Use of rnf144a protein in the preparation of a medicament for ameliorating listeria monocytogenes infection

Abstract

The application belongs to the technical field of antibacterial drug research, and particularly relates to a use of RNF144A protein in preparation of a drug for improving Listeria monocytogenes infection. It is verified through experiments that knocking down the RNF144A protein leads to reduction of Lm infection and innate immune response.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial drug research technology, specifically relating to the use of RNF144A protein in the preparation of drugs to improve Listeria monocytogenes infection. Background Technology

[0002] Listeria monocytogenes is a Gram-positive bacterium and the causative agent of listeriosis, with Listeria monocytogenes (Lm) being the most common. Lm is widespread in nature, resistant to low temperatures and high osmotic pressures, and is transmitted via the fecal-oral route, making it one of the most deadly foodborne pathogens. In healthy individuals, Listeria infection can cause mild gastroenteritis or be completely asymptomatic. However, immunocompromised individuals and pregnant women are more susceptible, and infection can lead to sepsis and meningitis. In late pregnancy, infection can cause septic miscarriage due to chorioamnionitis. In 2010, there were 23,000 cases of listeriosis worldwide, with over 5,000 deaths. The most recent major outbreak occurred in South Africa in 2018, with 1,060 cases and 216 deaths. Once listeriosis develops, the mortality rate is as high as 20-40%. Scholars have found that 562 cases of listeriosis were reported in my country from 2011 to 2017, a significant increase compared to the previous decade. Although large-scale outbreaks of listeriosis are rare in my country, the high contamination rate of Listeria in fast food poses a serious threat to public health. Therefore, studying the mechanism of interaction between the host and Listeria infection will help us further understand the relationship between Listeria and the host, and may provide new ideas and strategies for the clinical treatment of related diseases. Summary of the Invention

[0003] In view of the above technical problems, the present invention provides the use of RNF144A protein in the preparation of a drug to improve Listeria monocytogenes infection. Experimental results show that RNF144A protein promotes Lm infection and Lm-induced innate immune response in vivo.

[0004] The specific technical solution provided by this invention is as follows:

[0005] This invention provides the use of RNF144A protein in the preparation of a drug to improve Listeria monocytogenes infection.

[0006] Preferably, Listeria monocytogenes infection is improved by reducing the expression level of RNF144A protein.

[0007] Preferably, Listeria monocytogenes infection is improved by using an inhibitor of the RNF144A protein.

[0008] More preferably, the inhibitor of the RNF144A protein is A2 or A3;

[0009] The sense chain sequence of A2 is 5'-GAGUACCCAGUGGAGCAGAUGACAA-3', as shown in SEQ ID NO: 3; the antisense chain sequence is 5'-UUGUCAUCUGCUCCACUGGGUACUC-3', as shown in SEQ ID NO: 4;

[0010] The sense chain sequence of A3 is 5'-GCCUGAAACAGUAUGUUGAGCUCUU-3', as shown in SEQ ID NO: 5; the antisense chain sequence is 5'-AAGAGCUCAACAUACUGUUUCAGGC-3', as shown in SEQ ID NO: 6.

[0011] More preferably, the inhibitor of the RNF144A protein is used to prepare a drug that inhibits the secretion of IFN-β, IL-6, or TNF-α.

[0012] More preferably, the inhibitor of the RNF144A protein is used to prepare a drug that enhances the immune response to Listeria monocytogenes infection.

[0013] More preferably, the inhibitor of the RNF144A protein is used to prepare an expression promoter of LC 3-II.

[0014] Preferably, the inhibitor of the RNF144A protein is used to prepare a BECN1 expression promoter.

[0015] The present invention also provides a medicament for improving Listeria monocytogenes infection, comprising an expression inhibitor of the RNF144A protein, and a technically acceptable excipient or carrier.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention provides the use of RNF144A protein in the preparation of drugs to improve Listeria monocytogenes infection. Experimental results show that RNF144A protein promotes Lm infection and Lm-induced innate immune responses in vivo, and inhibits Lm-induced autophagosome accumulation. Simultaneously, Lm infection increases the interaction between RNF144A protein and BECN 1. Attached Figure Description

[0018] Figure 1 RNF144A deficiency prevented mice from being infected with Listeria monocytogenes, but reduced the innate immune response. (A) Immunoblot analysis was performed on lysed cells after stimulation of BMDMs with Listeria monocytogenes. (B) Wild-type and RNF144A-deficient mice were injected intravenously with HSV-1 (1×10⁻⁶) 7After 24 hours of PFU, the expression level of RNF144A in the liver, lungs, and spleen of mice was analyzed by Western blotting. (C) Wild-type (n=13) and RNF144A-deficient mice (n=12) were injected intravenously with Lm (1.5×10⁻⁶) 5 CFUs / mouse). Mouse survival rate during the 15-day infection period. (D-E) Quantitative bacterial load (1.5 × 10⁻⁶) in the liver (D) and spleen (E) of wild-type and RNF144A-deficient mice (n=4) on day 3 after Lm injection. 5 CFU / mouse). (F~H) Wild-type and RNF144A-deficient mice (n=3) were subjected to Lm (1.5×10 5 Twenty-four hours after infection with CFU / mouse, real-time quantitative PCR was used to detect IFN-β (F), Cxcl10 (G), and TNF-α (H) in the liver, spleen, and lungs of mice. Wild-type (I-K) and RNF144A-deficient mice (n=3) were infected with Lm (1.5×10⁻⁶) 5 Enzyme-linked immunosorbent assay (ELISA) of serum IFN-β (I), IL-6 (J), and TNF-α (K) 6 h after CFU / mouse tail vein infection. β-actin was used as a loading control in all immunoblotting assays. Data are representative of three independent experiments and are expressed as mean ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

