Method for reducing listeria monocytogenes infection by inhibiting host CypD protein synthesis

Abstract

The invention relates to the technical field of biology, in particular to a method for reducing listeria monocytogenes infection by inhibiting host CypD protein synthesis. Specifically, the invention finds that the proliferation of the listeria monocytogenes in cells can be obviously reduced and the growth activity of the cells is not influenced by inactivating or inhibiting the expression of the CypD protein (the coding gene is PPIF), and a basis is provided for preventing and treating the infection of the listeria monocytogenes by clinically utilizing a PPIF gene expression inhibitor or a CypD protein synthesis inhibitor. On the basis, on one hand, the invention provides application of a PPIF gene expression inhibitor or a CypD protein synthesis inhibitor in reduction of listeria monocytogenes infection. On the other hand, the invention provides an application of a vector for inactivating the PPIF gene or reducing expression of the PPIF gene or an engineered cell or a recombinant microorganism containing the vector in reduction of Listeria monocytogenes infection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for reducing Listeria monocytogenes infection by inhibiting the synthesis of host CypD protein. Background Technology

[0002] Listeria monocytogenes (Listeria monocytogenes for short) is a zoonotic intracellular parasite that can invade macrophages and proliferate intracellularly. When a host ingests contaminated food, Listeria monocytogenes can cross the intestinal epithelial barrier and enter the lamina propria, subsequently spreading via the lymphatic and hematogenous systems to the liver and spleen, where it proliferates. Listeria monocytogenes infection can induce meningoencephalitis and invade the placenta, leading to fetal infection, stillbirth, miscarriage, and neonatal infection. In humans, pregnant women, newborns, the elderly, and immunocompromised individuals are particularly vulnerable to severe infection.

[0003] Listeria monocytogenes is phagocytosed by phagosomes during host infection, and since it cannot proliferate normally within the phagosomes, it secretes a perforating toxin called listeriolysin O (LLO). This toxin disrupts the cell membrane and phagosome membrane, serving as a crucial mechanism for Listeria monocytogenes to evade the host's immune defenses. LLO is a 56 kDa secreted protein encoded by the hly gene, belonging to the cholesterol-dependent cytolysin (CDC) family, and is essential for Listeria infection.

[0004] Mitochondria, as important calcium ion regulators in host cells, have the ability to temporarily store and accumulate large amounts of calcium. 2+ The ability of mitochondrial calcium to maintain cellular calcium homeostasis is crucial. 2+ The level of mitochondrial calcium is crucial for maintaining the activity of tricarboxylic acid cycle enzymes and ATP synthesis, and also controls the mitochondrial pathway, including respiration, mitophagy / autophagy, and apoptosis. Among these, the mitochondrial membrane permeability transition pore (mPTP) is key to regulating mitochondrial calcium levels. 2+ One of the three main mechanisms of efflux. CypD is a key component of mPTP and participates in regulating mPTP-mediated cell death pathways.

[0005] Pathogenic microorganisms also influence mPTP opening through various mechanisms, thereby regulating host cell survival and death. Many bacteria can affect mitochondrial function and induce mPTP opening by secreting toxins or directly acting on host cell mitochondria, leading to host cell death. After Mycobacterium tuberculosis infects macrophages, it induces mPTP opening through the interaction of p53 with CypD and ANT1, resulting in mitochondrial depolarization and programmed cell death. Mycobacterium flexneri induces CypD-mediated mPTP opening and apoptosis by increasing mitochondrial Ca2+ load. When Shigella infects non-myeloid cells, it induces mPTP opening through Bnip3 and CypD, leading to necrosis, while simultaneously activating the NF-κB pathway to promote cell survival.

[0006] Listeria monocytogenes possesses a unique infection strategy that allows it to evade immune clearance and survive within the host. Similar to many pathogens, it enhances its adhesion, invasion, and spread by modulating the structure and function of host cell mitochondria, thereby altering the intracellular microenvironment. However, the role of CypD in Listeria monocytogenes infection requires further investigation. Summary of the Invention

[0007] Building upon existing technologies, the inventors discovered that the key virulence factor of Listeria monocytogenes, hemolysin O (LLO), can induce upregulation of CypD protein expression, and that the two interact. Furthermore, the inventors constructed CypD-deficient HeLa and THP-1 cells using CRISPR-Cas9 gene editing technology, thereby eliminating endogenous CypD protein. They found that in CypD-deficient HeLa and THP-1 cells, cell proliferation and growth activity were not affected, but the proliferative capacity of Listeria monocytogenes was significantly reduced.

[0008] Based on this, the present invention provides, on the one hand, the application of PPIF gene expression inhibitors or CypD protein inhibitors in reducing Listeria monocytogenes infection. On the other hand, the present invention provides the application of vectors that inactivate the PPIF gene or reduce PPIF gene expression, or engineered cells or recombinant microorganisms containing such vectors, in reducing Listeria monocytogenes infection.

[0009] In this regard, the present invention includes, but is not limited to, the following:

[0010] In one aspect, the present invention provides the use of PPIF gene expression inhibitors or CypD protein inhibitors in the preparation of medicaments for reducing Listeria monocytogenes infection.

[0011] In one aspect, the present invention provides the use of an inactivated PPIF gene or a vector that reduces PPIF gene expression, or a recombinant microorganism containing said vector, in the preparation of a medicament for reducing Listeria monocytogenes infection.

[0012] In one aspect, the present invention provides a method for reducing cell infection with Listeria monocytogenes in vitro, the method comprising inactivating the PPIF gene or reducing PPIF gene expression.

[0013] In one aspect, the method of the present invention includes the following steps: introducing into the cell an inactivated PPIF gene or a vector that reduces PPIF gene expression or a recombinant microorganism containing the vector.

