Clostridium difficile resistant lactococcus lactis as well as construction method and application thereof
By constructing Lactococcus lactis Lla-Cwp8 expressing the Cwp8 protein, the problems of drug resistance and intestinal imbalance caused by antibiotic treatment of Clostridium difficile infection were solved, and effective competition, exclusion and translocation of Clostridium difficile were achieved, reducing the risk of infection.
Patent Information
- Application Number
- CN202510862215.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-25
- Publication Date
- 2025-09-19
AI Technical Summary
In the existing technology, antibiotic treatment of Clostridium difficile infection easily leads to drug resistance and intestinal flora imbalance, and there is a lack of safe and effective treatment methods.
A Clostridium difficile-resistant Lactococcus lactis Lla-Cwp8 was constructed. The Cwp8 protein gene was amplified from its genome and expressed on plasmid pLEB124. The Cwp8 protein gene was then transferred into Lactococcus lactis F44 to express the adhesion protein and produce high levels of Nisin, thereby enhancing the competition, exclusion and translocation abilities against Clostridium difficile.
Lla-Cwp8 significantly reduces the survival rate of Clostridium difficile, reduces colon length, reduces the expression of virulence proteins TcdA and TcdB, and reduces the expression of immune factors TNF-α and IL-6, and has the effect of treating Clostridium difficile infection.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microorganisms and biomedicine, and particularly relates to a Clostridium difficile-resistant Lactococcus lactis and a construction method and application thereof. Background Art
[0002] The human intestine is home to a vast number of microorganisms, outnumbering the human body by 10 times. These microorganisms play a vital role in maintaining overall health and are closely linked to digestion, nutrition, immunity, and metabolism. However, in recent years, the overuse of antibiotics has led to disturbances in the intestinal flora. Disturbances in the intestinal flora can trigger a variety of intestinal diseases, with these diseases interacting and causally intertwined. Improving antibiotic-induced intestinal disturbances has become a research hotspot. Lactococcus lactis, a food fermentation agent and probiotic, is frequently consumed in large quantities by humans. Its 34-amino acid antimicrobial peptide, Nisin, exhibits broad antibacterial activity against a variety of Gram-positive pathogens, such as Listeria, Staphylococcus aureus, and Bacillus. Lactococcus lactis can improve intestinal flora disturbances and prevent and improve intestinal diseases by disrupting the cell wall structure of Gram-positive pathogens.
[0003] However, the degree to which Lactococcus lactis improves the intestinal environment is positively correlated with its ability to colonize the intestine. Current research has found that bacterial adhesion is related to bacterial surface proteins and the production of exopolysaccharides. Bacterial surface proteins can participate in adhesion to host cells or tissues, promoting bacterial colonization.
[0004] Clostridium difficile (CDI), a spore-forming, anaerobic, Gram-positive bacterium, is a leading cause of intestinal infections and diarrhea following antibiotic treatment worldwide. Diseases associated with CDI (CDI) range from mild diarrhea to pseudomembranous colitis and even life-threatening conditions, with increasing morbidity, severity, mortality, and healthcare costs. C. difficile has become the leading pathogen of hospital-acquired infections. Currently, the most common clinical treatment for C. difficile infection is antibiotics, which can lead to an imbalance in the intestinal flora, an increase in pathogens, and recurrent infections with drug-resistant C. difficile.
[0005] Therefore, there is an urgent need to construct a Lactococcus lactis that can treat Clostridium difficile infection without causing drug resistance and without safety risks. Summary of the Invention
[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Lactococcus lactis that is resistant to Clostridium difficile.
[0007] The second object of the present invention is to provide a method for constructing Lactococcus lactis that is resistant to Clostridium difficile.
[0008] The third object of the present invention is to provide the use of the above-mentioned Lactococcus lactis against Clostridium difficile in the preparation of drugs against Clostridium difficile infection.
[0009] The technical solution of the present invention is summarized as follows:
[0010] A method for constructing Clostridium difficile-resistant Lactococcus lactis, comprising the following steps: amplifying the Cwp8 protein gene from the Clostridium difficile genome, constructing the Cwp8 protein gene on the pLEB124 plasmid to obtain a recombinant plasmid pLEB124-Cwp8, and transferring the recombinant plasmid pLEB124-Cwp8 into Lactococcus lactis F44 to obtain a Clostridium difficile-resistant Lactococcus lactis, named Lla-Cwp8;
[0011] The nucleic acid sequence of the Cwp8 protein gene is shown in SEQ ID NO.1.