[0019] Figure 2RNF144A deficiency leads to decreased Lm infection and innate immune response at the cellular level. (A) CFUs obtained at specified time points from wild-type and RNF144A-deficient MEFs (MOI=10) infected with Lm. (B) CFUs obtained at 4 hours from wild-type and RNF144A-deficient BMDMs or PMs (MOI=10) infected with Lm. (C) Confocal microscopy images of Lm infected with wild-type and RNF144A-deficient MEFs 6 hours later. The left panel shows a merged view of DAPI (blue), Lm (red), actin (green), and the three channels. The middle panel shows a magnified view of the framed area in the merged view. Scale bar 10 μm (zoomed view). (K) The figure shows a statistical analysis of the total bacterial count and bacterial count per cell in all images (10 images per genotype). (D) Real-time quantitative PCR detection of wild-type and RNF144A-deficient BMDMs infected with Lm 8 hours later. (E) Wild-type and RNF144A-deficient BMDM cells were infected with Lm (MOI=10) for 24 h. The secretion of IFN-β, IL-6, and TNF-α was detected by ELISA. (F-H) Wild-type and RNF144A-deficient PM cells were infected with Lm cells for 8 h, and the secretion of IFN-β (F), Cxcl10 (H), and IL-6 (G) was detected by real-time PCR. (I) Wild-type and RNF144A-deficient PM cells were infected with Lm cells (MOI=10) for 24 h. The secretion of IFN-β, IL-6, and TNF-α was detected by ELISA. (J) Wild-type and RNF144A-deficient BMDM cells were infected with Lm cells (MOI=10) for a specified time, and then subjected to Western blotting. (L) PMA-THP1 cells were transfected with control siRNA (SC) or RNF144A-specific siRNA (A1, A2, and A3). Cells were lysed 24 h after transfection for Western blotting. (M) PMA-THP1 cells transfected with control siRNA (SC) or RNF144A-specific siRNA (A2). 24 h after transfection, cells were infected with Lm (MOI=10) and Lm load was measured. (N) PMA-THP1 cells transfected with control siRNA (SC) or RNF144A-specific siRNA (A2). 24 h after transfection, cells were infected with Lm (MOI=10) for 24 h, and IFN-β secretion was detected by ELISA. β-actin was used as a loading control in all immunoblotting assays. Data are representative of three independent experiments and are expressed as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001.

[0020] Figure 3RNF144A deficiency promoted Lm-induced autophagosome accumulation. (A) Wild-type and RNF144A-deficient MEFs were infected with Lm (MOI=10) for a specified time and then immunoblotting was performed. (B) LC3-II quantification. (C) Wild-type and RNF144A-deficient BMDM were infected with Lm (MOI=10) for a specified time and then immunoblotting was performed. (D) LC3-II quantification. (E) Wild-type and RNF144A-deficient MEFs were treated with PBS, Lm (MOI=10), or Lm and bafloxacin A1 (400 nM) for 2 hours and then immunoblotting was performed. (F) LC3-II quantification. (G) Wild-type and RNF144A-deficient MEFs were treated with C-176 (1 μM) or untreated for 24 hours and then infected with Lm (MOI=10) for 2 hours. Lysed cells were then immunoblotting performed. (H) LC3-II quantification. (I) STING-deficient (KO) THP1 cells were transfected with control siRNA (SC) or RNF144A-specific siRNA (A2). Twenty-four hours after transfection, cells were treated with Lm (MOI = 10) for a specified time. Cells were lysed for Western blotting. (J) LC3-II quantification. β-actin was used as a loading control in all Western blotting assays. Data are representative of three independent experiments and are expressed as mean ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

[0021] Figure 4RNF144A inhibits Lm-induced autophagosome accumulation. (A) HeLa cells were transfected with the vector (-) or RNF144A(+) plasmid. 24 h after transfection, cells were treated with Lm (MOI=10) for a specified time, followed by Western blotting. (B) LC3-II quantification. (C) HeLa cells were transfected with the vector (-) or RNF144A(+) plasmid. 24 h after transfection, cells were treated with PBS, Lm (MOI=10), or Lm (400 nM) for 2 h, followed by Western blotting. (D) LC3-II density. (E) HeLa-mCherry-LC3 cells were transfected with the vector (Vec) or RNF144A. 24 h after transfection, cells were infected with Lm (MOI=10) or uninfected for 4 h, followed by confocal microscopy. Scale bar 10 μm. The right figure shows LC3 spot quantification. (F) PMA-THP1 cells transfected with control siRNA (SC) or RNF144A-specific siRNA (A2 or A3). 24 h after transfection, cells were infected with Lm (MOI = 10) for a specified time, followed by lysis and Western blotting. (G) LC3-II quantification. (H) HeLa cells transfected with control siRNA (SC) or RNF144A-specific siRNA (A2). 24 h after transfection, cells were treated with Lm (MOI = 10) for a specified time, followed by Western blotting. (I) LC3-II quantification. (J) HeLa-mCherry-LC3 cells transfected with control siRNA (SC) or RNF144A-specific siRNA (A2). 24 h after transfection, cells were infected with Lm (MOI = 10) or uninfected for 4 h, followed by confocal microscopy. Scale bar 10 μm. The right figure shows LC3 spot quantification. (K) PMA-THP1 cells transfected with control siRNA (SC) or RNF144A-specific siRNA (A2). 24 h after transfection, cells were treated with DMSO, CQ (100 μM), bafloxacin A1 (400 nM), or NH4Cl (5 mM) for 4 h, respectively, followed by Western blotting. (N) LC3-II quantification. (L) HeLa-mCherry-LC3 cells transfected with control siRNA (SC) or RNF144A-specific siRNA (A2). 24 h after transfection, cells were treated with DMSO, CQ (100 μM), or bafloxacin A1 (400 nM) for 4 h, followed by confocal microscopy. Scale bar 10 μm. (M) LC3 spot quantification. β-actin was used as a loading control in all Western blotting assays. Data are representative of three independent experiments and are expressed as mean ± SD. *, p=<0.05, **, p<0.01, ***, p<0.001.

