Use of CD630_27900 gene in reducing tolerance to Clostridium difficile

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to the use of the CD630_27900 gene in reducing the tolerance of Clostridium difficile. Knocking out the CD630_27900 gene can reduce the tolerance of Clostridium difficile to acidic environments and antibiotics and reduce its cytotoxicity; the sequence of the CD630_27900 gene is shown in SEQ ID NO.1; the antibiotics include ampicillin, metronidazole, amoxicillin, vancomycin, norfloxacin, cefoxitin, clindamycin and kanamycin. The research results of the present invention suggest that the CD630_27900 gene can be used as a target for inhibiting the drug resistance and reducing the toxicity of Clostridium difficile, providing a new idea for the treatment of Clostridium difficile infection (CDI).

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the use of the CD630_27900 gene in reducing tolerance to Clostridium difficile. Background Art

[0002] Clostridioides difficile is an obligate anaerobic, spore-forming Gram-positive bacillus. It is widely distributed in the intestines of humans and animals, transmitted through the fecal-oral route, and is a conditional pathogen. Intestinal flora imbalance caused by the extensive use of antibiotics is the main cause of Clostridioides difficile Infection (CDI). Clinically, CDI can be divided into mild diarrhea to severe life-threatening complications, such as pseudomembranous colitis, intestinal perforation, and toxic megacolon. The pathogenicity of Clostridioides difficile is completely dependent on exotoxin A (Toxin A, TcdA) and exotoxin B (Toxin B, TcdB), which are enterotoxins and cytotoxins, respectively; after colonization of Clostridium difficile, TcdA and TcdB released into the extracellular space will act on intestinal epithelial cells through the cell wall, causing the loss of intestinal cell skeleton and destruction of the intestinal epithelial barrier, thereby causing related symptoms. Clostridium difficile has brought great challenges to the treatment of CDI due to its drug resistance and easy recurrence. The characteristics of drug resistance and persistent infection of Clostridium difficile indicate that the strain has developed a strong ability to adapt to the host intestinal environment during its interaction with the host, enabling it to survive and proliferate in the intestine.

[0003] The ability of Clostridium difficile to adapt to the host intestinal environment and escape host attack is closely related to its special cell wall composition and structure. Figure 1 As shown in the figure, the cell wall of Clostridium difficile is composed of polysaccharides, S layers and peptidoglycans attached to the cell wall. The S layer and polysaccharides that constitute the outermost layer of the cell wall are the first to be exposed to the host's innate immune system. The peptidoglycan structure located on the innermost side is an important skeleton molecule of the cell wall component of Clostridium difficile, and is composed of the basic units N-acetylmuramic acid (NAG) and N-acetylglucosamine (GlcNAc). Most bacteria adapt to the host environment and evade the host's defense mechanism by modifying the cell wall, especially deacetylation of cell wall peptidoglycan. The existence of cell wall deacetylation mechanism can be observed in Lactococcus lactis, Listeria monocytogenes and Streptococcus pneumoniae, and studies have shown that the lack of peptidoglycan N-deacetylation in Streptococcus pneumoniae and Listeria monocytogenes will change the cytotoxicity. Compared with other bacteria, the peptidoglycan structure of Clostridium difficile not only has a unique high-density peptide cross-linking, but also has a higher level of N-GlcNAc deacetylation (93-97.5%). This shows that N-GlcNAc deacetylation is very important for Clostridium difficile to maintain normal life activities.

[0004] The CD630_27900 gene of Clostridium difficile is located at the slpA-cwp66 locus, which contains a total of five genes responsible for encoding the main structure of the S layer and the translocase. In previous studies, the gene function of CD630_27900 has not been clarified. Only William.J et al. reported that the CD630_27900 gene belongs to the enzyme of the assumed LmbE family. According to ViarsS et al., the enzyme of the LmbE family is characterized by being able to hydrolyze the acetyl group in N-GlcNAc. In bacteria, N-GlcNAc is the basic unit of the cell wall structure, and the glycosylphosphatidylinositol membrane anchor in parasites is also derived from its anabolism. Therefore, the enzyme of the LmbE family has become a target for the development of drugs for the treatment of various bacteria and parasitic diseases. Therefore, the present invention describes for the first time that the CD630_27900 gene can significantly reduce the antibiotic resistance, pH tolerance and cytotoxicity of Clostridium difficile, and can be used as a potential target for the treatment of Clostridium difficile infection. Summary of the invention

[0005] To achieve the above object, the present invention provides the use of CD630_27900 gene in reducing the tolerance of Clostridium difficile. Specifically, the sequence of the CD630_27900 gene is shown in SEQ ID NO.1, and the tolerance is tolerance to acidic environment and antibiotic drugs.

[0006] Furthermore, the antibiotic drugs include ampicillin, metronidazole, amoxicillin, vancomycin, norfloxacin, cefoxitin, clindamycin and kanamycin.