[0014] In one aspect, the vector of the present invention comprises a pX459 plasmid or a LentiCRISPR V2 plasmid containing sgRNA targeting the PPIF gene.

[0015] And / or, the recombinant microorganism is selected from lentiviruses or adenoviruses.

[0016] In one aspect, the microorganism is selected from lentiviruses or adenoviruses. Preferably, the microorganism is a lentivirus.

[0017] In one aspect, the sgRNA targeting the PPIF gene described in this invention is selected from SEQ ID NO:3, 4, or 5.

[0018] In one aspect, the cells described in this invention are human cells, preferably human mononuclear cells or human cervical cancer cells.

[0019] The PPIF gene expression inhibitor or CypD protein inhibitor described in this invention is an interfering nucleic acid, such as siRNA, shRNA, miRNA, or antisense nucleic acid (ASO). Furthermore, this invention can also utilize gene editing technology to reduce PPIF gene expression or CypD protein synthesis in host cells.

[0020] In one aspect, the nucleotide sequence of the PPIF gene described in this invention is shown in SEQ ID NO:1.

[0021] SEQ ID NO:1 (NCBI Registry Number: NM_005729.4)

[0022]

[0023] In one aspect, the amino acid sequence of the CypD protein of the present invention is shown in SEQ ID NO:2.

[0024] SEQ ID NO:2 (NCBI Registry Number: NP_005720.1)

[0025] MLALRCGSRWLGLLSVPRSVPLRLPAARACSKGSGDPSSSSSSSGNPLVYLDVDANGKPLGRVVLELKADVVPKTAENFRALCTGEKGFGYKGSTFHRVIPSFMCQAGDFTNHNGTGGKSIYGSRFPDENFTLKHVGPGVLSMANAGPNTNGSQFFICTIKTDWLDGKHVVFGHVKEGMDVVKKIESFGSKSGRTSKKIVITDCGQLS

[0026] In another aspect, the present invention provides a cell obtained by the method described in the present invention.

[0027] The cells described in this invention are selected from HeLa or THP-1 cells. The model cells of this invention may not express CypD protein at all, and can serve as an important cell model for studying the interaction between Listeria monocytogenes and host cell CypD protein or the effects of Listeria monocytogenes on host cell mitochondria.

[0028] The beneficial effects of the present invention include at least the following:

[0029] This invention is the first to discover and verify the relationship between host cell CypD protein and Listeria monocytogenes infection. Specifically, inactivation or inhibition of CypD protein expression can significantly reduce the proliferation of Listeria monocytogenes in cells without affecting cell growth activity. This provides a basis for the clinical use of PPIF gene expression inhibitors or CypD protein synthesis inhibitors to prevent and treat Listeria monocytogenes infection. Attached Figure Description

[0030] Figure 1 The pX459-sgRNA recombinant plasmid was successfully constructed. The images show the recombinant plasmid maps of pSL446(A), pSL447(B), and pSL448(C); and (D) the results of colony PCR screening for positive clones.

[0031] Figure 2 The LentiCRISPR V2-sgRNA recombinant plasmid was successfully constructed. The images show the recombinant plasmid maps of pSL469(A), pSL470(B), and pSL471(C); and (D) the results of colony PCR screening for positive clones.

[0032] Figure 3 The results show that the CypD-deficient polyclonal cell population was successfully constructed. The analysis includes: (A) sequencing results of the CypD-KO HeLa polyclonal cell population; (B) Western blot analysis of CypD protein expression levels in the CypD-KO HeLa polyclonal cell population; (C) sequencing results of the CypD-KO THP-1 polyclonal cell population; and (D) Western blot analysis of CypD protein expression levels in the CypD-KO THP-1 polyclonal cell population.

[0033] Figure 4 The results showed that CypD-deficient monoclonal cells were successfully screened. The analysis included: (A) sequencing results of CypD-KO HeLa monoclonal cells; (B) Western blot analysis of CypD protein expression levels in CypD-KO HeLa monoclonal cells; (C) sequencing results of CypD-KO THP-1 monoclonal cells; and (D) Western blot analysis of CypD protein expression levels in CypD-KO THP-1 monoclonal cells.

[0034] Figure 5 The results show that CypD deficiency does not affect normal cell growth.

[0035] Figure 6 The proliferation ability of Listeria monocytogenes in CypD-deficient cells was significantly reduced. Specifically, (A) the proliferation ability of Listeria monocytogenes in CypD-KO HeLa cells; (B) the proliferation ability of Listeria monocytogenes in CypD-KO THP-1 cells. **: P < 0.01; ***: P < 0.001.

[0036] Figure 7 This shows mitochondrial Ca2+ after CypD deficiency. 2+ The levels were significantly elevated. Among them, (A) a representative histogram of intracellular Rhod-2 AM fluorescence intensity detected by flow cytometry after Listeria monocytogenes infection of CypD-KO HeLa cells; (B) quantitative analysis of fluorescence intensity; (C) a representative histogram of intracellular Rhod-2 AM fluorescence intensity detected by flow cytometry after Listeria monocytogenes infection of CypD-KO THP-1 cells; (D) quantitative analysis of fluorescence intensity. *: P<0.05; **: P<0.01; ***: P<0.001. Detailed Implementation Plan

[0037] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0038] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0040] 1. Strains, plasmids, and cells

[0041] The strains include: Listeria monocytogenes wild-type strain EGD-e.

[0042] The plasmids included: CRISPR-Cas9 vector plasmid pX459, lentiviral vector plasmid LentiCRISPR V2, packaging plasmid psPAX2, and envelope plasmid PMD2.0G; the cells included: HeLa cells (human cervical cancer cells, from the China Center for Type Culture Collection, accession number: GDC0009) and THP-1 cells (human monocytes, from the China Center for Type Culture Collection, accession number: GDC0100).