[0022] Figure 5This is an interaction between RNF144A and BECN1. (A, B) HEK293T cells were transfected with the specified plasmids. 24 h after transfection, immunoprecipitation (IP) and Western blotting (IB) were performed as instructed. (C) HeLa cells were transfected with the flag-BECN1 and HA-RNF144A plasmids. 24 h after transfection, immunofluorescence was performed using anti-flag (green) and anti-HA (red) plasmids. Cell nuclei were stained with DAPI. (D) Pixel intensity distribution along the white line, scale bar 10 μm. (E) PMA-THP1 cells were infected with Lm for a specified time, and then the cell lysates were analyzed using the specified method using immunoprecipitation (IP) and Western blotting (IB). (F) BMDMs were infected with Lm for a specified time, and then the cell lysates were analyzed using the specified method using immunoprecipitation (IP) and Western blotting (IB). (G) RNF144A-deficient (KO) MEF cells were transfected with Flag-RNF144A. 24 h after transfection, cells were infected with Lm or uninfected for 4 h, and then the colocalization of RNF144A and BECN1 was detected by in situ PLA. RNF144A-BECN1 complex, red; nucleus, blue. Scale bar 10 μm. (H) Schematic diagram of full-length BECN1. BH3, Bcl-2 homolog 3; CCD, double helix region; ECD, evolutionarily conserved domain. HEK293T cells were transfected with the specified plasmid. 24 h post-transfection, cell lysates were subjected to immunoprecipitation (IP) and Western blot (IB) assays as instructed (I). (J) Schematic diagram of full-length RNF144A. RING, a very interesting new gene; IBR, intermediate between RING; TM, transmembrane. HEK293T cells were transfected with the specified plasmid. 24 h post-transfection, cell lysates were subjected to immunoprecipitation (IP) and Western blot (IB) assays as instructed (K). These data are representative of three independent experiments.

[0023] Figure 6RNF144A promotes BECN1 degradation. HEK293T cells were transfected with HA-BECN1, resulting in increased RNF144A plasmid levels (0, 0.2, 0.4, 0.6 μg). Immunoblotting was performed on cell lysates 24 hours after transfection. (B) HEK293T cells were transfected with either the vector (-) or RNF144A (+) plasmid. 24 hours after transfection, cells were treated with CHX (50 μg / ml) for a specified time, and then immunoblotting was performed on cell lysates. (C) HEK293T cells were transfected with Flag-RNF144A. 24 hours later, cells were treated with DMSO, MG132 (20 μM), NH4Cl (5 mM), or 3-MA (2 mM) for 6 hours, respectively, followed by immunoblotting after lysis. (D1) PMA-THP1 cells were transfected with control siRNA (SC) or RNF144A-specific siRNA (A2). Twenty-four hours after transfection, cells were infected with Lm (MOI = 10) for a specified period, followed by Western blotting (IB) of cell lysates. BECN1 expression was quantified using β-actin, as shown in (D2). (E1) Wild-type (WT) and RNF144A-deficient (KO) MEF cells were infected with Lm (MOI = 10) for a specified time, followed by Western blotting. BECN1 expression was quantified using β-actin, as shown in (E2). (F1) Wild-type (WT) and RNF144A-deficient (KO) BMDM cells were infected with Lm (MOI = 10) for a specified time, followed by Western blotting. BECN1 expression was quantified using β-actin, as shown in (F2). (G1) Wild-type (WT) and RNF144A-deficient (KO) BMDM mice were treated with CHX (50 μg / ml) for a specified time, followed by immunoblotting of cell lysates. BECN1 expression was quantified by β-actin, see (G2). (HI) Wild-type (WT) and RNF144A-deficient (KO) PMa-THP1 mice were infected with Lm (MOI = 10) for a specified time, followed by immunoblotting. LC3-II expression was quantified by β-actin, as shown in (I2). (J1) Wild-type (WT) and RNF144A-deficient (KO) mice were either injected intravenously with PBS (n = 2) or infected with Lm (n = 3, 1.5 × 10⁵ CFU / mouse) for 2 days, followed by immunoblotting of liver and spleen. LC3-II and BECN1 expression levels are shown in (J2–J5). β-actin was used as a loading control in all immunoblotting assays. Data are representative of three independent experiments and are expressed as mean ± SD. *, p = < 0.05, **, p < 0.01.