[0007] Furthermore, the CD630_27900 gene negatively regulates the cytotoxicity of Clostridium difficile and its tolerance to acidic environments and antibiotics.

[0008] Furthermore, the method of reducing the cytotoxicity of Clostridium difficile and its tolerance to acidic environments and antibiotics includes the steps of knocking out the CD630_27900 gene or inhibiting the expression of the CD630_27900 gene.

[0009] Furthermore, the step of knocking out the CD630_27900 gene includes:

[0010] S1. Connect the CD630_27900-arm fragment to the pMTL-BY vector linearized by NotI-HF to obtain a knockout plasmid. The sequence of the CD630_27900-arm fragment is shown in SEQ ID NO.2;

[0011] S2, transforming the knockout plasmid into Escherichia coli to obtain recombinant Escherichia coli;

[0012] S3, co-culturing the recombinant Escherichia coli with Clostridium difficile to perform homologous recombination;

[0013] S4. Screening of Clostridium difficile CD630_27900 gene knockout transformants.

[0014] Furthermore, the Escherichia coli is Escherichia coli CA434.

[0015] Another object of the present invention is to prepare a therapeutic drug for diseases caused by Clostridium difficile infection by using a preparation for targeted knockout or inhibition of the CD630_27900 gene, wherein the preparation can reduce the cytotoxicity of Clostridium difficile and its tolerance to acidic environments and antibiotics. The present invention has the following beneficial effects:

[0016] The research results of the present invention found that by knocking out the CD630_27900 gene, Clostridium difficile's tolerance to all common antibiotics tested (8 types) was reduced. At the same time, compared with the wild type, the mutant strain is more sensitive to acidic environments, and these sensitivity changes can be restored by gene complementation. Interestingly, the autolysis rate of the ΔCD630_27900 strain is significantly lower than that of the wild-type strain, and the amount of toxin release is significantly reduced. In summary, after the loss of the CD630_27900 gene, Clostridium difficile's tolerance to acidic environments and antibiotics is significantly reduced, suggesting that this gene can be used as a potential target for the development and control of Clostridium difficile infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the cell wall structure of Clostridium difficile. The outermost layer of the cell wall of Clostridium difficile is cell wall polysaccharide, the middle layer is the S layer structure, and the innermost layer is the peptidoglycan structure, which is close to the cell membrane.

[0018] Figure 2 The construction of CD630_27900 gene targeting plasmid and complementing plasmid. A: Connect the CD630_27900 gene homology arm to the pMTL-BY plasmid to obtain the CD630_27900 gene targeting plasmid pYJY1. B: Connect the pyrF gene with promoter to the pMTL82151 plasmid to obtain the pyrF complementing plasmid pYJY2-S. C: Connect the CD630_27900 gene with promoter to the pYJY2-S plasmid to obtain the pyrF and CD630_27900 gene double complementing plasmid pYJY2. pyrF' is the homologous gene of pyrF of CD630 strain (the gene comes from Clostridium beijerinickii NCIMB 8052).

[0019] Figure 3To construct the ΔpyrFΔCD630_27900 mutant based on the ACE method. A: RHA (right homology arm) and LHA (left homology arm) on the pMTL-BJ1 plasmid recombined with the CD630 genome homologous genes under 5-FOA pressure. The primer pair HW736 / HW735 was used to screen out colonies resistant to 5-FOA. B: Schematic diagram of the construction of a single-exchange strain. The longer RHA (right homology arm) on the pMTL-YJY1 plasmid has a higher recombination rate and tends to exchange with the ΔpyrF genome at the RHA site to produce a single-exchange strain. C: Schematic diagram of the construction of a double-exchange mutant. The obtained single-exchange strain was spread on BHIS medium containing 5-FOA to screen for double-exchange mutant colonies resistant to 5-FOA. The LHA (Left-side Homology Arm) of the single-exchange mutant recombined with the LHA on the genome to produce a double-exchange mutant (ΔpyrFΔCD630-27900). The double-crossover mutants were screened using primer pair HW797 / HW798, and Tm represents chloramphenicol sulfamethoxazole.

[0020] Figure 4 Screening of ΔCD630_27900 mutant strains. 24 colonies were randomly selected for single exchange genotype and wild genotype detection to screen pure single exchange strains. Bands appeared in Figure A, and no bands were amplified in Figure B, indicating that the colonies were pure single exchange strains. The screening results are shown in Figures A and B. A: Screening of single exchange strains in the homologous arms of the CD630_27900 gene, the target band size was 2681bp. B: Screening of the wild type of the CD630_27900 gene, the wild type band size was 1513bp, and the mutant band size was 805bp. C: After purification of the mixed strain with 20 channels in Figure B, 24 colonies were randomly selected for mutant strain screening, and the obtained band size was 805bp, which was consistent with the mutation characteristics. D: The comparison results after sequencing showed that the ΔCD630_27900 gene was successfully deleted. WT: wild type strain; M: 1kb plus maker.

