveals a novel role of dynamic miRNA-mRNA regulation in immune response to bacteria infection, and provides a new insight into the underlying molecular regulatory mechanism of Drosophila innate immune responses.
Unexpected role of the IMD pathway in Drosophila gut defense against Staphylococcus aureus
2018, Biochemical and Biophysical Research Communications
In this study, fruit fly of the genus Drosophila is utilized as a suitable model animal to investigate the molecular mechanisms of innate immunity. To combat orally transmitted pathogenic Gram-negative bacteria, the Drosophila gut is armed with the peritrophic matrix, which is a physical barrier composed of chitin and glycoproteins: the Duox system that produces reactive oxygen species (ROS), which in turn sterilize infected microbes, and the IMD pathway that regulates the expression of antimicrobial peptides (AMPs), which in turn control ROS-resistant pathogens. However, little is known about the defense mechanisms against Gram-positive bacteria in the fly gut. Here, we show that the peritrophic matrix protects Drosophila against Gram-positive bacteria S. aureus. We also define the few roles of ROS in response to the infection and show that the IMD pathway is required for the clearance of ingested microbes, possibly independently from AMP expression. These findings provide a new aspect of the gut defense system of Drosophila, and helps to elucidate the processes of gut-microbe symbiosis and pathogenesis.
Identification and expression analysis of IκB and NF-κB genes from Cyclina sinensis
2016, Fish and Shellfish Immunology
Citation 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.
With the increasing economic importance of Cyclina sinensis aquaculture, interest in its defense mechanisms against pathogenic infection has grown in recent years. Inhibitor of nuclear factor-kappaB (IκB) and nuclear factor-kappaB (NF-κB) are proteins with central roles in many important physiological and pathological processes, such as innate immune responses. In this study, we identified CsIκB and CsNF-κB genes from a C. sinensis transcriptome library. In healthy adult clams, CsIκB and CsNF-κB genes were widely expressed in various tissues and highly expressed in hemocytes. Further, the expression levels of these genes were significantly increased in hemocytes challenged by Vibrio anguillarum, Micrococcus luteus and poly I:C. Inhibition of CsMyD88 expression by RNAi technology significantly altered the mRNA expression patterns of CsIκB and CsNF-κB as measured using quantitative real-time PCR. These results collectively indicated that the NF-κB signaling pathway, including CsIκB and CsNF-κB genes, might be involved in early innate immune responses and may be regulated by a MyD88-dependent signaling pathway in C. sinensis.

View all citing articles on Scopus

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.

View full text

Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract

Highlights

Abstract

Introduction

Section snippets

Purification and characterization of the Drosophila larva-derived tissue extract

Purification and characterization of the Drosophila larva-derived tissue extract that potentiates Toll pathway activation

Discussion

Funding

Author contributions

Conflict of interest

Acknowledgments

The host defense of Drosophila melanogaster

Annu. Rev. Immunol.

The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections

Nat. Rev. Immunol.

The Drosophila IMD pathway in the activation of the humoral immune response

Dev. Comp. Immunol.

The Drosophila imd signaling pathway

J. Immunol.

The Drosophila Toll signaling pathway

J. Immunol.

Dual activation of the Drosophila toll pathway by two pattern recognition receptors

Science

Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein

Nature

Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors

Cell

A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway

Proc. Natl. Acad. Sci. U. S. A.

A Spatzle-processing enzyme required for toll signaling activation in Drosophila innate immunity

Dev. Cell

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