[0012] The above construction method constructs a Clostridium difficile-resistant Lactococcus lactis.
[0013] The application of the above-mentioned Lactococcus lactis that resists Clostridium difficile in the preparation of drugs that resist Clostridium difficile infection.
[0014] Advantages of the present invention:
[0015] The surface of the Clostridium difficile-resistant Lactococcus lactis Lla-Cwp8 can express adhesion protein and can produce Nisin at a high level.
[0016] Experiments have shown that the Lla-Cwp8 of the present invention has strong competition, exclusion, and translocation capabilities against Clostridium difficile in vitro, and can effectively reduce the survival rate of Clostridium difficile and reduce colon shortening in vivo. At the same time, Lla-Cwp8 can significantly reduce the expression of virulence proteins TcdA and TcdB and the expression of immune factors TNF-α and IL-6, and has a therapeutic effect on Clostridium difficile infection. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 To determine the adhesion of Lla-Cwp8 to polystyrene and mucin models;
[0018] Figure 2 To measure the adhesion of Lla-Cwp8 to cell models;
[0019] A: Observation of F44 adhesion to the cell model under an inverted fluorescence microscope;
[0020] B: Observation of Lla-Cwp8 adhesion to the cell model under an inverted fluorescence microscope;
[0021] C: Bacterial count results after Lla-Cwp8 and F44 adhered to the cell model.
[0022] Figure 3 The competition, exclusion, and translocation effects of Lla-Cwp8 on Clostridium difficile in cell models;
[0023] A: Competitive ability test of Lla-Cwp8 and F44 against Clostridium difficile in cell models;
[0024] B: Lla-Cwp8 and F44 were tested for their ability to repel Clostridium difficile in a cell model;
[0025] C: Detection of the translocation ability of Lla-Cwp8 and F44 against Clostridium difficile in a cell model.
[0026] Figure 4 The competition, exclusion, and translocation effects of Lla-Cwp8 on Clostridium difficile in a mouse intestinal tissue model;
[0027] A: Competitive ability test of Lla-Cwp8 and F44 against Clostridium difficile in mouse intestinal tissue model;
[0028] B: Detection of the ability of Lla-Cwp8 and F44 to repel Clostridium difficile in a mouse intestinal tissue model;
[0029] C: Detection of the translocation ability of Lla-Cwp8 and F44 against Clostridium difficile in a mouse intestinal tissue model.
[0030] Figure 5 The effect of Lla-Cwp8 in treating Clostridium difficile infection in mouse models. Specific implementation methods
[0031] The present invention will be further described below with reference to specific embodiments.
[0032] Example 1
[0033] Lactococcus lactis F44 is deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 30924; deposit date: June 11, 2024.
[0034] Example 2
[0035] A method for constructing Lactococcus lactis that is resistant to Clostridium difficile comprises the following steps:
[0036] (1) Bacterial genome extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract Clostridium difficile 630
[0037] (Commercial product) genome, using this genome as a template, primers cwp8-F (SEQ ID NO. 2) and cwp8-R (SEQ ID NO. 3) were designed based on the nucleotide sequence of gene cwp8 encoding the cell wall protein Cwp8 of Clostridium difficile 630, and PCR was performed to amplify the Cwp8 protein gene (SEQ ID NO. 1);
[0038] The upstream and downstream primer sequences are:
[0039] cwp8-F (SEQ ID NO.2): 5'-atgttaagcaacaaaaagagaagtatg-3'
[0040] cwp8-R (SEQ ID NO.3): 5'-ctacactatgtcttacatattaacgc-3'
[0041] (2) The PCR product of the Cwp8 protein gene in step (1) was purified and recovered using a universal DNA purification and recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the purified and recovered product of this step was double-digested with restriction endonucleases BamHI and SmaI. The digested product was treated with a universal DNA purification and recovery kit to obtain a digested fragment (Cwp8 protein gene fragment).