[0024] Figure 7The RING domain is crucial for the role of RNF144A in Lm infection. (A1) RNF144A-deficient (KO) MEF cells were transfected with vector (-), RNF144A (WT) plasmid, or its mutant. After 24 h, cells were infected with Lm (MOI=10) for 2 h, and then the cell lysates were analyzed by immunoblotting. BECN1 expression was quantified by β-actin, see (A2). (B) RNF144A-deficient (KO) MEF cells were transfected with vector (-), RNF144A (WT), or its mutant. After 24 h, cells were treated with MG132 (20 μM) for 6 h, and then the cell lysates were analyzed by immunoprecipitation (IP) and immunoblotting (IB). (C) Immunoblotting detection of BECN1 ubiquitination in vitro. BECN1 and wild-type RNF144A or its mutant were rapidly translated in vitro, and then biotin-ubiquitin E1 and E2 were added for in vitro ubiquitination detection. Anti-ubi assay for BECN1 ubiquitination. (D) RNF144A-deficient (KO) MEF cells were transfected with vector (-), RNF144A (WT), or its mutant. After 24 h, cells were infected with Lm (MOI=10) for 2 h, and then the cell lysates were analyzed by Western blotting. (E) RNF144A-deficient (KO) MEF cells were transfected with vector (Vec), RNF144A (WT), or its mutant. After 24 h, cells were infected with Lm (MOI=10) for 4 h, and colony formation assays were performed. (F1) RNF144A-deficient (KO) MEF cells were transfected with vector (Vec), RNF144A (WT), or its mutant. After 24 h, cells were infected with Lm (MOI=10) for 6 h, and then observed by confocal microscopy. The left panel shows a combined view of DAPI (blue), actin (green), Lm (red), and the three channels. The middle panel shows a magnified view of the framed area in the combined view. Scale bar 10 μm (magnified view). (F2) shows statistical analysis of total bacterial count (top) and bacterial count per cell (bottom) across all images (10 images per genotype). (A) β-actin was used as a loading control in all immunoblotting assays. Data are representative of three independent experiments and are expressed as mean ± SD. **, p < 0.01.

[0025] Figure 8 In the presence of C-176, Lm induced an innate immune response in wild-type and RNF144A-deficient MEFs. Wild-type (WT) and RNF144A-deficient (KO) MEFs were treated with C-176 (1 μM) or untreated for 24 hours, followed by infection with Lm (MOI = 10) for 8 hours. Cell lysates were then used for real-time PCR detection. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] 1. Laboratory animals

[0028] RNF144A protein-deficient mice were purchased from Shanghai Model Biotechnology Co., Ltd. Mice were housed in an environment with food and water, maintaining a 12-hour light / 12-hour dark cycle. All animal procedures were performed according to guidelines approved by the Animal Care Committee of Xinxiang Medical University, China. Age- and sex-matched wild-type (WT) and RNF144A protein-deficient (KO) mice were used in the experiment.

[0029] 2. cDNA construction and reagents

[0030] Human RNF144A (NM_014746) and its deletion mutants were amplified by PCR using cDNA from THP1 cells, and then cloned into the pcDNA3 vector (Invitrogen). All RNF144A deletion mutants were constructed by PCR and subcloned into the pcDNA3 vector. Human BECN1 was obtained from China National Biotec Group (CNBG), and mutants were constructed using the Fast Site-Directed Mutagenesis Kit (KM101, TIANGEN).

[0031] The following antibodies were used for immunoblotting or immunoprecipitation: anti-Flag (F3165, Sigma-Aldrich), anti-HA (901515, Biolend), anti-Myc (66004-1-Ig, Proteintech), anti-p-TBK1 (5483T, Cell Signaling Technology), anti-TBK1 (CSB-PA024154LA01HU, Flarbio), anti-p-IRF3 (4947, Cell Signaling Technology), anti-IRF3 (11312-1-AP, Proteintech), anti-RNF144A (26144-1-AP, Proteintech; anti-BECN1 (11306-1-AP, Proteintech), anti-LC3 (MBL, PM036), anti-Lm (Abcam, ab35132), and anti-Ubi (sc-8017, Santa Cruz). Biotechnology), anti-Ubi-K48 (05-1307, Millipore), and anti-β-actin (60008-1, Proteintech).

[0032] PMA (S1819) was purchased from Beyotime Biotechnology. C-176 (S6575) was obtained from Selleck. Bafilomycin A1 (11038) was purchased from Cayman Chemical. Chloroquine (C6628), 3-MA (M9281), and NH4Cl (A9434) were purchased from Sigma-Aldrich. MG132 (474790) was purchased from Millipore.

[0033] 3. Cell culture, transfection, and stimulation

[0034] HEK293T and THP1 cells were provided by the Stem Cell Bank of the Chinese Academy of Sciences. HeLa and HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM). THP1 cells were grown in RPMI 1640. PMA-THP1 cells refer to THP1 cells pretreated with 100 ng / ml PMA for 24 h. All cells were cultured at 37°C and 5% CO2 with 10% FBS (Gibco), 4 mM 1-glutamine, 100 μg / ml penicillin, and 100 U / ml streptomycin. HeLa, HEK293T, THP1, and MEF cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

[0035] 4. Preparation of BMDM, PM and MEF cells

[0036] BMDM preparation: Mouse bone marrow cells were isolated, cultured with 30% L929 supernatant and 70% complete culture medium (i.e., 10% serum culture medium), and the medium was changed every 2 days. Cells were harvested on the 7th day.

[0037] PM cell preparation: Mice were intraperitoneally injected with 2.5 ml of 4% Brewers TG solution. Four days later, the mice were sacrificed, and the peritoneum was exposed under aseptic conditions, keeping it intact. 5 ml of pre-chilled PBS was drawn up with a syringe and injected into the peritoneum without removing the needle. The peritoneum was gently massaged with a finger, and the saturated cells were aspirated. The cells were then rinsed once with fresh, pre-chilled PBS. The cells were centrifuged at 1000 rpm for approximately 5 minutes, and the supernatant was discarded. Cell counting was performed. Cells were seeded into 24-well plates at 1 × 10⁶ cells per well. 6 After 6 hours, the non-adherent suspended cells were washed away, and the remaining cells were peritoneal macrophages.