[0043] Culture conditions for plasmid-containing strains: After streaking on LB solid medium with antibiotics, invert the culture medium and incubate at 37°C. After isolating single clones, culture the single clones in LB liquid medium with antibiotics at 37°C in a shaker to obtain single clone culture.

[0044] Culture conditions for Listeria monocytogenes EGD-e strain: After streaking on antibiotic-free BHI plates, the culture was placed in a 37°C incubator for static incubation. Monoclonal bacterial suspensions were obtained by shaking culture in antibiotic-free BHI liquid medium at 37°C.

[0045] HeLa cell culture conditions: HeLa cells were cultured statically in a 37°C constant temperature cell culture incubator containing 5% CO2 and cultured in DMEM medium containing 10% fetal bovine serum.

[0046] THP-1 cell culture conditions: static culture in a 37°C constant temperature cell culture incubator containing 5% CO2, and cultured in RPMI-1640 medium containing 10% fetal bovine serum.

[0047] 2. Test instruments

[0048] The shaker was purchased from Hualida Experimental Equipment Company.

[0049] The benchtop centrifuge was purchased from Eppendorf, Germany.

[0050] The single-channel pipette was purchased from Eppendorf, Germany.

[0051] The vortex mixer was purchased from Eppendorf, Germany.

[0052] The electronic balance was purchased from Sartorius, Germany.

[0053] The ultrasonic cell disruptor was purchased from Ningbo Xinzhi Bio-instrument Co., Ltd.

[0054] The ice maker was purchased from Ningbo Grant Refrigeration Equipment Manufacturing Co., Ltd.

[0055] The protein electrophoresis apparatus was purchased from Bio-Rad Laboratories, USA.

[0056] The pure water system was purchased from EPED.

[0057] The real-time PCR instrument was purchased from Bio-Rad Laboratories, USA.

[0058] The transfer apparatus was purchased from GenScript.

[0059] The Smart Chemi chemiluminescence imaging system was purchased from Saizhi Technology Co., Ltd.

[0060] The metal bath thermostat was purchased from Thermo Fisher Scientific.

[0061] The PCR amplification instrument was purchased from Eppendorf GmbH, Germany.

[0062] The nucleic acid electrophoresis system was purchased from Beijing Bayi Instrument Factory.

[0063] The multi-functional microplate reader was purchased from Bertek Instruments, Inc., USA.

[0064] 3. Test reagents

[0065] RPMI-1640 medium was purchased from Gibco.

[0066] PMA was purchased from Shanghai Sangon Biotech Co., Ltd.

[0067] T4 PNK was purchased from NEB.

[0068] T4 DNALigation was purchased from NEB.

[0069] Polybrene was purchased from Beyotime Biotechnology Co., Ltd.

[0070] Puromycin was purchased from Solarbio.

[0071] Example 1: Construction of gene knockout plasmid

[0072] 1.1 Due to the high transfection efficiency of HeLa cells, the pX459 vector was used to directly transfect cells to achieve gene knockout in the experiment of constructing a CypD-deficient HeLa cell line. We first designed a specific sgRNA sequence targeting the CypD-encoding gene PPIF and cloned it into the pX459 vector to construct the pX459-sgRNA recombinant plasmid. Subsequently, the recombinant plasmid was transfected into HeLa cells, and a cell population stably expressing Cas9 protein and the sgRNA targeting the PPIF gene was obtained through puromycin selection. Under the guidance of the sgRNA, the Cas9 protein can generate a double-strand break (DSB) at a specific site in the PPIF genome. Cells are prone to frameshift mutations when repairing this break, ultimately achieving the deletion of CypD protein.

[0073] 1.1.1 Design and Synthesis of sgRNA

[0074] Download the human PPIF gene sequence from the NCBI database, open http: / / crispor.tefor.net / , input the sequence, select the correct species and Cas9 protein, and click "submit" to generate the sgRNA sequence. The generated sgRNA sequence information includes the sgRNA sequence, PAM, specificity score, cleavage efficiency score, and other specific information. Select the three best sgRNAs for subsequent experiments (Table 1). To enable the sgRNA to ligate to the sticky ends of the vector after enzyme digestion, add five bases "CACCG" to the 5' end of the upstream primer and four bases "AAAC" to the 5' end of the downstream primer, and add one base "C" to the 3' end. Finally, send the designed primer sequences to the biotechnology company for synthesis.

[0075] Table 1 sgRNA Sequence

[0076]

[0077] 1.1.2 Synthesis of Oligonucleotide Double Strands

[0078] The synthesized forward and reverse oligonucleotide single-stranded primer powders were diluted to a final concentration of 100 μM and then annealed to synthesize oligonucleotide double strands. The PCR reaction system and procedure are detailed in Tables 2 and 3.

[0079] Table 2 PCR reaction system

[0080]

[0081] Table 3 PCR reaction procedure

[0082]

[0083] 1.1.3 Enzyme digestion

[0084] The pX459 vector was linearized by restriction endonuclease BbsI. The digestion system and procedure are detailed in Table 4. After digestion, the vector was recovered and purified, and the concentration was detected using an ELISA plate.

[0085] Table 4 Enzyme digestion reaction system and procedure

[0086]

[0087] 1.1.4 Enzyme-linked

[0088] The synthesized oligonucleotide double strands were diluted 200-fold using ddH2O, and then ligated with the linearized vector. The enzyme ligation system and procedure are detailed in Table 5.

