Application of fruit fly Tet protein in preparation of host IMD antibacterial immune defense product

Abstract

The application discloses the field of biotechnology, and relates to application of a fruit fly Tet protein in preparation of a host IMD antibacterial immune defense product, wherein the fruit fly Tet protein or a functional fragment thereof mediates host IMD antibacterial immune defense in a non-catalytic activity-dependent manner, the product is a reagent for regulating antibacterial immune defense mediated by a host IMD signal pathway, the reagent promotes the combination of a Rel protein and a downstream antibacterial peptide gene promoter by combining with an NF-kappa B transcription factor Rel protein, thereby regulating the transcription induction of the antibacterial peptide gene; the product is a reagent for evaluating the antibacterial immune defense capability of a host IMD signal pathway, the reagent evaluates the immune response state of the host to gram-negative bacteria infection by detecting the expression level of the Tet protein or the functional fragment thereof in fat body tissue or the interaction level of the Tet protein or the functional fragment thereof and the Rel protein; the application discloses a non-catalytic function of Tet in fruit fly innate immune transcription regulation, and widens the understanding of gene regulation mediated by Tet.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the application of Drosophila Tet protein in mediating host IMD antimicrobial immune defense in a non-catalytically activity-dependent manner. Background Technology

[0002] Innate immunity provides the first line of defense against microbial infections and is conserved from insects to mammals. In all metazoans, activation of innate immunity must be both rapid and precisely regulated. A core regulatory aspect of innate immunity is transcriptional control: immune signaling pathways ultimately converge on transcription factors that rapidly bind to pre-configured enhancers and promoters to initiate, amplify, and resolve gene expression programs adapted to the threat of infection. This transcriptional logic is not solely executed by DNA-binding factors. Instead, it relies on a broader nuclear environment constructed of cofactors, remodeling complexes, and epigenetic regulators that authorize DNA access, coordinate the dynamics of RNA polymerase II, and finely regulate the magnitude and duration of the response.

[0003] Drosophila melanogaster has become the primary animal model for elucidating the principles of innate immunity due to its easily manipulated genetics and a range of well-defined microbial challenges and detection methods. Two major pathways centered on nuclear factor κB (NF-κB) dominate the systemic antimicrobial immune defense in Drosophila. The Toll pathway primarily responds to Gram-positive bacteria and fungi, while the immunodeficiency (IMD) pathway is activated by many Gram-negative bacteria and some Gram-positive bacteria possessing diaminopimelic acid (DAP)-type peptidoglycans. In the Drosophila IMD signaling pathway, upstream pattern recognition is mainly mediated by peptidoglycan recognition proteins (PGRPs). PGRP-LC on the plasma membrane and PGRP-LE in the cytoplasm are key sensors for sensing DAP-type peptidoglycans, driving IMD signal transduction through connective modules and caspases. Downstream, the IκB kinase (IKK) complex and death-associated ced-3 / Nedd2-like caspase (Dredd) cleave the NF-κB family transcription factor Relish (Rel), which contains an N-terminal Rel homology domain (called... ) and C-terminal ankyrin repeat sequences (called The complex protein of Rel. Cleavage of Rel releases its A fragment translocates to the nucleus and binds to the κB motif to activate the transcription of antimicrobial peptide (AMP) genes, such as Attacin (Att), Cecropin (Cec), and Diptericin (Dpt). Loss-of-function or gain-of-function mutations within this pathway produce easily measurable phenotypes in AMP transcription and post-infection host survival, providing a sensitive detection method for molecules that regulate antimicrobial immune responses in Drosophila.

[0004] Although the topology of the IMD pathway is relatively well-defined, little is known about the nuclear co-regulatory systems that shape Rel target selectivity, enhance its occupancy at relatively weak κB sites, or maintain transcriptional “capacity” during or after microbial invasion. Pioneering work has shown that chromatin remodeling complexes and histone-modifying enzymes play roles in supporting the expression of downstream immune genes in IMD. These studies collectively demonstrate that immune loci exist in different chromatin states, and that transcription factors rely on resident cofactors to achieve efficient promoter clearance, productive elongation, and timely shutdown. These ideas naturally raise the question of which nuclear proteins are essential partners of Rel at AMP loci, and whether their functions are enzymatic, structural, or both.

[0005] Ten-eleven translocation (Tet) family proteins are Fe(II) / α-ketoglutarate-dependent dioxygenases that catalyze the stepwise oxidation of 5mC to 5hmC, 5fC, and 5caC, thereby initiating an active DNA demethylation pathway. In vertebrates, Tet family proteins (TET1 / 2 / 3) are crucial for epigenomic remodeling during development, lineage specialization, immunity, and disease. However, accumulated evidence over recent decades has expanded the functional scope of TET beyond catalyzing DNA demethylation. TET proteins can bind to the transcription machinery through protein-protein interactions, anchoring co-regulatory factors such as O-GlcNAc transferases (OGTs), histone deacetylase (HDAC) complexes, or chromatin “readers” to specific genomic regions. They can influence the pausing and release of RNA polymerase II, stabilize enhancer-promoter contacts, and cooperate with precursor factors to finely regulate transcriptional activity. Some of these activities persist in catalytically impaired mutants, highlighting the structural role of TET in chromatin, which can be separated from DNA oxidation itself. These non-catalytic functions of TET are particularly relevant in Drosophila because the DNA methylation profile in Drosophila is very sparse compared to mammals. The Drosophila genome encodes a single TET ortholog (Tet, also known as DNA 6mA demethylase, DMAD), and genetic studies have linked it to neuronal development, behavior, and other physiological processes. The relative lack of typical DNA methylation in Drosophila supports the hypothesis that the role of Tet in this organism may be disproportionately non-catalytic, or may extend to RNA-protein interactions independent of 5mC oxidation.