[0038] MEF preparation: Pregnant mice aged 12.5–14.5 days were used, and offspring were removed under aseptic conditions. The embryonic head, internal organs, and limbs were removed. The torso was placed in another petri dish containing PBS and washed at least three times with PBS to thoroughly remove red blood cells. The mouse embryonic torso was cut into 1 mm pieces.3 The following fragments were placed in centrifuge tubes. After digestion with an appropriate amount of trypsin, the cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and an appropriate amount of culture medium was added. The cells were then repeatedly pipetted 20 times. The cells were then incubated in a 37°C, 5% CO2, saturated humidity incubator.

[0039] 5. Immunoprecipitation and Western blot detection

[0040] For the immunoprecipitation experiment, first prepare 1×Lysis buffer (500 μl of weak RIPA lysis buffer + 1% PMSF per well). Discard the original cell culture medium of the pretreated cells and place them on ice. Add 500 μl of 1×Lysis buffer to each well, gently shake to mix, and incubate on ice for about 20 min for lysis. After lysis, use a pipette to remove cell debris from the bottom of the cell culture plate, collect and transfer to pre-chilled EP tubes, balance and centrifuge at 12000 rpm for 20 min at 4°C. After centrifugation, add 40 μl of supernatant to the corresponding Input group EP tube, vortex to mix, and place in a metal bath at 100°C for 10 min, then store at -20°C. Transfer all remaining supernatant to the IP group EP tubes, shake on a rotary shaker for 60 min, add 25 μl of Protein A / Gbeads to each EP tube, and then incubate overnight at 4°C. On the second day, balance the IP samples and centrifuge them at 3000 rpm for 1 minute in both directions at 4°C. After centrifugation, discard the supernatant. Add 500 μl of 1× washing buffer to each EP tube, shake and wash on a rotary shaker for 1 minute, then centrifuge again for 1 minute. Repeat the washing process three times. Add 20 μl of 1× loading buffer and boil at 100°C for 10 minutes before performing the immunoblotting experiment.

[0041] 6. Real-time PCR

[0042] Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. All gene transcripts were quantified by real-time PCR using a SYBR Green qPCR Master Mix system (Applied Biosystems) with a 7500 Fastreal-time PCR system. Two [units / items / etc.] were used. -△△Ct The relative refractive index was calculated using this method. The primers used for real-time PCR are as follows:

[0043] Mouse ifn-: Forward, 5'-TCCTGCTGTGCTTCTCCACCACA-3', as shown in SEQ ID NO: 7; Reverse, 5'-AAGTCCGCCCTGTAGGTGAGGTT-3', as shown in SEQ ID NO: 8;

[0044] Mouse cxcl10: Forward, 5'-ATCATCCCTGCGAGCCTATCCT-3', as shown in SEQ ID NO: 9; Reverse, 5'-GACCTTTTTTGGCTAAACGCTTTC-3', as shown in SEQ ID NO: 10;

[0045] Mouse tnf: Forward, 5'-CGTAGGCGATTACAGTCACGG-3', as shown in SEQ ID NO: 11; Reverse, 5'-GACCAGGCTGTCGCTACATCA-3', as shown in SEQ ID NO: 12;

[0046] Mouse il6: Forward, 5'-GCTACCAAACTGGATATAATCAGGA-3', as shown in SEQ ID NO: 13; Reverse, 5'-CCAGGTAGCTATGGTACTCCAGAA-3', as shown in SEQ ID NO: 14;

[0047] Mouse GAPDH: Forward, 5'-ACGGCCGCATCTTCTTGTGCA-3', as shown in SEQ ID NO: 15; Reverse, 5'-ACGGCCAAATCCGTTCACACC-3', as shown in SEQ ID NO: 16.

[0048] 7. ELISA

[0049] BMDM-infected mice for 24 hours, and the supernatant was collected to detect IFN-β (KE00187, Proteintech) and TNF-α (88-7246-88, Thermo Fisher Scientific). Six hours after infection with BMDM, serum from 8-week-old wild-type mice was collected, and the levels of IFN-β (R&D), TNF-α (88-7223-88, Thermo Fisher Scientific), and IL-6 (88-7064-88, Thermo Fisher Scientific) were measured by ELISA.

[0050] 8. Interfering RNA

[0051] The RNF144A-Stealth-RNAi siRNA was designed by Invitrogen BLOCKiT RNAi Designer. The small interfering RNA (siRNA) sequence used is as follows:

[0052] A1, positive, 5'-GCAAGUGCAGUAAAGGUGACGACGA-3', as shown in SEQ ID NO: 1; negative, 5'-UCGUCGUCACCUUUACUGCACUUGC-3', as shown in SEQ ID NO: 2;

[0053] A2, forward, 5'-GAGUACCCAGUGGAGCAGAUGACAA-3', as shown in SEQ ID NO: 3; reverse, 5'-UUGUCAUCUGCUCCACUGGGUACUC-3', as shown in SEQ ID NO: 4;

[0054] A3, forward, 5'-GCCUGAAACAGUAUGUUGAGCUCUU-3', as shown in SEQ ID NO: 5; reverse, 5'-AAGAGCUCAACAUACUGUUUCAGGC-3', as shown in SEQ ID NO: 6.

[0055] The silencing selection negative control siRNA was purchased from Invitrogen (catalog number 4390843). PMA-THP1 cells were transfected with siRNA using Lipofectamine 2000 according to the manufacturer's instructions. Cells were used for further experiments 24 hours after transfection.

[0056] 9. Lm infection and bacterial load count

[0057] Listeria monocytogenes with an MOI of 10 was added to the cells, centrifuged at 1500 rpm for 5 min, and incubated at 37°C for 1 h. The culture medium was then removed, and fresh culture medium was added, along with gentamicin (50 μg / ml). To determine the number of bacteria entering the cells, they were treated with 100 μg / ml gentamicin (Sigma-Aldrich, 1045-41-0) at 37°C for 30 min to kill extracellular bacteria. The infected cells were then washed three times with PBS and lysed in distilled water using 0.05% Triton X-100 (Sigma-Aldrich, 9036-19-5). After serial dilution, the cells were inoculated onto BHI agar plates, and CFU were counted after 36 h.