[0042] The enzyme digestion reaction system is: BamHI 1.5μL, SmaI 1.5μL, Cut Smart Buffer 5μL, Cwp8 protein gene fragment 42μL. The enzyme digestion reaction conditions are: 25℃ water bath for 2h, then 37℃ water bath for 2h;
[0043] (3) Plasmid pLEB124 (commercial product) was double-digested with restriction endonucleases BamHI and SmaI. The enzyme digestion reaction system was the same as that in step (2) of this example, wherein the Cwp8 protein gene fragment was replaced by plasmid pLEB124 to obtain a linearized pLEB124 plasmid;
[0044] (4) The enzyme digestion product in step (2) and the enzyme digestion product in step (3) were connected to obtain the recombinant plasmid pLEB124-Cwp8. The connection method was T4 DNA ligase connection. The connection reaction system was:
[0045] 7 μL of Cwp8 protein gene fragment, 1 μL of linearized pLEB124 plasmid, 1 μL of T4 ligase buffer, and 1 μL of T4 ligase. Incubate the ligation reaction at 22°C for 2 hours.
[0046] (5) The recombinant plasmid pLEB124-Cwp8 obtained in step (4) was transformed into Escherichia coli TG1 (commercially available) by the calcium chloride transformation method. After recovery, it was spread on an LB medium plate containing erythromycin (200 μg / ml) resistance. Single colonies growing on the plate were picked for colony PCR verification. If the PCR product had an electrophoresis band at 1896 bp, it indicated that the transformant was a positive clone. The positive colony was cultured in LB liquid medium overnight, and the plasmid was extracted and sent for sequencing. The correct plasmid was the vector containing Cwp8, which was named pLEB124-Cwp8.
[0047] (6) Cultivating the transformants sequenced correctly in step (5) and extracting the plasmid using a plasmid extraction kit;
[0048] (7) The recombinant plasmid pLEB124-Cwp8 obtained in step (6) was introduced into Lactococcus lactis F44 by electroporation, wherein the electroporation voltage was 2.5 kV. After recovery, the strain was spread on a plate containing erythromycin-resistant seed culture medium, and a single colony grown on the plate was selected for colony PCR verification. If the PCR product had an electrophoretic band at 1896 bp, it was a Clostridium difficile-resistant Lactococcus lactis Lla-Cwp8 (hereinafter referred to as Lla-Cwp8). The verified strain was cultured and stored.
[0049] The composition of the seed culture medium containing erythromycin resistance is: yeast powder 15g / L, peptone 15g / L, sucrose 15g / L, potassium dihydrogen phosphate 20g / L, sodium chloride 1.5g / L, magnesium sulfate 0.15g / L, pH 7.2, and erythromycin concentration is 5μg / mL.
[0050] Example 3
[0051] 1. Determination of adhesion of Lla-Cwp8 to mucin model
[0052] Take 100 μl of 10 mg / mL porcine gastric mucin-PBS solution and add it to a polystyrene 96-well plate and fix it at 4°C for 20 hours. Wash off the unfixed porcine gastric mucin with PBS. At the same time, culture Lla-Cwp8 to the logarithmic phase and adjust the OD600 to nm = 1, added to a polystyrene 96-well plate fixed with porcine gastric mucin, incubated at 30°C for 3 hours, washed with PBS to remove the unattached strains, stained with 100 μl crystal violet solution per well, and then decolorized with 100 μl 33% acetic acid aqueous solution for 30 minutes, dried, and then measured at OD583. nm Determine absorbance, see Figure 1 .
[0053] 2. Experimental study on the adhesion of Lla-Cwp8 to polystyrene models
[0054] Lla-Cwp8 was cultured to the logarithmic phase and adjusted to OD600. nm = 1, added to a polystyrene 96-well plate, incubated at 30°C for 3 hours, washed with PBS to remove the unattached strains, stained with 100 μl crystal violet solution per well, and then decolorized with 100 μl 33% acetic acid aqueous solution for 30 minutes, dried, and then measured at OD583. nm Determine absorbance, see Figure 1 .
[0055] Example 4
[0056] 1. Adhesion force counting experiment of Lla-Cwp8 in HT-29 cell model
[0057] The Lla-Cwp8 bacterial suspension was resuspended in fresh 2% fetal bovine serum cell culture medium and diluted. HT-29 cells were cultured in a 12-well plate, washed twice with PBS at pH = 7.5, and 0.5 ml of the diluted Lla-Cwp8 cell suspension was added to each well. Lla-Cwp8 and HT-29 cells were co-incubated at 37°C, 10% CO2 for 2 hours. After incubation, the cells were washed 5 times with PBS at pH = 7.5 to remove unbound bacteria. 0.5 ml of Triton X-100 was added, and the cells were repeatedly pipetted and incubated at room temperature for 30 minutes. The cell suspension was serially diluted, and the number of adherent bacteria was counted by spot counting. See Figure 2 C.