[0006] Whether and how Drosophila tet participates in immune transcription has not been fully explored to date. In particular, it is unclear whether tet interacts with NF-κB signaling in Drosophila, as suggested by the interactions between mammalian TETs and NF-κB family members in macrophages (Huang et al., 2019; Cobo et al., 2022; Lv et al., 2024) and other immune cells (Carrillo-Jimenez et al., 2019; Morante-Palacios et al., 2022; Lin et al., 2024). Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of Drosophila Tet protein in the preparation of antimicrobial immune defense products for host IMD.

[0008] The present invention achieves the above objectives through the following technical solutions: A first aspect of the invention provides the use of Drosophila Tet protein in the preparation of a product for host IMD antimicrobial immune defense, said Drosophila Tet protein or a functional fragment thereof mediating host IMD antimicrobial immune defense in a non-catalytically activity-dependent manner.

[0009] As a further optimization of the present invention, the product is a reagent for regulating host IMD signaling pathway-mediated antimicrobial immune defense. This reagent promotes the binding of the NF-κB transcription factor Rel protein to the promoter of downstream antimicrobial peptide genes by binding to the NF-κB transcription factor Rel protein, thereby regulating the transcriptional induction of antimicrobial peptide genes.

[0010] As a further optimization of the present invention, the product is a reagent for evaluating the host's antibacterial immune defense capability mediated by the IMD signaling pathway. This reagent assesses the host's immune response status to Gram-negative bacterial infection by detecting the expression level of Tet protein or its functional fragment in adipose tissue, or the interaction level between Tet protein or its functional fragment and Rel protein.

[0011] As a further optimization of the present invention, the amino acid sequence of the fruit fly Tet protein is shown in SEQ ID NO.1; the functional fragment of the fruit fly Tet protein contains amino acid residues 865-1560 of SEQ ID NO.1.

[0012] As a further optimization of the present invention, the antimicrobial peptide gene includes at least one of Attacin A, Cecropin A1, or Diptericin; The Gram-negative bacteria include at least one of Erwinia carotenoides subsp. carotenoides 15 (Ecc15) or Serratia marcescens.

[0013] As a further optimization of the present invention, the Tet protein or its functional fragments have no significant effect on antibacterial immune defense mediated by the Toll signaling pathway, which is activated by the Gram-positive bacterium Enterococcus faecalis.

[0014] As a further optimization of the present invention, the host is a fruit fly.

[0015] A second aspect of the present invention provides a method for in vitro screening of candidate substances capable of regulating antibacterial immune defense mediated by the Drosophila IMD signaling pathway for non-therapeutic purposes, comprising: (1) Provide an interaction system between Tet protein or its functional fragment with Rel binding ability and Rel protein; (2) Contact the candidate material with the interaction system; (3) Detect the effect of the candidate substance on the interaction between Tet protein and Rel protein or detect the effect of the candidate substance on the binding of Rel protein to the promoter of downstream antimicrobial peptide gene; the antimicrobial peptide gene includes at least one of Attacin A, Cecropin A1 or Diptericin; Among them, candidate substances that can enhance or inhibit the interaction between Tet protein and Rel protein, or that can enhance or inhibit the binding of Rel protein to the promoter of downstream antimicrobial peptide genes, were identified as positive substances that can regulate the antimicrobial immune defense mediated by the IMD signaling pathway.

[0016] As a further optimization of the present invention, the interacting system is selected from at least one of the following (1)-(3): (1) Protein extract from Drosophila fat body cells; (2) Drosophila S2 cells co-expressing Tet and Rel proteins; (3) Contains purified His-Tet protein and In vitro binding system for proteins.

[0017] A third aspect of the present invention also provides a kit for detecting the IMD signaling pathway in Drosophila, comprising the following components: An RNA interference construct for reducing Tet protein expression in Drosophila fat bodies; A detection reagent for detecting the expression level of at least one antimicrobial peptide gene downstream of the IMD signaling pathway, wherein the antimicrobial peptide gene is selected from Attacin A, Cecropin A1, or Diptericin; and Detection reagents for detecting the interaction between Tet and Rel proteins.

[0018] As a further optimization of the present invention, the RNA interference construct is a Tet RNAi strain, selected from at least one of the Vienna Drosophila Resource Center strains #102273 or #110549, for hybridization with the fat body-specific Gal4 driver *yolk-Gal4* or *r4-Gal4*.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention reports a previously undescribed function of the Tet protein in Drosophila melanogaster: mediating host antimicrobial innate immune defense through a mechanism independent of its enzymatic activity. Studies have demonstrated that gene silencing of Tet in the Drosophila fat body impairs activation of the post-infection immunodeficiency (IMD) signaling pathway, leading to defective antimicrobial peptide (AMP) induction and increased susceptibility in Drosophila. Furthermore, catalytically active mutants of Tet retain normal immune defense against bacterial pathogens, indicating that its enzymatic activity is non-essential for antimicrobial immune defense in Drosophila. Mechanistically, the Tet protein physically binds to the nuclear factor κB (NF-κB) transcription factor Rel in the IMD signaling pathway and promotes the binding of Rel to its downstream AMP gene. In summary, this invention reveals a non-catalytic function of Tet in the transcriptional regulation of innate immunity in Drosophila, broadening our understanding of Tet-mediated gene regulation. Attached Figure Description

[0020] Figure 1 The fat body-specific silencing of Tet provided by this invention affects the immune defense of flies infected with Ecc15.

[0021] Figure 2 The silencing of Tet in the fat body provided by this invention does not affect the fertility and body weight of fruit flies.

[0022] Figure 3 The silencing of Tet in the fat body provided by this invention weakens the defense of Drosophila against S. marcescens infection.

[0023] Figure 4 In the Toll antibacterial immune defense system for fruit flies provided by the present invention, silencing of the Tet gene in the fat body is not necessary.