[0058] 10. Confocal microscope

[0059] After treatment, HeLa and MEF cells were fixed in PBS with 4% PFA, infiltrated with Triton X-100, and then blocked with 1% BSA. Cell nuclei were stained with 4,6-diamino-2-phenylindole (DAPI). Images were taken using a Nikon A1R microscope.

[0060] 11. In-situ PLA

[0061] Cells were seeded on cell culture slides and grown, then infected with Lm for 4 hours. Cells were fixed with 4% PFA for 15 minutes and washed three times with PBS. Afterward, cells were permeabilized with Triton X-100 on ice for 5 minutes and washed three times with PBS. Blocking buffer was then applied at 37°C for 1 hour, followed by incubation with primary antibody at 4°C overnight. The next day, cells were washed three times with washing buffer, incubated with PLA probe at 37°C for 2 hours, washed three times with washing buffer, incubated with ligase at 37°C for 2 hours, washed three times with washing buffer, incubated with amplification buffer containing polymerase at 37°C for 1 hour and 40 minutes. After final washing, cell nuclei were stained with DAPI, and the cell culture slides were mounted upside down on a glass slide for microscopic imaging and analysis.

[0062] 12. Correlation test between RNF144A and BECN1

[0063] BECN1, RNF144A and their mutants were expressed using the TNT Rapid Coupled Transcription / Translation System Kit (Promega, L1171).

[0064] result

[0065] 13.1 RNF144A deficiency prevents mice from being infected with LM bacteria but reduces the innate immune response.

[0066] To investigate the role of RNF144A in bacterial infection, we used Lm as an infection model of Gram-positive intracellular bacteria.

[0067] First, we examined the expression pattern of RNF144A after Lm infection. Figure 1 As shown in Figure A, the immunoblotting results revealed that *Limosa pudica* (Lm) induces RNF144A expression at the protein level, suggesting an association between RNF144A and Lm infection. Next, we explored the effect of RNF144A on Lm infection in an animal model. RNF144A-deficient mice were generated using CRISPR / Cas9 technology, and immunoblotting confirmed the presence of RNF144A deficiency in multiple organs at the protein level. Figure 1 B). Wild-type and RNF144A-deficient mice were infected with Lm, and their survival rates were assessed. Notably, RNF144A-deficient mice had a higher survival rate compared to wild-type mice. Figure 1 C). Consistent with this observation, compared to the wild-type group, Lm infection was reduced in the liver and spleen of RNF144A-deficient mice, such as the number of colony-forming units (cfu). Figure 1 As shown in D~E).

[0068] To explore the mechanism by which RNF144A deficiency prevents death in infected mice, we first evaluated the innate immune response in wild-type and RNF144A-deficient mice after infection with *L. m.*. Surprisingly, RNF144A deficiency led to reduced production of Ifnb, Cxcl10, and Tnf in the liver, spleen, and lungs of infected mice. Figure 1 F~H). Furthermore, in the serum of Lm-infected mice, RNF144A deficiency led to a significant decrease in the secretion of IFN-β, IL-6, and TNF-α. Figure 1 The results (I-K) suggest that RNF144A has a positive effect on the immune response induced by Lm.

[0069] 13.2 At the cellular level, RNF144A deficiency leads to reduced Lm infection and innate immune response.

[0070] Further investigation was conducted into the role of RNF144A in Lm infection at the cellular level. Bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM), as well as non-immune mouse embryonic fibroblasts (MEF), were isolated from wild-type and Rnf744a-deficient mice and then infected with Lm cells. Intracellular bacterial load and innate immune response were assessed. Among all three cell types examined, Rnf144a deficiency resulted in a reduction in CFU (crystal fibroblasts) in Lm cells. Figure 2 (A and 2B).

[0071] To confirm this observation, we used an anti-Lm antibody and directly imaged intracellular Lm infection in MEFs using confocal microscopy. Figure 2 C and Figure 2 As shown in K, compared with wild-type MEFs, the production of Lm in Rnf144a-deficient MEFs was significantly reduced after Lm infection (indicated by red dots). Then, the host's innate immune response to Lm was assessed. Compared with wild-type BMDMs, the response of RNF144A-deficient BMDMs to Lm infection showed reduced production of IFN-β, IL-6, and CXCL10 (…). Figure 2 D). Therefore, RNF144A deficiency leads to reduced secretion of IFN-β, IL-6, and TNF-α after Lm infection. Figure 2 E). Similar results were obtained in Lm-infected wild-type and RNF144A-deficient PMs. Figure 2 F~I).

[0072] Furthermore, in LM-infected BMDMs, RNF144A deficiency led to a significant decrease in the activation of IRF3 and TBK1. Figure 2 J). Finally, we investigated the role of RNF144A in Lm infection using siRNA. Figure 2 As shown in Figure L, both A2 and A3 inhibit endogenous RNF144A expression, with A2 exhibiting higher efficiency than A3 in suppressing RNF144A expression. Therefore, A2 and A3 were used in the following experiments. Consistent with results observed in RNF144A-deficient cells, knockdown of RNF144A by A2 resulted in reduced Lm infection and innate immune response. Figure 2 M and 2N).

[0073] In summary, our findings suggest that RNF144A deficiency in response to Lm infection leads to cellular restriction of Lm infection and suppression of the innate immune response.