[0089] Table 5 Enzyme ligation reaction system and procedure

[0090]

[0091] 1.1.5 Transformation and Colony Validation

[0092] 1.1.5.1 Conversion

[0093] (1) Take the competent cells DH5α out of the -80℃ freezer and place them on ice until they just thaw;

[0094] (2) Add 10 μL of enzyme ligation product to DH5α and gently tap the tube wall to mix it.

[0095] (3) Place DH5α on ice for 30 minutes;

[0096] (4) Heat shock at 42℃ for 90s, then place on ice for 2min;

[0097] (5) Add 1 mL of antibiotic-free LB liquid medium to DH5α, mix well, and place at 37℃ with shaking at 200 rpm for 1 h.

[0098] (6) Centrifuge the bacteria at 4500 rpm for 5 min, retain 100 μL of supernatant to resuspend the precipitate, spread the bacterial solution on LB solid medium containing Amp resistance with a spreader, spread until dry, and then invert and place in a 37℃ incubator for static culture.

[0099] (7) Observe the colony growth after incubation overnight.

[0100] 1.1.5.2 Colony Validation

[0101] Single colonies were picked up with a pipette tip and added to 10 μL of LB liquid medium containing Amp. After mixing by pipetting, colony PCR was performed using HA-pCMV vector primers. The PCR reaction system and procedure are shown below. After amplification, 1% agarose gel electrophoresis was used to detect the presence of bands at the target fragment size. ddH2O was used as a negative control. Positive clones with correct bands were streaked onto LB Amp solid medium to isolate single colonies, cultured overnight, and then single colonies were picked for amplification culture before being sent to a biotechnology company for sequencing. After successful sequencing, the bacteria were preserved by mixing 50% sterile glycerol with the bacterial culture at a 1:1 ratio, adding it to cryovials, and storing at -80°C.

[0102] Colony PCR system:

[0103]

[0104] Colony PCR reaction procedure:

[0105]

[0106] Using pX459 plasmid as a vector, a double-stranded sgRNA fragment was successfully amplified and annealed using specific primers containing the BbsI restriction site sequence. pX459 was linearized with BbsI restriction endonuclease and then ligated to the sgRNA fragment using T4 DNA ligase to construct pX459-sgRNA recombinant plasmids. The obtained recombinant plasmids were named pSL446, pSL447, and pSL448, respectively. Figure 1 AC). After heat transformation into *E. coli*, single colonies were selected as templates for colony PCR verification, using pX459 vector primers (primers: pX459-F: TGTTAGAGAGATAATTGGAATTAATTTGACTGT; pX459-R: CTAGAGCCATTTGTCTGCAGAATTGG). The PCR amplification products were detected by 1% agarose gel electrophoresis using the DL 2000 DNA Marker as a standard. The results showed clear bands in lanes 1-6. Figure 1 D) indicates that the sgRNA was successfully inserted. Further sequencing results showed that the inserted sequence was accurate, indicating that the three pX459-sgRNA recombinant plasmids were successfully constructed.

[0107] 1.2 Since THP-1 cells are suspension cells, the transfection efficiency of liposome transfection is relatively low. Therefore, this experiment used a lentiviral packaging system, which can more efficiently introduce exogenous genes into THP-1 cells, thereby improving the efficiency of gene editing. In this experiment, we selected the lentiviral vector LentiCRISPR V2 as the vector. Since both THP-1 and HeLa cells are human cells, the sgRNA used for THP-1 gene deletion cells was the same as that used for HeLa cells, as shown in Table 1. This experiment first constructed the LentiCRISPR V2-sgRNA recombinant plasmid. Subsequently, the recombinant plasmid was used to infect THP-1 cells using the lentiviral packaging system. After selection with puromycin, a cell line stably expressing Cas9 and sgRNA was obtained, thereby achieving CypD knockout.

[0108] 1.2.1 Enzyme digestion

[0109] LentiCRISPR V2 was linearized using the restriction endonuclease BsmBI. The digestion system and procedure are detailed in Table 6. After digestion, the enzyme was recovered and purified, and the concentration was determined. The specific steps are as follows:

[0110] (1) The PCR products were separated by electrophoresis in a gel containing 1% agarose, and the specific bands were detected by a UV imaging system;

[0111] (2) Cut off the strips of the correct size, place them in a 1.5 mL EP tube, and weigh the gel.

[0112] (3) Add binding liquid GMB at a volume of 3 times its weight, place in a 56°C water bath and heat until the glue is completely dissolved. During this process, use a vortex mixer to accelerate the dissolution.

[0113] (4) Transfer the entire solution to the adsorption column, let it stand at room temperature for 1 min, then centrifuge at 12000 rpm for 30 s and discard the waste liquid.

[0114] (5) Add 600 μL of WB washing solution to the adsorption column, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0115] (6) Repeat step (5);

[0116] (7) Isolate the adsorption column at 12000 rpm for 2 min;

[0117] (8) Place the adsorption column into a new 1.5 mL EP tube, open the adsorption column cap, and place it in a 60 °C oven for 2 min to allow the ethanol to evaporate completely.

[0118] (9) Place the clean ddH2O in a 68℃ metal bath and heat it in advance;

[0119] (10) Add 50 μL ddH2O to the adsorption column, let it stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 1 min;

[0120] (11) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect DNA concentration and store it in a -20°C refrigerator.

[0121] Table 6 Enzyme digestion reaction system and procedure

[0122]

[0123] 1.2.2 Enzyme-linked

[0124] Oligonucleotide duplexes were synthesized and diluted using the same method described above. The diluted oligonucleotide duplexes were then ligated to a linearized vector. The enzyme ligation system and procedure are detailed in Table 7.

[0125] Table 7 Enzyme ligation reaction system and procedure

[0126]

[0127] 1.2.3 Transformation and Colony Validation

[0128] The transformation and colony validation methods are the same as described above.