[0058] 2. Adhesion staining experiment of Lla-Cwp8 in HT-29 cell model
[0059] The Lla-Cwp8 bacterial suspension was resuspended in fresh 2% fetal bovine serum cell culture medium and diluted. HT-29 cells were cultured in a 12-well plate, washed twice with PBS (pH 7.5), and 0.5 ml of the diluted Lla-Cwp8 cell suspension was added to each well. Lla-Cwp8 and HT-29 cells were incubated at 37°C in 10% CO₂ for 2 hours. After incubation, the plates were washed five times with PBS (pH 7.5) to remove unbound bacteria.
[0060] Fix with 4% paraformaldehyde (PFA) for 15 minutes at room temperature and wash three times with pre-chilled PBS. Add 50 μl of hybridization solution (formulation: 900 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.01% sodium dodecyl sulfate (SDS), and 30% formamide) containing 6 ng / μl of fluorescent probe (fluorescein isothiocyanate (FITC)-labeled EUB 338 probe) and incubate at 35°C for 16 hours. Wash 10 times with wash solution (64 mM NaCl, 20 mM Tris-HCl, pH 8.0, 0.01% SDS) at 37°C and three times with pre-chilled PBS. Block with PBS containing 3% (w / v) bovine serum albumin (BSA) for 1 hour at room temperature. Incubate with Goat Anti-Rabbit IgG (H+L) antibody and PBS at a volume ratio of 1:200 at 4°C overnight, then wash three times with PBS. Then incubate with Rabbit Anti-MUC5AC antibody and PBS at a volume ratio of 1:1000 for 1 hour, and then wash three times with PBS. Incubate with DAPI (4',6-diamidino-2-phenylindole) dye and PBS at a volume ratio of 1:2000 for 5 minutes, and then wash three times with PBS. Observe under a fluorescence inverted microscope (eyepiece magnification 10x, objective magnification 40x). Figure 2 B, F44 was used to replace Lla-Cwp8 in this example, and the other conditions were the same as in this example, and the samples were observed under a fluorescent inverted microscope. Figure 2 A.
[0061] Example 5
[0062] Verification of the competition, exclusion, and translocation abilities of Lla-Cwp8 (F44 as control group) against Clostridium difficile 630 in a Caco-2 cell model
[0063] Lla-Cwp8 and Clostridium difficile 630 were cultured to the logarithmic phase, and Caco-2 cells (commercial products) were cultured in DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin-streptomycin double antibody + 1% glutamine under 5% CO2 conditions to a cell density of 1×10 6 The logarithmic phase Lla-Cwp8 and Clostridium difficile 630 bacterial suspensions were added to the pre-cultured Caco-2 cell plate and incubated for 2 hours to perform a cell model competition experiment. After the end, 0.5 ml of Triton X-100 was added, and the cells were repeatedly pipetted and incubated at room temperature for 30 minutes. The cell suspension was serially diluted and plated on CCFA Clostridium difficile culture medium for spot counting. Figure 3 A.
[0064] Lla-Cwp8 and Clostridium difficile 630 were cultured to the logarithmic phase, and Caco-2 cells (commercial products) were cultured in DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin-streptomycin double antibody + 1% glutamine under 5% CO2 conditions to a cell density of 1×10 6 The logarithmic phase Lla-Cwp8 bacterial suspension was added to the pre-cultured caco-2 cell plate and incubated for 2 hours. Then the logarithmic phase Clostridium difficile 630 bacterial suspension was added and incubated for 2 hours to perform the cell model rejection experiment. After the end, 0.5ml Triton X-100 was added, and the suspension was repeatedly pipetted and incubated at room temperature for 30 minutes. The cell suspension was diluted in a gradient manner and plated on CCFA Clostridium difficile culture medium for spot counting. Figure 3 B.
[0065] Lla-Cwp8 and Clostridium difficile 630 were cultured to the logarithmic phase, and Caco-2 cells (commercial products) were cultured in DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin-streptomycin double antibody + 1% glutamine under 5% CO2 conditions to a cell density of 1×10 6 Cells / ml. Add the 630 Clostridium difficile suspension to the pre-cultured Caco-2 cell plate and incubate for 2 hours. After 2 hours of incubation, add the logarithmic phase Lla-Cwp8 bacterial suspension to conduct a cell model translocation experiment. After the end, add 0.5ml Triton X-100, repeatedly pipette and incubate at room temperature for 30 minutes. The cell suspension is serially diluted and spread on CCFA Clostridium difficile culture medium for spot counting. Figure 3 C.