[0024] Figure 5 The Tet provided by this invention modulates the antibacterial immune defense of fruit flies in a manner independent of catalytic activity.

[0025] Figure 6 The interaction group of Tet protein in the fat body of Drosophila provided by the present invention.

[0026] Figure 7 The Tet provided by this invention is combined with Rel physics in a domain-dependent manner.

[0027] Figure 8 The present invention provides Tet to enhance the binding of Rel to its downstream AMP gene.

[0028] Figure 9 The Tet protein provided by this invention does not affect the binding affinity between the Dl and Drs promoter regions. Detailed Implementation

[0029] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0030] 1. Materials In this invention, all experimental materials used, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0031] The amino acid sequence of the Drosophila Tet protein is shown in SEQ ID NO.1, and the CDS sequence encoding the Drosophila Tet protein is shown in SEQ ID NO.2.

[0032] 1.1 Fruit fly strains and breeding All fruit fly strains were reared on standard corn flour-yeast-agar medium (6.65% (w / v) corn flour, 7.15% (w / v) glucose, 5% (w / v) yeast, 0.66% (w / v) agar, 2.2% (w / v) parabens and 3.4 ml / L propionic acid) at 25°C under a 12h light / 12h dark cycle.

[0033] Before genetic hybridization, fruit flies and The strain has been backcrossed for at least 5 generations. The strain was purchased from the Bloomington Fruit Fly Stock Center.

[0034] The fruit flies used in the infection experiments were confirmed to be free of Wolbachia infection and known major fruit fly-associated pathogens, including Noravirus, DAV, microsporidia, and other detectable microbial contaminants.

[0035] For the RNA interference experiment, two Tet RNAi lines (Tet RNAi #1 and Tet RNAi #2) were purchased from the Vienna Drosophila Resource Center (#102273 and #110549) and hybridized with fat body-specific drivers *yolk-Gal4* or *r4-Gal4*.

[0036] The control fruit flies were the offspring of *yolk-Gal4* or *r4-Gal4* and GFP RNAi fruit flies.

[0037] Catalytically deactivated Tet strains , The *r4-Gal4* were purchased from the Bloomington Fruit Fly Stock Centre (#606061, #55778 and #33832).

[0038] The *yolk-Gal4* and GFP RNAi strains have been previously described and are available to the public (Cai et al., 2022 DOI: 10.1016 / j.celrep.2022.111795l; Wu et al., 2025 DOI: 10.1016 / j.ibmb.2025.104422).

[0039] 1.2 Drosophila S2 cells 1.2.1 Preparation of cell culture medium (1) Basic culture medium: Add 1% penicillin and streptomycin to Sf-900 III SFM medium, and aliquot 45mL into 50mL centrifuge tubes and store at 4℃.

[0040] (2) Complete culture medium: Add 5 mL (10%) fetal bovine serum to the basal culture medium and store at 4°C.

[0041] 1.2.2 Culture and passage of S2 cells (1) Incubation temperature The temperature is consistent with that used for fruit fly culture.

[0042] (2) S2 cell passage Cells cultured for 3-4 days (density 2×10⁻⁶) 7 After that, the cells were transferred to fresh complete culture medium at a ratio of 1:3.

[0043] 1.2.3. S2 Cell Cryopreservation and Thawing 1.2.3.1 Cell cryopreservation (1) Collecting cells: Centrifuge the cultured cells at low speed for 3 min (1000 rpm), discard the upper culture medium and retain the cell pellet.

[0044] (2) Preparation of cryopreservation cell solution: Add 1 mL of culture medium, 800 μL of serum and 200 μL of DMSO, mix gently with pipette tip and dispense into cryopreservation tubes, label with cryopreservation date and cell name.

[0045] (3) Cell preservation: First, store at 4℃ for a short time, then store at -20℃ for 1 hour, then transfer to a -80℃ refrigerator overnight, and finally transfer to liquid nitrogen for storage.

[0046] 1.2.3.2 Cell resuscitation (1) Rapid thawing: Quickly remove the frozen cells from liquid nitrogen and transfer the frozen cells to a water bath at 30°C to thaw.

[0047] (2) Cell activation: Wipe the cryovial with 75% ethanol, transfer the cells from the tube to a 6cm culture dish in the operating table, and add 5mL of culture medium. Incubate at 27℃ for 30min to activate the cells.

[0048] (3) Cell culture: Centrifuge the cultured cells at low speed for 3 minutes (1000 rpm), discard the upper culture medium and retain the cell pellet.

[0049] (4) Subsequent culture: After resuspending the cells in 5 mL of fresh culture medium, transfer them to a 6 cm culture dish. Observe the cell status in time. If the cells are growing well, transfer them to a 10 cm culture dish for further culture and continue to passage them according to the passage method.

[0050] 1.3 Antibodies (1) Primary antibodies: Rabbit anti-Tet (1:1000, Thermo Fisher, Cat#PA5-144404), mouse anti-β-tubulin (1:3000, Cowin, Cat#CW0098M), mouse anti-Flag (1:2000, Merck, Cat#F3165), rabbit anti-Flag (1:1000, Merck, Cat#F7425), rabbit anti-Myc (1:3000, Medical & Biological Laboratories, Cat#562), mouse anti-His antibody (1:3000, Santa Cruz, Cat#sc-57598), mouse anti-GST antibody (1:3000, Santa Cruz, Cat#sc-138), mouse anti-H3 (1:3000, Cell Signaling, Cat#4499S) and mouse anti-Dl (1:1000, Developmental Studies Hybridoma Bank, Cat#7A4). (2) Secondary antibodies: goat anti-mouse IgG H&L (1:5000, Abcam, Cat#ab150078) and goat anti-rabbit IgG H&L (1:5000, Abcam, Cat#ab6789).