[0129] 1.2.4 Lentiviral Packaging

[0130] (1) HEK293T cells were prepared at a rate of 5 × 10⁻⁶. 5 The plasmids were evenly seeded at a density of 1 / mL in 6-well plates, and the successfully constructed LentiCRISPR V2-sgRNA recombinant plasmid, as well as psPAX2 and pMD2.0G plasmids, were prepared in advance.

[0131] (2) When the cell density reaches 70%-80%, transfection is performed. The recombinant plasmid, psPAX2, and PMD2.0G plasmid are mixed with jetPRIME buffer according to the system in Table 3.9. Then, jetPRIMEreagent is added, mixed, and incubated for 10 minutes. Finally, the incubated mixture is added to the cells.

[0132] (3) 18 hours after transfection, discard the original culture medium and replace it with fresh complete culture medium and continue culturing;

[0133] (4) Three days after transfection, collect the supernatant containing the virus, filter it with a 0.45 μM filter, and aliquot the filtered lentivirus into 1 mL / tubes. It can be used directly for subsequent experiments or stored in a -80 degree freezer.

[0134] Table 8 Transfection System

[0135]

[0136] Using the LentiCRISPR V2 plasmid as a vector, a double-stranded sgRNA fragment was successfully amplified and annealed using specific primers containing the BsmBI restriction site sequence. The vector was linearized with BsmBI restriction endonuclease, and then ligated to the sgRNA fragment using T4 DNA ligase to construct LentiCRISPR V2-sgRNA recombinant plasmids. The obtained recombinant plasmids were named pSL469, pSL470, and pSL471, respectively. Figure 2 AC). After heat transformation into *E. coli*, colony PCR was performed using LentiCRISPR V2 vector primers (primers: LentiCRISPR V2-F: AGACAGAATCCATTCGATTAGTGAAC; LentiCRISPR V2-R: GTACACGACATCACTTTCCCAGTTT). Single colonies were selected as templates for colony PCR verification. The PCR amplification products were detected by 1% agarose gel electrophoresis using the DL2000 DNA Marker as a standard. The results showed clear bands of consistent size in lanes 1-3 and 5-7. Figure 2 D) indicates that the sgRNA was successfully inserted. The sample was then sent to the company for sequencing. The sequencing results showed that the inserted sequence was accurate, indicating that the three LentiCRISPR V2-sgRNA recombinant plasmids were successfully constructed.

[0137] Example 2: Construction of CypD-deficient cell lines

[0138] 2.1 Construction of CypD-deficient HeLa cell lines

[0139] 2.1.1 Transfection

[0140] (1) HeLa cells were prepared at a ratio of 2 × 10⁻⁶ 5 The cells were evenly seeded in the 24-well plate at a density of cells / mL;

[0141] (2) The successfully constructed pX450-sgRNA recombinant plasmid was transfected into cells at a rate of 0.5 μg per well. A non-transfected group was also included as a blank control. The specific steps are as follows:

[0142] (a) Transfection should be performed when the cell density after plating reaches 60%-80%;

[0143] (b) Take 1.5 mL EP tubes and add 200 μL jetPRIME buffer to each tube;

[0144] (c) Add 2 μg of the target plasmid to each tube and mix well;

[0145] (d) Add 4 μL jetPRIMEreagent, vortex to mix, then briefly separate and incubate for 10 min.

[0146] (e) Remove the cell culture plate and gently add the incubated mixture into the cell culture wells;

[0147] (f) Follow-up experiments were conducted 24 hours after transfection.

[0148] 2.1.2 Screening for positive cells with puromycin

[0149] (1) 24 h after transfection, the culture medium was replaced with a complete culture medium containing a final concentration of 3 μg / mL puromycin;

[0150] (2) After the cells in the blank control die, the concentration of puromycin is halved;

[0151] (3) After the cells have grown to a sufficient density, extract the cell DNA and total protein, and continue to culture and passage the remaining cells.

[0152] (4) Design upstream and downstream primers before and after the sgRNA region of the PPIF gene, use the extracted cellular DNA as a template for PCR amplification, and send the amplification products to the company for sequencing. At the same time, the expression level of CypD protein is detected by Western blot.

[0153] (5) If the sequencing results of the PCR product show mutations such as double peaks, frameshifts or base deletions in the sgRNA region, and Western blot analysis shows that the expression level of CypD protein is lower than that of wild cells, it indicates that the genome of some cells has been edited, indicating that there are positive cells in the polyclonal cell population where the target gene has been knocked out.

[0154] 2.1.3 Monoclonal cell isolation

[0155] (1) Collect the successfully verified polyclonal cells into a 15mL centrifuge tube and centrifuge at 700rpm for 3min;

[0156] (2) Discard the supernatant, add fresh DMEM medium containing 10% FBS to resuspend the cells, and adjust the cell density to 10 cells / mL by cell counting;

[0157] (3) Use a 96-well plate, seed 100 μL of cell suspension into each well to ensure that there is only one cell per well on average. Place the culture plate in a 37°C, 5% CO2 incubator and observe the cell growth regularly.

[0158] (4) Observe the growth of cell clones under a microscope at 5-7 days. If more than one cell cluster is found, discard the well; if only one cell cluster is found, keep the well for subsequent verification.

[0159] (5) Transfer well-grown monoclonal cells to 24-well plates, continue culturing and gradually expand them to 6-well plates or T25 culture flasks to provide sufficient cell quantity for subsequent validation experiments.

[0160] 2.1.4 Monoclonal cell validation

[0161] Cellular DNA and total protein were extracted from monoclonal cells and verified by PCR sequencing and Western blot, respectively.