[0066] Example 6
[0067] Verification of the competition, exclusion, and translocation abilities of Lla-Cwp8 (F44 as control group) against Clostridium difficile 630 in a mouse intestinal tissue model
[0068] Lla-Cwp8 and Clostridium difficile 630 were cultured to the logarithmic phase, and fresh mouse colon tissue of about 1 cm in length was collected. The logarithmic phase Lla-Cwp8 and Clostridium difficile 630 bacterial suspensions were added to the surface of the prepared mouse colon tissue and incubated for 2 hours to perform an intestinal tissue competition experiment. After the end, the cell suspension was spread on CCFA Clostridium difficile culture medium and counted. Figure 4 A.
[0069] Lla-Cwp8 and Clostridium difficile 630 were cultured to the logarithmic phase, and fresh mouse colon tissue of about 1 cm in length was collected. The logarithmic phase Lla-Cwp8 bacterial suspension was added to the surface of the prepared mouse colon tissue and incubated for 2 hours. Then, the logarithmic phase Clostridium difficile 630 bacterial suspension was added and incubated for 2 hours to perform the intestinal tissue model rejection experiment. After the end, the cell suspension was spread on CCFA Clostridium difficile culture medium and counted. Figure 4 B.
[0070] Lla-Cwp8 and Clostridium difficile 630 were cultured to the logarithmic phase, and fresh mouse colon tissue was collected at the same time. The logarithmic phase Clostridium difficile 630 bacterial suspension was added to the surface of the prepared mouse colon tissue and incubated for 2 hours. Then, the logarithmic phase Lla-Cwp8 bacterial suspension was added and incubated for 2 hours to perform the intestinal tissue translocation experiment. After the end, the cell suspension was spread on CCFA Clostridium difficile culture medium and counted. Figure 4 C.
[0071] Example 7
[0072] Mouse modeling and treatment plan: Mice (C57BL / 6N, (6-8 weeks) male mice) were given continuous drinking of mixed antibiotic water (kanamycin 0.8 mg / ml; gentamicin 0.07 mg / ml; polymyxin 0.1135 mg / mg; vancomycin 0.09 mg / ml; metronidazole 0.43 mg / ml) for 7 days, sterile water for 2 days, and clindamycin (100 mg / kg) was injected once on the 10th day. Clostridium difficile 630 (10 8 cfu / mL) (commercial product), and Lactococcus lactis Lla-Cwp8 or F44 bacterial solution (10 9 cfu / mL) (as the control group) and then samples were collected.
[0073] Mouse colon length detection:
[0074] The colon of mice treated with Lla-Cwp8 or F44 was collected, and the colon length was measured using a vernier caliper and recorded.
[0075] Clostridium difficile survival rate test:
[0076] About 1 g of feces from mice treated with Lla-Cwp8 or F44 was collected, dissolved in PBS, and spread on CCFA Clostridium difficile culture medium for spot counting.
[0077] Toxins TcdA and TcdB were detected using the Clostridium difficile toxin A ELISA kit and the Clostridium difficile toxin B ELISA kit;
[0078] Immune factors TNF-α and IL-6 were detected using immune factor ELISA kits. Figure 5 .
Claims
1. A method for constructing Lactococcus lactis that is resistant to Clostridium difficile, characterized in that The method comprises the following steps: amplifying the Cwp8 protein gene from the Clostridium difficile genome, constructing the Cwp8 protein gene on the pLEB124 plasmid to obtain a recombinant plasmid pLEB124-Cwp8, and transferring the recombinant plasmid pLEB124-Cwp8 into Lactococcus lactis F44 to obtain a Clostridium difficile-resistant Lactococcus lactis, named Lla-Cwp8; The nucleic acid sequence of the Cwp8 protein gene is shown in SEQ ID NO.
1.
2. A Clostridium difficile-resistant Lactococcus lactis constructed by the construction method of claim 1.
3. Use of the anti-Clostridium difficile Lactococcus lactis according to claim 2 in the preparation of an anti-Clostridium difficile infection drug.