[0051] 2. Method In this invention, all methods used are conventional methods known to those skilled in the art unless otherwise specified.

[0052] 2.1 Determination of bacterial strains, fruit fly survival rate, and bacterial load. Systemic infection experiments were conducted using Gram-negative bacteria *Serratia marcescens* and *Erwinia carotenoides* subsp. *carotene* 15 (Ecc15), and Gram-positive bacteria *Enterococcus faecalis*. *S. marcescens* and *E. faecalis* were purchased from the China General Microbiological Culture Collection Center (#1.1215 and #1.2135), while Ecc15 was kindly provided by Dr. Dominique Ferrandon's research group (Institute of Molecular and Cellular Biology, France).

[0053] For infection, overnight bacterial cultures were centrifuged to precipitate and resuspended in sterile phosphate-buffered saline (PBS) to a final concentration of OD600 = 1. Under CO2 anesthesia, 3-5 day old adult male fruit flies were selected, and each fly was injected with 4.6 nmol of bacterial suspension or an equal volume of PBS solution. The injected fruit flies were incubated at 29°C, and survival rates were monitored daily. Fruit flies that died within 2 hours of injection (<5% of the total number) were not included in the statistics.

[0054] Each survival experiment contained approximately 50 fruit flies per genotype and was repeated three times. Data from these replicates are presented as mean plus standard error. For fruit flies with a *yolk-Gal4* genetic background, female adults were used for bacterial infection experiments. In other cases, both male and female fruit flies were collected for infection experiments.

[0055] Bacterial load assay: Drosophila were collected at specified time points, immersed in 75% ethanol, and allowed to evaporate for 2 min on a Drosophila handling pad. Subsequently, the Drosophila were homogenized in 100 μl of sterile PBS and serially diluted. Finally, 100 μl of each dilution was plated on LB agar plates and incubated overnight at 30°C. Data were collected from 21 biological replicates and pooled.

[0056] 2.2 RT-qPCR Analysis Quantitative reverse transcription polymerase chain reaction (RT-qPCR) experiments were performed according to the previously described protocol. Briefly, total RNA was extracted from cultured Drosophila S2 cells, dissected tissue, or whole Drosophila using TRIzol reagent (Thermo Fisher, Cat#15596026), following the manufacturer's instructions. RNA integrity was assessed using a Bioanalyzer 2100. cDNA was synthesized from 1 µg of total RNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix kit (TransGen, Cat#AT341-01). Quantitative PCR was performed on a Light Cycler 480 using the SYBR Green One-Step kit (TransGen, Cat#AQ211-01). Relative expression levels were calculated using the ΔΔCt method and normalized with the housekeeping gene ribosomal protein L32 (RpL32) as an internal control.

[0057] The primers used in the RT-qPCR experiment are shown in Table 1.

[0058] Table 1. Primers used in RT-qPCR experiments

[0059] Real-time quantitative PCR reaction system: 2×SYBR Premix EX 10uL; cDNA 2uL; upstream primer (10uM) 0.8uL; downstream primer (10uM) 0.8uL; pure water 6.4uL; Real-time quantitative PCR reaction conditions: 98℃ for 2 minutes; 40 cycles (98℃ for 10 seconds; 60℃ for 10 seconds; 68℃ for 30 seconds); 95℃ for 15 seconds; 65℃ for 5 seconds.

[0060] 2.3 Western blot assay Western blot experiments were performed according to the previously described protocol. In brief, protein extracts were prepared by homogenizing dissected fat bodies or cultured Drosophila S2 cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, and 1 mM benzyl sulfonyl fluoride (PMSF). The lysis buffer was centrifuged at 14,000 g for 20 min at 4 °C. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher, Cat#23225).

[0061] Equal amounts of protein (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked for 1 h in 5% skim milk in PBST (PBS containing 0.1% Tween-20) buffer, and then incubated with the specified primary and secondary antibodies at room temperature for 1 h. Development was performed on a Bio-Rad platform using an enhanced chemiluminescent substrate (Tiangen, Cat#PA112-02).

[0062] 2.4 Immunoprecipitation co-IP and GST pull-down Cultured Drosophila S2 cells were transfected with the designated expression plasmid according to the previously described method. After 48 h, cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5% Triton X-100, and 1 mM PMSF). For in vivo samples, fat body tissue was dissected from designated Drosophila, and protein lysis buffer was prepared as above. After centrifugation at 14,000 g for 20 min at 4 °C, one-tenth of the supernatant was collected as the “input” sample. The remaining supernatant was incubated overnight at 4 °C with the designated antibody and agarose beads for immunoprecipitation.

[0063] For the GST pull-down experiment, the purified protein (His-Tet and...) The sample was incubated with GST beads (Millipore, Cat#G0924) in lysis buffer at 4°C for 2 h. Subsequently, the sample was washed three times (for a total of 1 h) at 4°C with wash buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.5% Triton X-100, and 10% glycerol). Western blotting was then performed using the specified antibody. IP efficiency was assessed using 20% ​​immunoprecipitate (IP sample), and 80% was used for co-IP detection.

[0064] 2.5 Detection of dual-luciferase reporter gene in Drosophila S2 cells For RNAi treatment, Drosophila S2 cells were first incubated with double-stranded RNA (dsRNAs) according to a prior protocol. Briefly, dsRNA was synthesized using the T7 RiboMAX Express large-scale RNA production system (Promega, Cat#P1320). 3 μg of dsRNA from each sample was added to S2 cells without fetal bovine serum (FBS). After 1 h, FBS was added to these cells to a final concentration of 10% dsRNA. After 48 h of treatment, cells were transfected with the Att-luciferase reporter plasmid and the Renilla luciferase plasmid as a transfection efficiency control. 36 h after transfection, cells were collected and lysed in 100 μL of Passive Lysis Buffer (Promega, Cat#E1960). After centrifugation, 20 μL of the supernatant was used to measure firefly or Renilla luciferase activity. Data were collected from three independent biological replicates.