[0162] 2.2 Construction of CypD-deficient THP-1 cell line

[0163] 2.2.1 THP-1 cell resuscitation

[0164] (1) Preheat the RPMI-1640 complete medium containing 10% FBS to 37°C in advance;

[0165] (2) Take out a tube of THP-1 cells from liquid nitrogen and quickly place it in a 37°C water bath and shake it to melt it quickly.

[0166] (3) When the ice has melted down to the size of a mung bean, put the cryovial into a centrifuge and centrifuge at 700 rpm for 3 minutes.

[0167] (4) Wipe the opening of the cryovial with a squeezed alcohol-soaked cotton ball, let it dry slightly, and then open the cap.

[0168] (5) Carefully aspirate the supernatant, add 1 mL of complete culture medium to the cryovial, and gently pipette to resuspend the cells;

[0169] (6) Add the cell suspension to a T25 cell culture flask and then add 3 mL of complete culture medium;

[0170] (7) Place the cells in a constant temperature incubator at 37°C and 5% CO2 concentration and incubate statically. Observe the cell adhesion after 24 hours.

[0171] 2.2.2 THP-1 cell passage

[0172] (1) After the cells have grown to full capacity, collect all the cells into a 15mL centrifuge tube, put it into a centrifuge, and centrifuge at 700rpm for 3min.

[0173] (2) Discard the supernatant and add 2 mL of RPMI-1640 complete culture medium to gently resuspend the cells into a single-cell suspension;

[0174] (3) Take half of the culture medium and add it to a new T75 culture flask, then add 6 mL of complete culture medium;

[0175] (4) Place the cells in a 37°C, 5% CO2 incubator for static culture.

[0176] 2.2.3 THP-1 cell plating

[0177] (1) The collected and centrifuged cell pellet was resuspended in 6 mL of cell culture medium to form a single-cell suspension;

[0178] (2) Clean the blood cell counting chamber with alcohol and dry it. Place the coverslip on top of the counting chamber.

[0179] (3) Take 10 μL of cell suspension and spray the liquid along the edge of the coverslip so that all the liquid is absorbed;

[0180] (4) Place the counting board under a microscope to observe, count, and calculate;

[0181] (5) The cells were arranged at a ratio of 1×10 6 After diluting the cells at a density of cells / mL, PMA was added to a final concentration of 100 ng / mL to induce THP-1 cells to differentiate into macrophages. After thorough mixing, the cells were added to cell culture plates, 0.5 mL per well in a 24-well plate. After gently shaking to mix, the plates were placed in a cell culture incubator for static culture.

[0182] 2.2.4 Lentiviral infection

[0183] (1) Select THP-1 cells in good growth condition, collect them into 15mL centrifuge tubes, centrifuge at 700rpm for 3min, discard the supernatant, resuspend the cells in RPMI-1640 complete medium, and dilute the cells to 5×10⁻⁶. 5 Density per unit / mL;

[0184] (2) Remove the frozen lentivirus from the -80℃ freezer and thaw it on ice;

[0185] (3) Take 1 mL of THP-1 cell suspension into a centrifuge tube, add polybrene, an infection-promoting reagent with a final concentration of 8 μg / mL, mix gently, then add 1 mL of lentivirus, mix well, and add to a 6-well plate. Simultaneously, set up THP-1 wild-type cells without lentivirus as a blank control;

[0186] (4) After centrifuging the cell plate at 500g / min for 1h, place it in an incubator and incubate for 3h. Then add 1mL of complete culture medium containing 8μg / mL polybrene and continue to incubate overnight.

[0187] 2.2.5 Screening for positive cells with puromycin

[0188] (1) On the second day after lentivirus infection, cells were collected into centrifuge tubes, centrifuged, and the supernatant was discarded. Fresh complete culture medium was then used for further culture.

[0189] (2) After 3 days, change the medium and add complete culture medium containing 1 μg / mL puromycin for screening;

[0190] (3) After all the cells in the blank control have died, replace the culture medium with a complete culture medium containing 0.5 μg / mL puromycin;

[0191] (4) After the cells have proliferated to a sufficient density, extract the cell DNA and total protein, and continue to culture the remaining cells;

[0192] (5) Using cellular DNA as a template, PCR amplification was performed using PPIF gene primers, and the product was sent to the company for sequencing. The expression level of CypD protein was detected by Western blot.

[0193] (6) If the sequencing results of the PCR product show mutations such as double peaks, frameshifts or base deletions in the sgRNA region, and Western blot analysis shows that the expression level of CypD protein is lower than that of wild cells, it indicates that the genome of some cells has been edited, indicating that there are positive cells in the polyclonal cell population where the target gene has been knocked out.

[0194] 2.2.6 Monoclonal cell isolation and identification

[0195] Refer to the aforementioned method for the isolation and identification of CypD-deficient THP-1 monoclonal cells.

[0196] Because HeLa cells have high transfection efficiency, the constructed pX459-sgRNA recombinant plasmid was directly transfected into cells using the jetPRIMEreagent transfection reagent. Puromycin was added 24 hours after transfection for selection, resulting in a polyclonal cell population. For THP-1 cells, which are more difficult to transfect, a lentiviral infection method was used. The constructed LentiCRISPR V2-sgRNA recombinant plasmid was packaged into lentivirus and used to infect THP-1 cells. Puromycin selection was then performed to obtain a polyclonal cell population. To verify the gene editing effect, cellular DNA was extracted for PCR amplification (primers: PPIF-F: TTCAAAGCACTTCACGCTCCGC; PPIF-R: CCTTGAGCTTTCTCCTCCACG) and sequencing. The results showed that HeLa cells (… Figure 3 A) and THP-1 cells ( Figure 3C) The sequencing peaks of the sgRNA target site of the DNA showed a double-peak signal and base deletion, indicating that the genomic sequence of the PPIF gene had been edited. Further analysis of CypD protein expression levels using Western blot revealed that, compared to wild-type cells, CypD-deficient HeLa cells (… Figure 3 B) and THP-1 cells ( Figure 3 CypD protein expression was significantly reduced in both D) and THP-1 cells. These results indicate that polyclonal populations of CypD-deficient HeLa and THP-1 cells were successfully constructed and can be further screened for monoclonal cells.