[0074] 13.3 RNF144A inhibits Lm-induced autophagosome accumulation.

[0075] Next, we explored the possible mechanism by which Rnf 144a regulates Lm infection. Notably, Lm induces autophagy, and autophagy restricts Lm intracellular growth in the early stages of primary infection. Therefore, we investigated the effect of Rnf 144a deficiency on Lm-triggered autophagy by detecting microtubule-associated protein 1 light chain 3 (MAP1LC3 / LC3)-II levels using Western blotting. In Lm-infected MEF and BMDM, Rnf 144a deficiency led to an increase in LC3-II levels, indicating that Rnf 144A inhibits Lm-induced LC3-II accumulation. Figure 3 (A-D). To differentiate the effects on autophagosome accumulation and degradation, we pretreated cells with bafloxacin A1 (BafA1), which impaired lysosomal activity and allowed us to assess LC3-II / autophagosome formation. Figure 3 As shown in E-F, Rnf144a deficiency increased Lm-induced LC3-II accumulation to a similar degree in the presence or absence of BafA1, indicating that RNF144A regulates autophagosome formation but not lysosomal degradation. Given that RNF144A-deficient cells exhibit impaired Lm-triggered innate immune responses, we wanted to know whether the effect of RNF144A on autophagosome formation is a result of its regulation of innate immune signaling pathways.

[0076] To eliminate the influence of innate immune response, we blocked innate signaling using the STING inhibitor C-176. In the presence of C-176, Rnf144a deficiency no longer reduced Listeria-induced innate immune response. Figure 8 However, Lm stimulation still increases autophagosome formation. Figure 3 G~H).

[0077] Next, we explored the role of RNF144A in autophagosome formation in STING-deficient THP1. RNF144A knockdown led to increased LC3-II accumulation, indicating that RNF144A inhibits LM-induced autophagy, independent of STING and STING-mediated signaling pathways. Figure 3 I~J).

[0078] To confirm the role of RNF 144A in autophagy, we further investigated the effect of RNF 144A overexpression on LC3-1I accumulation. HeLa cells were transfected with RNF 144A or a control plasmid, followed by Lm infection. Figure 4 As shown in A-B, overexpression of RNF144A inhibited Lm infection-induced LC3-II accumulation, and after Lm infection, BafAl did not alter the reduction in LC3-II caused by RNF144A overexpression. Figure 4 (C-D) indicates that RNF 144A inhibits LM-induced autophagosome formation. Further studies in HeLa cells expressing mCherry-LC3, performed by confocal microscopy, demonstrated that RNF 144A overexpression led to a reduction in LC3-II punctate formation, confirming the negative regulation of LC3-II 225 accumulation by RNF 144A.

[0079] Next, we further investigated the role of RNF 144A in Lm-induced autophagy using a knockdown strategy. In Lm-infected PMA-THP 1 cells, knockdown of RNF 144A via A2 or A3 enhanced LC3-II accumulation, as shown by Western blotting. Figure 4 D~E).

[0080] Furthermore, based on the efficiency of A2-transfected cells in inhibiting RNF 144A expression, A2-transfected cells exhibited higher LC3-II levels than A3-transfected cells. Figure 4 F~G). In HeLa cells, immunoblotting and confocal microscopy also showed that RNF 144A knockdown increased LC 3-II accumulation (F~G). Figure 4 H~J).

[0081] Finally, to further determine whether RNF 144A regulates LC 3-II accumulation by affecting autophagosome formation or by modulating lysosomal activity, three different types of lysosomal inhibitors, including BafA1, CQ, and NH4Cl, were used. Results from the RNF 144A knockdown experiment indicated that RNF 144A negatively regulates Lm-induced autophagosome formation, and Western blot analysis showed LC 3-II levels (…). Figure 4 K and Figure 4 N). Similar results were obtained in confocal microscopy measurements, showing the formation of LC 3-II spots (N). Figure 4 L and Figure 4 In summary, these results indicate that RNF 144A inhibits Lm-induced autophagosome accumulation.

[0082] 13.4 Interaction between RNF144A and BECN 1

[0083] To investigate the target proteins of RNF 144A in autophagosome formation, an immunoprecipitation assay was used to transfect RNF 144A along with a series of important ATGs (including ATG 3, ATG 4B, ATG 5, ATG 7, ATG 10, and BECN 1) into human embryonic kidney (HEK) 293T cells. The immunoprecipitation results showed that among these ATGs, only BECN 1 co-precipitated with RNF 144A, indicating that RNF 144A binds to BECN 1. Figure 5 A and 5B). Confocal microscopy confirmed this association through the co-localization of RNF 144A and BECN 1 in HeLa cells. Figure 5 (C~D).

[0084] Next, in PMA-THP 1 and BMDM cells, endogenous RNF144A-BECN 1 interaction was observed in the absence of Lm infection, and this interaction was enhanced by Lm infection. Figure 5 E and 5F).

[0085] In addition, in situ proximity assays (PLA) were performed to verify the interaction between RNF 144A and BECN 1. RNF144A-deficient MEFs were transfected with Flag-RNF144A, followed by Lm infection or no infection. Using anti-Flag and anti-BECN 1 antibodies, we observed co-localization of RNF 144A and BECN 1, as shown by red dots, which was enhanced by Lm infection. Figure 5 G).

[0086] Finally, domain mapping experiments showed that only full-length BECN 1 is associated with RNF 144A, and the TM domain in the N-terminus of RNF 144A is crucial for the RNF 144A-BECN 1 interaction. Figure 5 In summary, our findings indicate that RNF 144A interacts with BECN 1, and Lm infection increases this interaction.

[0087] 13.5. RNF144A promotes the degradation of BECNI.