[0065] Detailed information on the oligomers used for dsRNA synthesis is shown in Table 2.

[0066] Table 2. Information on Tet dsRNA oligomers

[0067] 2.6 mRNA decay detection Drosophila S2 cells were first treated with GFP (control) or Tet dsRNA as described above. Cells were then stimulated with heat-inactivated Ecc15 for 6 h, followed by treatment with 50 μg / mL actinomycin D (Sigma-Aldrich, Cat#A9415-10MG). Total RNA was extracted at specified time points (0, 0.5, 1, 2, and 4 h) after actinomycin D treatment, and the mRNA levels of AttA, CecA1, and Dpt were monitored by RT-qPCR. Data were collected from three independent biological replicates.

[0068] 2.7 Identification of Tet interactomes using IP-LC-MS / MS Immunoprecipitation and liquid chromatography-tandem mass spectrometry (IP-LC-MS / MS) were performed according to the previous protocol with slight modifications. Specifically, dissected fat bodies were homogenized in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5% Triton X-100, and 1 mM PMSF). After centrifugation at 14,000 g for 20 min at 4°C, the supernatant was collected and immunoprecipitated with anti-Tet antibody as described above. IgG was used as the control sample. After immunoprecipitation, the samples were washed three times (for a total of 1 h) at 4°C with wash buffer (500 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10% glycerol, and 0.5% Triton X-100), and then incubated with trypsin (Thermo Fisher, Cat#90057) at 37°C for 30 min. The samples were then desalted using a Pierce™ C-18 column (Thermo Fisher, Cat#89870) and analyzed by LC-MS / MS to identify the Tet interactome. LC-MS / MS data were processed using Thermo Proteome Discovery software (version 1.4.1.14) and compared with the UniProt-Drosophila database. Raw data are available online at Mendeley Data (https: / / data.mendeley.com / preview / mzbgtn757z?a=debe54c7-6974-49cd-84a9-0aeed61afd81).

[0069] 2.8 ChIP-qPCR Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) experiments were performed according to previously published protocols. Specifically, fat bodies were dissected from 100 Drosophila and incubated in 10 mL of ice-cold swelling buffer (0.1 M Tris-HCl, pH 7.5, 10 mM KOAc, 15 mM MgOAc, 1% NP-40, and 1 mM PMSF). The mixture was homogenized for 2 min using a loose-fitting Dounce homogenizer, fixed with 1% formaldehyde for 10 min, and the fixation was terminated with 125 mM glycine. After centrifugation at 1,000 g for 10 min at 4 °C, the pellet was resuspended in 10 mL of fresh swelling buffer and filtered through 70 μm and 40 μm cell filters, respectively. Samples were centrifuged at 1,000 g for 10 min at 4 °C to obtain nuclear precipitate, which was then lysed using lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1 mM dithiothreitol (DTT), and 1 mM PMSF). Chromatin was sheared to approximately 300 bp using sonication. Immunoprecipitation was performed using anti-Rel antibody (IgG was used in the control samples). DNA was purified and analyzed by qPCR using primers amplifying flanking sequences of specified gene promoters. Primers used in the ChIP-qPCR experiments are shown in Table 3.

[0070] Table 3. Primers used in ChIP-qPCR experiments

[0071] ChIP-qPCR reaction system: 10µL of 2×SYBR Green qPCR Master Mix; 1µL of forward primer (10µM); 1µL of reverse primer (10µM); 1µL of ChIP DNA template; ddH2O to 20µL.

[0072] ChIP-qPCR reaction conditions: Pre-denaturation: 95℃, 3 min; 45 amplification cycles: 95℃ for 10 s, 60℃ for 30 s; Melting curve: 95℃ for 15 s → 60℃ for 60 s → increase to 95℃ at 0.3℃ / s, and continuously collect fluorescence.

[0073] 2.9 Extraction of cytoplasmic and nuclear proteins Cytoplasmic and nuclear protein extraction was performed according to a previous protocol with slight modifications. Briefly, dissected fat bodies were lysed in 200 μL of cytoplasmic buffer (10 mM Tris-HCl, pH 7.5, 60 mM KCl, 1 mM EDTA, 0.075% NP40, 1 mM DTT, and 1 mM PMSF) and incubated on ice for 10 min. After centrifugation at 1,000 g for 5 min, the supernatant was collected as the cytoplasmic fraction. The nuclear pellet was resuspended in 100 μL of nuclear buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM PMSF, and 10% glycerol) and incubated on ice for 10 min. After centrifugation at 15,000 g for 10 min, the supernatant was collected as the nuclear fraction.

[0074] 2.10 Gel migration assay using EMSA detection An oligonucleotide probe containing a Rel binding site (GGGGAAGAAC) from the AttA promoter was synthesized. The binding reaction was performed using a 20 μL volume containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 5% glycerol, 0.1% NP-40, 50 ng / μL poly(dI-dC), and 50 ng of FAM-labeled probe. The purified... (5 μg) was incubated with increasing concentrations of Tet protein (0, 1, 2, 5 μg) at room temperature for 20 min. Subsequently, the reaction mixture was incubated with FAM-labeled Att probe (50 ng) at room temperature for another 20 min.

[0075] For the competition assay, a 40-fold molar excess of unlabeled probe (2 μg) was added to the binding reaction. The protein-DNA complexes were separated by electrophoresis at 100 V for 90 min on a 6% non-denaturing polyacrylamide gel at 4°C. After electrophoresis, the complexes were transferred to a positively charged nylon membrane using a semi-dry transfer instrument, UV cross-linked, and detected using a chemiluminescent nucleic acid detection module (Thermo Fisher, Cat#89880) according to the manufacturer's instructions. The chemiluminescent signal was captured using a ChemiDoc imaging system.