[0197] Cells were diluted to approximately one cell per well using a limiting dilution method and seeded in 96-well plates for culture. Wells containing only a single cell cluster were selected using a microscope for further culture. DNA and total protein were extracted from monoclonal cells and subjected to PCR amplification, sequencing, and Western blot analysis. Sequencing results showed that CypD knockout HeLa cells (… Figure 4 A) and THP-1 cells ( Figure 4 The sequencing peaks of the sgRNA target region in C) all showed single-peak signals and single-base deletions, indicating successful editing of the HeLa and THP-1 genes. Western blot results showed that the CypD-knockout HeLa ( Figure 4 B) and THP-1 Figure 4 D) CypD protein expression was completely absent in all monoclonal cells. These results indicate that the CypD-KO HeLa and CypD-KO THP-1 monoclonal cell lines were successfully constructed.

[0198] Example 3: Performance Validation of CypD-Deficient Cell Lines

[0199] 3.1 Cell growth curve determination

[0200] (1) CypD-KO cells and wild-type cells were mixed at a ratio of 1×10⁻⁶. 4 The culture medium was evenly inoculated into 24-well plates at a density of 0.5 mL per well. Three parallel groups were set up at each time point, and the plates were continuously cultured in a constant temperature incubator at 37℃ and 5% CO2.

[0201] (2) Cell density was measured every 24 hours using a hemocytometer;

[0202] (3) Count the cells for 7 consecutive days and use GraphPad Prism 9.0 to plot the proliferation curve with culture time as the x-axis and cell density as the y-axis.

[0203] Wild-type cells and CypD-deficient cells in normal logarithmic growth phase were selected and seeded at 1×10⁶ cells per well in a 24-well cell culture plate.4 Cell counts were performed on 100 cells over 7 consecutive days, and growth curves were plotted. Results showed that the growth trends of wild-type and CypD-deficient cells were consistent in both HeLa and THP-1 cells: after 3-4 days of culture, cells entered the logarithmic growth phase, and the proliferation rate significantly increased; after 5-6 days, the cell proliferation rate gradually decreased, possibly due to high cell aggregation density and nutrient depletion in the culture medium. In conclusion, there was no significant difference in the growth curves between the two groups of cells. Figure 5 This indicates that the absence of CypD does not affect the in vitro proliferation capacity of HeLa and THP-1 cells.

[0204] 3.2 Intracellular proliferation assay of Listeria monocytogenes in HeLa cells after CypD deletion

[0205] (1) WT HeLa and CypD-KO HeLa cells were mixed at a ratio of 2×10⁻⁶. 5 The EGD-e colonies were evenly spread in a 24-well plate at a density of 0.5 mL / mL. On the same night as the plate was spread, a single colony of EGD-e was picked and placed in 5 mL of BHI liquid medium and cultured with shaking at 37°C and 200 rpm.

[0206] (2) After culturing for 12 hours, take 1 mL of bacterial solution, centrifuge at 5000 rpm for 2 min, wash twice with sterile PBS, and adjust the bacterial solution concentration to 0.6 at OD600 nm.

[0207] (3) Discard the culture medium in the cell culture wells, gently rinse the cells three times with preheated PBS, dilute the prepared bacterial culture with DMEM cell culture medium without fetal bovine serum, mix well by pipetting, and add it to the cell plate at a ratio of MOI=200:1. After adding, place it in a cell culture incubator at 37℃ for culture.

[0208] (4) After culturing for 1 hour, gently wash the cells 3 times with preheated PBS. Dilute 50 mg / mL gentamicin with DMEM cell culture medium at a ratio of 1:1000, add it to the culture plate, and continue culturing for 1 hour. Then replace it with DMEM complete culture medium containing a final concentration of 5 μg / mL gentamicin.

[0209] (5) Wash the cells three times with sterile PBS at 2h and 6h after infection. Then add 500μL of pre-chilled lysis buffer (0.25% Trypsin-EDTA: sterile ddH2O = 1:4) to each well and lyse on ice for 15min. Collect the samples into 1.5mL EP tubes, shake to mix and then perform 10-fold serial dilution.

[0210] (6) After vortexing and mixing, take 10 μL and inoculate it onto a BHI plate. Perform three replicates for each sample.

[0211] (7) Finally, place the BHI plates in a 37°C incubator and incubate overnight. Count the single colonies the next day.

[0212] 3.3 Intracellular proliferation assay of Listeria monocytogenes in THP-1 cells after CypD deletion

[0213] WT THP-1 and CypD-KO THP-1 cells were divided at a ratio of 1×10 6 THP-1 cells were evenly seeded at a density of 0.5 mL / well in a 24-well plate and induced to differentiate into macrophages. The next day, cells were infected with EGD-e (MOI = 5:1). The steps for inducing THP-1 cells to differentiate into macrophages are as follows:

[0214] (a) The collected and centrifuged cell pellet was resuspended in 6 mL of cell culture medium to form a single-cell suspension;

[0215] (b) Clean the hemocytometer with alcohol and dry it, then place the coverslip over the counting chamber;

[0216] (c) Take 10 μL of cell suspension and aspirate the liquid along the edge of the coverslip to ensure that all the liquid is absorbed.