[0088] It is well known that K48-linked ubiquitination typically leads to the degradation of target proteins. Therefore, we investigated the stability of BECN1 in the presence of RNF144A overexpression.

[0089] like Figure 6 As shown in Figure A, overexpression of RNF144A in HEK293T cells promoted BECN1 degradation in a dose-dependent manner. Even in the presence of the protein synthesis inhibitor cycloheximine (CHX), RNF144A overexpression still led to decreased BECN1 protein expression, indicating that RNF144A increased BECN1 degradation. Figure 6 B). Intracellular proteins are primarily degraded via the ubiquitin-proteasome system or the autophagy-lysosome system. To determine which system RNF144A uses to regulate BECN1 degradation, we treated cells with the proteasome inhibitor MG132, the autophagy inhibitor 3-MA, or the lysosome inhibitor NH4Cl. We found that only MG132 treatment restored BECN1 expression, indicating that RNF144A promotes BECN1 degradation in a ubiquitin-proteasome-dependent manner. This is consistent with our finding that RNF144A increases K48-linked BECN1 ubiquitination. Figure 6 C).

[0090] Next, we investigated the effect of endogenous RNF144A on BECN1 degradation. In Lm-infected PMA-THP1 cells, RNF144A knockdown led to enhanced BECN1 expression. Figure 6 D1–D2). Similarly, in Lm-infected MEF and BMDM, RNF144A-deficient cells showed increased BECN1 expression compared to wild-type cells. Figure 6 E1、 Figure 6 E2, Figure 6 F1 Figure 6 F2).

[0091] Furthermore, under CHX treatment, the BECN1 protein level in RNF144A-deficient cells remained higher than that in control wild-type cells. Figure 6G1-G2). Then, RNF144A-deficient THP1 cells were generated using CRISPR to further investigate the role of RNF144A in autophagy regulation. Western blotting showed that RNF144A expression was not detected in RNF144A-deficient PMA-THP1 cells. Figure 6 H). Compared with wild-type PMA-THP1 cells, RNF144A-deficient cells showed increased BECNI expression and LC3-II accumulation levels after Lm infection. Figure 6 H and Figure 6 I1、 Figure 6 I2).

[0092] To further confirm these results, we investigated the effects of RNF144A on BECN1 degradation and LC3-II accumulation in mouse organs. Wild-type and RNF144A-deficient mice were infected with Lm for 2 days. In the liver and spleen, RNF144A deficiency led to increased BECN1 expression and LC3-II accumulation. Figure 6 J1~J5).

[0093] In summary, our results indicate that RNF144A promotes the degradation of BECN1.

[0094] 13.6 The RING domain is crucial for the role of RNF144A in Lm infection.

[0095] C20 / 23A and C198A mutants of RNF144A have been reported as E3 ligase-deficient mutants of RNF144A with RING18.21 domain disruption.

[0096] Next, we used these two mutants to explore the importance of the RING domain in RNF144A-regulated Lm infection. Wild-type RNF144A or its mutants were transfected into RNF144A-deficient MEFs, followed by Lm infection or no infection of cells. Here, we observed that neither the C20 / 23A nor the C198A mutants of RNF144A promoted BECN1 degradation and ubiquitination as much as wild-type RNF144A. Figure 7 A1, A2 and Figure 7 B). Consistent with intracellular phenomena, the C20 / 23A and C198A mutants of RNF144A had no effect on the ubiquitination of BECN1 in the in vitro ubiquitination system. Figure 7 C).

[0097] Furthermore, neither the RNF144C20 / 23A nor the C198A mutants suppressed LC3-II accumulation as much as the wild-type RNF144A. Figure 7 D).

[0098] Finally, in colony formation and confocal microscopy experiments, we observed that transfection with wild-type RNF144A promoted Lm infection, while the C20 / 23A and C198A mutants of RNF144A did not. Figure 7 E and Figure 7 F1, F2, F3).

[0099] In summary, these findings suggest that the integrity of the ring domain is crucial for RNF144A to enhance Lm infection.

[0100] The above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Use of an RNF144A protein in the preparation of a drug to improve Listeria monocytogenes infection.

2. The use according to claim 1, characterized in that, Improve Listeria monocytogenes infection by reducing the expression level of RNF144A protein.

3. The use according to claim 1, characterized in that, Improve Listeria monocytogenes infection by using an inhibitor of the RNF144A protein.

4. The use according to claim 3, characterized in that, The inhibitor of the RNF144A protein is A2 or A3; The positive chain sequence of A2 is 5'-GAGUACCCAGUGGAGCAGAUGACAA-3', and the negative chain sequence is 5'-UUGUCAUCUGCUCCACUGGGUACUC-3'; The positive chain sequence of A3 is 5'-GCCUGAAACAGUAUGUUGAGCUCUU-3', and the negative chain sequence is 5'-AAGAGCUCAACAUACUGUUUCAGGC-3'.

5. The use according to claim 3, characterized in that, The inhibitor of the RNF144A protein is used to prepare drugs that inhibit the secretion of IFN-β, IL-6, or TNF-α.

6. The use according to claim 3, characterized in that, The inhibitor of the RNF144A protein is used to prepare a drug that enhances the immune response to Listeria monocytogenes infection.

7. The use according to claim 3, characterized in that, The inhibitor of the RNF144A protein is used to prepare an expression promoter for LC 3-II.

8. The use according to claim 3, characterized in that, The inhibitor of the RNF144A protein is used to prepare a BECN1 expression promoter.

9. A drug for improving Listeria monocytogenes infection, characterized in that, It includes an expression inhibitor of the RNF144A protein as described in claim 1, and a scientifically acceptable excipient or carrier.

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