[0076] 2.11 Statistical Analysis Unless otherwise stated, all experiments were performed using at least three independent biological replicates. Data are presented as mean plus standard error. Statistical analysis was performed using GraphPad Prism 9. Survival curves were analyzed using the Log-Rank test. Multiple group comparisons were performed using one-way ANOVA followed by Tukey's post-hoc test. A p-value less than 0.05 was considered statistically significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

[0077] 3. Results 3.1 Tet is essential for the antibacterial immune defense of Drosophila IMD. Previous studies have shown that complete loss-of-function mutations in Tet lead to pupal lethality. To circumvent this developmental requirement of Tet and to investigate whether Tet is involved in the antimicrobial immune defense of Drosophila, this invention employs tissue-specific RNA interference (RNAi) technology to selectively silence Tet in the adult fat body, the main tissue / organ in Drosophila responsible for the production of systemic antimicrobial peptides. Two independent Tet RNAi transgenic lines were hybridized with the adult fat body-specific driver *yolk-Gal4* (referred to as yolk>Tet RNAi #1 and yolk>Tet RNAi #2, respectively), with age-matched yolk>GFP RNAi Drosophila serving as a non-targeted control. Western blot analysis confirmed a specific reduction in Tet protein levels in the fat body of both RNAi lines. Figure 1 A).

[0078] Importantly, Tet knockdown confined to the fat body does not affect adult fertility or body weight. Figure 2 (A and 2B) indicate that silencing Tet in the fat body does not produce significant physiological defects that could interfere with subsequent immune challenges.

[0079] Then, these Tet RNAi and control Drosophila were exposed to systemic infection with *Erwinia carotenoides* subsp. *carotenoides* 15 (Ecc15), a Gram-negative bacterium widely used to activate the immunodeficiency (IMD) signaling pathway in Drosophila. Notably, the Tet-silenced Drosophila exhibited accelerated mortality after Ecc15 challenge, dying much faster than the control Drosophila. Figure 1 B). Quantitative analysis of independent experimental cohorts showed that the median survival time of Tet RNAi animals after Ecc15 infection was reduced by approximately 70% to 80%. Figure 1 C). A key control is that injection of sterile PBS solution alone did not produce a difference in mortality between genotypes ( Figure 1(D) confirmed that the enhanced susceptibility was specifically attributed to bacterial infection rather than injection trauma. Consistent with impaired immune defenses, colony-forming unit (CFU) counts at 1 and 2 days post-infection showed that the bacterial load in Tet-silenced fruit flies was higher than in controls. Figure 1 E) indicates that its ability to limit pathogen proliferation is impaired.

[0080] To assess whether the observed phenotypes reflected transcriptional activation defects in the IMD pathway, the expression of downstream typical antimicrobial peptide (AMP) genes was then quantified. In control animals, Ecc15 infection induced strong transcriptional induction of Attacin A (AttA), Cecropin A1 (CecA1), and Diptericin (Dpt), with mRNA levels increasing 5 to 40-fold at different time points (3, 6, and 12 h post-infection). Figure 1 FH). In contrast, silencing Tet weakened this response, reducing AMP induction by nearly 25% to 60% in all three genes tested. Figure 1 This profound transcriptional defect provides a molecular explanation for the impaired bacterial clearance and increased mortality observed in Tet silent fruit flies.

[0081] To extend these findings, the fruit flies were then attacked with another Gram-negative pathogen, *Serratia marcescens*, which also activates IMD-dependent immunity in fruit flies (Neyen et al., 2014). Similar to the results obtained using Ecc15, Tet knockdown weakened the survival rate of fruit flies infected with *S. marcescens*. Figure 3 AB), bacterial clearance rate ( Figure 3 C) and AMP gene induction ( Figure 3 D-3F).

[0082] In addition, genetic experiments were performed using another fat body-specific driver, *r4-Gal4*. Consistent results were obtained when observing Drosophila survival, bacterial load, and AMP induction after Ecc15 infection. Figure 3 In summary, these complementary infection models demonstrate that Tet is essential for effective IMD signaling in Drosophila to ensure a robust antimicrobial response to pathogen challenge.

[0083] 3.2 Tet is not essential in Toll-mediated antimicrobial immunity. After establishing that Tet is crucial for Drosophila IMD-mediated antimicrobial immune defense, the question remains whether its function extends to the parallel Toll signaling pathway, which constitutes another major branch of Drosophila innate immunity.

[0084] To address this issue, Tet RNAi and control fruit flies were then challenged with *Enterococcus faecalis*, a Gram-positive bacterium that primarily activates the adult Toll signaling pathway rather than the IMD pathway. In stark contrast to the susceptibility observed when challenged with pathogens activating IMD, Tet RNAi and control animals exhibited statistically indistinguishable survival dynamics. Figure 4 A) indicates that Tet is not necessary for fruit flies to resist this Toll-dependent infection challenge.

[0085] Consistent with survival data, quantification of bacterial load showed no significant difference between Tet-silenced and control fruit flies at 1 or 2 days post-infection. Figure 4 B). To examine whether Toll pathway activation proceeds normally at the transcriptional level in the absence of Tet, the induction of typical Toll-responsive AMP genes was then measured. Following E. faecalis challenge, the expression of Drosomycin (Drs) and Metchnikowin (Mtk) was strongly increased and reached comparable levels in both Tet RNAi and control Drosophila. Figure 4 (C and 4D) indicate that the Toll-dependent transcriptional response remains intact when Tet is depleted in the fat body.

[0086] In summary, these findings reveal the pathway specificity of Tet immune function: while IMD signaling absolutely requires Tet, Tet is not essential in Toll-mediated antimicrobial immunity.