[0217] (d) Observe and count the counting instruments under a microscope;

[0218] (e) The cells were arranged at a ratio of 1×10 6 After diluting the cells at a density of cells / mL, PMA was added to a final concentration of 100 ng / mL to induce THP-1 cells to differentiate into macrophages. After thorough mixing, the cells were added to cell culture plates, 0.5 mL per well in a 24-well plate. After gently shaking to mix, the plates were placed in a cell culture incubator for static culture.

[0219] 3.4 Flow cytometry detection of mitochondrial calcium ion levels

[0220] (1) Following the aforementioned cell plating method, HeLa cells and THP-1 cells were evenly seeded into 6-well plates, 2 mL per well, and incubated overnight in an incubator.

[0221] (2) Cells were infected with Listeria monocytogenes, and an uninfected group was set up as a control.

[0222] (3) Use serum-free culture medium to dilute Rhod-2 AM to a final concentration of 5 μM working solution and mix thoroughly; discard the culture medium in the cells 2 h after infection, wash the cells with PBS, then add 200 μL of 0.25% Trypsin-EDTA to digest the cells, collect the cells in 1.5 mL EP tubes, and centrifuge at 700 rpm for 3 min.

[0223] (4) Carefully aspirate the supernatant, add 500 μL of Rhod-2 AM working solution to each tube, gently resuspend the cells, place the EP tubes in a 37°C cell culture incubator and incubate in the dark for 30 minutes. At the same time, set up an unstained control group to detect cell autofluorescence. After incubation, discard the staining solution and wash the cells twice with sterile PBS.

[0224] (5) Resuspend the cells in PBS to a density of 1×10⁻⁶. 6 A single-cell suspension of cells / mL was prepared, and 500 μL of the sample was used for analysis. 10,000 cells were collected for each sample. The fluorescence signal intensity was analyzed using FlowJo software.

[0225] Using CRISPR-Cas9 gene editing technology, we successfully constructed CypD-deficient HeLa and THP-1 cell lines to investigate the effect of endogenous CypD protein alterations on the intracellular proliferation capacity of Listeria monocytogenes. In this experiment, CypD-deficient cells were infected with Listeria monocytogenes, and samples were collected at 2 and 6 hours after infection. Bacterial proliferation was detected by plate counting. The results showed that compared with wild-type HeLa cells, the number of Listeria monocytogenes colonies in CypD-KO HeLa cells was significantly reduced. Figure 6 A). Furthermore, the same results were observed in THP-1 cells ( Figure 6 B). In summary, the absence of CypD significantly inhibited the proliferation of Listeria monocytogenes in HeLa and THP-1 cells, suggesting that Listeria monocytogenes may assist in self-infection by regulating CypD.

[0226] To further investigate the mechanism by which CypD affects Listeria monocytogenes infection, this study combined the mitochondrial calcium ion fluorescent probe Rhod-2 AM with flow cytometry to detect changes in mitochondrial calcium ion concentration before and after infection in CypD gene-deleted cells. The results showed that, under uninfected conditions, CypD-KO HeLa cells had significantly higher mitochondrial calcium ion concentrations before and after infection. 2+ The level was significantly higher than in wild-type cells, indicating that the absence of CypD protein would impair mitochondrial calcium metabolism. 2+ Elevated levels; Listeria monocytogenes infection induces mitochondrial Ca2+ levels in wild-type HeLa cells. 2+ The level increased significantly, while the mitochondrial Ca of CypD-KO HeLa cells... 2+ The level further increased ( Figure 7 A). Similar results were also observed in THP-1 cells. Figure 7 B). The above results indicate that the loss of CypD protein not only increases mitochondrial calcium levels but also... 2+ Accumulation, and also further increased mitochondrial calcium levels induced by Listeria monocytogenes infection. 2+The elevated levels suggest that Listeria monocytogenes may alter mitochondrial calcium levels by regulating CypD protein expression. 2+ The level of the body affects its own survival.

[0227] 3.5 Data Statistics and Analysis

[0228] All data used in this study were processed using Graphpad Prism 9.0 software and analyzed using Student's t-test. Results are expressed as mean ± SD, ns indicates no statistical difference, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

Claims

1. Use of a PPIF gene expression inhibitor or a CypD protein inhibitor for the manufacture of a medicament for reducing Listeria monocytogenes infection.

2. Use of a vector inactivating a PPIF gene or reducing PPIF gene expression or a recombinant microorganism comprising said vector for the manufacture of a medicament for reducing Listeria monocytogenes infection.

3. A method for reducing cell infection with Listeria monocytogenes in vitro, characterized in that, The method comprises inactivating a PPIF gene or reducing PPIF gene expression.

4. The method of claim 3, wherein, It comprises the following steps: introducing into said cell a vector inactivating a PPIF gene or reducing PPIF gene expression or a recombinant microorganism comprising said vector.

5. Use according to claim 2 or method according to claim 4, characterized in that, The vector comprises a pX459 plasmid or a LentiCRISPR V2 plasmid containing a sgRNA targeting the PPIF gene; and / or, the recombinant microorganism is selected from a lentivirus or an adenovirus.

6. Use or method according to claim 5, characterized in that, The sgRNA targeting the PPIF gene is selected from SEQ ID NO: 3, 4 or 5.

7. The method according to claim 3, said cell being a human cell, preferably a human monocyte or a human cervical cancer cell.

8. Use according to claim 1 or 2 or method according to claim 3, characterized in that, The nucleotide sequence of the PPIF gene is shown in SEQ ID NO:

1.

9. Use according to claim 1, characterized in that, The amino acid sequence of the CypD protein is shown in SEQ ID NO:

2.

10. A cell, comprising: The cell is obtained from the method according to any one of claims 3 to 9.

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