[0087] 3.3 Tet regulates Drosophila innate immunity independently of its dioxygenase activity. Tet family proteins have been widely identified as Fe(II) / α-ketoglutarate-dependent dioxygenases that catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby initiating an active DNA demethylation pathway. Although Drosophila Tet retains its characteristic catalytic domain and is classically annotated as a 5mC dioxygenase, the Drosophila genome exhibits very low levels of typical DNA methylation compared to vertebrate systems. This unique epigenetic landscape prompts us to critically examine whether the enzymatic activity of Tet is indeed essential for its immune function, or whether it may function through non-catalytic mechanisms in this context.

[0088] To rigorously test catalytic requirements, this invention utilizes a catalytically inactivated Tet allele in which the conserved HxD metal-binding triplet within the double-stranded β-helix (DSBH) catalytic core is replaced by YRA residues (referred to as...). These amino acid substitutions eliminated coordination of the essential Fe(II) cofactor and eliminated detectable 5mC oxidation activity in biochemical assays, while structural studies showed that the overall protein structure remained intact.

[0089] Next to The mutant fruit flies were infected with Ecc15 or S. marcescens.

[0090] The results showed that The survival dynamics of the animals after Ecc15 challenge were statistically indistinguishable from those of the control animals. Figure 5 (A-5C), indicating that the catalytic activity of Tet is not essential for host resistance to bacterial infection. Bacterial load measurements at 1 and 2 days post-infection also showed no significant difference between genotypes. Figure 5 D and 5E). Furthermore, the transcriptomic profile of the AMP gene shows that, following bacterial infection, AttA, CecA1, and Dpt are... Induced to a similar level in both control and control fruit flies ( Figure 5 (F-5K). These genetic complementation experiments demonstrate that Tet's significant contribution to antimicrobial immunity in Drosophila is independent of its dioxygenase activity.

[0091] 3.4. Tet interaction group in Drosophila fat body To identify potential candidate partners that may mediate the non-catalytic role of Tet in the antimicrobial immune defense of Drosophila IMD, the interactionome of Tet proteins in the Drosophila fat body was analyzed by immunoprecipitation and liquid chromatography-tandem mass spectrometry (IP-LC-MS / MS). Figure 6 A). Parallel samples treated with control IgG were used as negative controls to distinguish specific interactors from background contaminants. This invention applied a stringent enrichment threshold at least twice that of the IgG control to define high-confidence Tet-interacting proteins, ultimately identifying 53 proteins that met this criterion (…). Figure 6 B and Table S2).

[0092] To gain a deeper understanding of the functions of these Tet-related proteins / peptides, gene ontology (GO) enrichment analysis and pathway classification were performed using standard bioinformatics tools. The analysis revealed significant enrichment of proteins involved in several key biological processes. Figure 6 C). Furthermore, Relish (Rel, accession number: Q94527), an NF-κB transcription factor that is a core effector of the IMD signaling pathway, was observed to be highly enriched in Tet immunoprecipitates. Figure 6 (D and Table 4).

[0093] Table 4. Tet Interaction Group Information Table

[0094] 3.5 Tet integrates with Rel physics in a domain-dependent manner. The co-enrichment of Rel and Tet in unbiased proteomics screening prompted this invention to verify this interaction using orthogonal biochemical methods and to study its functional significance in immune gene regulation.

[0095] First, co-immunoprecipitation (co-IP) experiments were performed in the fat bodies of Drosophila before and after Ecc15 infection. For example... Figure 7 As shown in Figure A, using anti-Tet antibody immunoprecipitation, Flag-labeled Tet was successfully co-precipitated from fat body lysates. (N-terminal region of the Rel protein), while control IgG failed to reduce levels above background. Reciprocal immunoprecipitation experiments using anti-Flag antibodies also co-precipitated Tet (). Figure 7 B). These bidirectional co-IP results indicate that Tet and A stable complex was formed in the fat body cells. Notably, after Ecc15 attack, The endogenous interactions were unaffected. Figure 7 A and 7B).

[0096] To map the region responsible for Tet / Rel interactions, this invention generates a low-complexity region covering the N-terminus (amino acids 1-864, referred to as...). ), the middle region (amino acids 865-1560, referred to as ) and C-terminal catalytic core (amino acids 1561-2921, referred to as Tet truncated body ( Figure 7 C). In the co-IP experiment in Drosophila S2 cells, it was shown that... Retained Combining abilities, and or No detectable interactions were generated above the background. Figure 7 D). These data indicate that Tet is integrated with Rel physics in a region-dependent manner.

[0097] Finally, this invention conducted a GST pull-down experiment and found that purified His-Tet and Proteins directly bind to each other ( Figure 7 E).

[0098] 3.6 Tet promotes the binding of Rel to downstream AMP genes. Given the physical interaction between Tet and Rel in adiposome cells, this invention first hypothesizes that Tet promotes the occupancy of Rel at the κB regulatory site within the AMP locus, thereby driving strong transcriptional activation during the antibacterial immune response in Drosophila. Figure 8 A).

[0099] To verify this hypothesis, following Ecc15 infection, the levels of endogenous AMP gene promoters in dissected fat bodies were measured using chromatin immunoprecipitation combined with quantitative PCR (ChIP-qPCR). Enrichment. In Tet RNAi fat bodies, compared to controls, these AMP gene regulatory regions showed increased activity. Enrichment decreased by approximately 25% to 50%, while recruitment at non-target background sites remained at low levels and essentially unchanged. Figure 8 B-8D). As a pathway-specific control, the occupation of Dorsal (Dl) at the Drosomycin (Drs) promoter was then examined, a typical target of the Toll signaling pathway. Importantly, Tet RNAi did not affect the binding of Dl at the Drs locus ( Figure 9 A), which is consistent with the non-essentiality of Tet in Toll-mediated immune defense in Drosophila.

[0100] To further evaluate the functional consequences of Tet-mediated enhanced Rel-DNA binding, this invention employed luciferase reporter gene assays, in which multiple tandemly linked κB sites (Rel-binding sites, RBS) were placed upstream of a minimal promoter to drive luciferase expression (referred to as RBS-Luc). In Drosophila S2 cells, overexpression alone... This resulted in approximately 14-fold activation of the RBS-Luc reporter gene compared to baseline levels. Figure 8 E). Notably, Tet and Co-expression of these two factors synergistically increased luciferase activity by more than 28-fold, exceeding the additive effect of either factor acting alone. Figure 8 E).

[0101] Furthermore, Tet's silence weakened [the situation]. Driven reporter gene activation ( Figure 8 F), supporting Tet in A model where downstream processes act to facilitate transcriptional output. To directly test whether Tet affects... Regarding DNA binding ability, this invention uses purified [material] in the presence or absence of Tet protein. Electrophoretic mobility shift analysis (EMSA) was performed. Fluorescently labeled oligonucleotide probes containing a conserved κB motif from the Att promoter were used. As expected, individual... It showed binding to the κB probe, producing a characteristic migration band ( Figure 8 G).

[0102] It is worth noting that Tet and Co-expression of Tet significantly enhanced the strength of the protein-DNA complex, indicating that in the presence of Tet... Increased affinity Figure 8 G). This enhancement was eliminated when an excess of unlabeled competing probes was added, confirming... Specificity of interaction ( Figure 8 G). These EMSA results provide direct biochemical evidence that Tet enhances... Binding to the target DNA sequence.

[0103] 4. Conclusion The above studies demonstrate that Tet in Drosophila is an essential non-catalytic co-regulatory factor in antimicrobial innate immunity. By physically binding to Rel and promoting its binding to the regulatory region of AMP genes, Tet ensures a rapid and efficient transcriptional response to infection, a process independent of its dioxygenase activity. These findings extend the functional scope of Tet beyond DNA demethylation and elucidate a general principle of inducible gene control: enzymes typically studied for their chemical reactions can also act as architects of transcription, organizing factors at the right time and place to translate transient signals into powerful gene expression programs.

[0104] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of Drosophila Tet protein in the preparation of antimicrobial immune defense products for host IMD, characterized in that, The Drosophila Tet protein or its functional fragment mediates host IMD antimicrobial immune defense in a non-catalytic activity-dependent manner.

2. The application according to claim 1, characterized in that, The product is a reagent used to regulate host IMD signaling pathway-mediated antimicrobial immune defense. This reagent promotes the binding of the NF-κB transcription factor Rel protein to the promoter of downstream antimicrobial peptide genes by binding to the Rel protein, thereby regulating the transcriptional induction of antimicrobial peptide genes.

3. The application according to claim 1, characterized in that, The product is a reagent for assessing the host's antimicrobial immune defense capabilities mediated by the IMD signaling pathway. This reagent assesses the host's immune response to Gram-negative bacterial infections by detecting the expression level of Tet protein or its functional fragments in adipose tissue, or the interaction level between Tet protein or its functional fragments and Rel protein.

4. The application according to any one of claims 1-3, characterized in that, The amino acid sequence of the fruit fly Tet protein is shown in SEQ ID NO.1, and the functional fragment of the fruit fly Tet protein contains amino acid residues 865-1560 of SEQ ID NO.

1.

5. The application according to any one of claims 1-4, characterized in that, The antimicrobial peptide gene includes at least one of Attacin A, Cecropin A1, or Diptericin; The Gram-negative bacteria include at least one of Erwinia carotenoides subsp. carotenoides 15 (Ecc15) or Serratia marcescens.

6. The application according to any one of claims 1-5, characterized in that, The host is the fruit fly.

7. A method for in vitro screening of candidate substances for antibacterial immune defense mediated by the Drosophila IMD signaling pathway for non-therapeutic purposes, characterized in that, include: (1) Provide an interaction system between Tet protein or its functional fragment with Rel binding ability and Rel protein; (2) Contact the candidate material with the interaction system; (3) Detect the effect of the candidate substance on the interaction between Tet protein and Rel protein or detect the effect of the candidate substance on the binding of Rel protein to the promoter of downstream antimicrobial peptide gene; the antimicrobial peptide gene includes at least one of Attacin A, Cecropin A1 or Diptericin; Among them, candidate substances that can enhance or inhibit the interaction between Tet protein and Rel protein, or that can enhance or inhibit the binding of Rel protein to the promoter of downstream antimicrobial peptide genes, were identified as positive substances that can regulate the antimicrobial immune defense mediated by the IMD signaling pathway.

8. The method according to claim 7, characterized in that, The interacting system is selected from at least one of the following (1)-(3): (1) Protein extract from Drosophila fat body cells; (2) Drosophila S2 cells co-expressing Tet and Rel proteins; (3) Contains purified His-Tet protein and In vitro binding system for proteins.

9. A kit for detecting the IMD signaling pathway in Drosophila, characterized in that, It contains the following ingredients: An RNA interference construct for reducing Tet protein expression in Drosophila fat bodies; A detection reagent for detecting the expression level of at least one antimicrobial peptide gene downstream of the IMD signaling pathway, wherein the antimicrobial peptide gene is selected from Attacin A, Cecropin A1, or Diptericin; and Detection reagents for detecting the interaction between Tet and Rel proteins.

10. The reagent kit according to claim 9, characterized in that, The RNA interference construct is a Tet RNAi strain, selected from at least one of strains #102273 or #110549 from the Vienna Drosophila Resource Center, and is used for hybridization with the fat body-specific Gal4 driver *yolk-Gal4* or *r4-Gal4*.

Images