[0021] Figure 5 The horizontal axis represents the measurement time, and the vertical axis represents the OD 600 * indicates P < 0.05, ** indicates P < 0.01, and n indicates P > 0.05.

[0022] Figure 6The sensitivity of CD630, ΔCD630_27900, and ::CD630_27900 to ampicillin (A), metronidazole (B), amoxicillin (C), vancomycin (D), norfloxacin (E), cefoxitin (F), clindamycin (G), and kanamycin (H) was determined. Compared with wild-type CD630, ΔCD630_27900 was significantly sensitive to all tested antibiotics. The sensitivity of ::CD630_27900 was restored after complementation of the mutant strain. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001, and n indicates P>0.05.

[0023] Figure 7 These are the sensitivity results of CD630, ΔCD630_27900, and ::CD630_27900 strains to different pH values.

[0024] Figure 8 The cytotoxicity test of CD630 and ΔCD630_27900, A1 and B1 are the Vero cell morphology without toxin, A2 and B2 are the toxicity of undiluted CD630 and ΔCD630_27900 supernatant toxin on Vero cells, A2-A8 are the dilutions of 10 1 ~10 6 The toxicity of CD630 supernatant toxin to Vero cells was 10 times that of B2-B8. 1 ~10 6 The toxic effect of ΔCD630_27900 supernatant toxin on Vero cells was fold higher. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0026] The main culture medium and reagents used in the embodiments of the present invention are:

[0027] BHIS (Brain Heart infusion, BHI) medium: brain heart extract powder 38.5g / L, L-cysteine ​​(L-cysteine) 1g / L, yeast extract (Yeast extract) 5g / L. Luria-Bertani (LB) medium: tryptone (Tryptone) 10g / L, sodium chloride (Nacl) 10g / L, yeast extract 5g / L. Metronidazole, vancomycin, amoxicillin, kanamycin, cefoxitin, norfloxacin, chloramphenicol, ampicillin, clindamycin, D-cycloserine, thiamphenicol, etc. were purchased from Solarbio (Beijing); restriction endonucleases XhoI (R0146V), NotI-HF (R3189S), HindIII (R0104V) were purchased from New England BioLabs (NEB, Beijing); DNA recovery kit, plasmid extraction kit, ClonExpress Multis One Step Cloning were purchased from VazymeBiotech (Nanjing), and all primer synthesis and sequencing were completed by Sangon Biotech Co., Ltd. (Sangon, Shanghai).

[0028] The strains and culture conditions used in the examples of the present invention are:

[0029] Clostridium difficile CD630 was purchased from the American Type Culture Collection (ATCC), Escherichia coli CA434 strain was preserved by our laboratory, and Escherichia coli NEB Express was purchased from NEB. Clostridium difficile CD630 was cultured in BHIS medium, anaerobic environment, at 37°C. Escherichia coli was cultured in Luria-Bertani (LB) medium, at 37°C.

[0030] The carrier used in the embodiment of the present invention is:

[0031] pMTL82151: Recorded in Hong W, Zhang J, Cui G, et al.Multiplexed CRISPR-Cpf1-Mediated Genome Editing in Clostridium difficile toward the Understanding ofPathogenesis of C.difficile Infection[J]. ACS synthetic biology, 2018, 7(6):1588–1600.

[0032] Example 1: Construction of CD630_27900 gene knockout plasmid and complementing plasmid

[0033] 1 Methods

[0034] 1.1 Primer design

[0035] Table 1 Primer sequence information

[0036]

[0037] 1.2CD630_27900 gene knockout plasmid

[0038] In order to obtain a CD630_27900 gene-deficient strain, a targeting plasmid must be constructed first. This plasmid construction requires the insertion of two target fragments into the pMTL82151 vector. The first fragment is the pyrF homologous gene with a promoter, and the second target fragment is the upstream and downstream homologous arms of the CD630_27900 gene. Using the genome of Clostridium beijerinckii NCIMB 8052 strain as a template, the pyrF gene and its promoter were amplified using the primer pair HW601 / HW602 to obtain a 2875bp fragment. The amplified target fragment was connected to the PmeI linearized pMTL82151 plasmid using the ClonExpress MultiS One Step Cloning Kit, and the recombinant plasmid pMTL-BY was obtained after screening with the HW606 / HW605 primer pair. The CD630_27900 gene targeting plasmid can be obtained by inserting the CD630_27900 gene homology arm into the pMTL-BY plasmid. The construction process is as follows: using the CD630 (NC_009089) genome as a template, PCR amplifying the upstream and downstream homology arms of the CD630_27900 gene, respectively, and obtaining the complete homology arm CD630_27900-arm fragment (shown in SEQ ID NO.2) by overlapping PCR. After agarose gel electrophoresis, the fragments are recovered and the fragments are purified by ClonExpress Multis One Step The CD630_27900-arm fragment was connected to the pMTL-BY vector linearized with NotI-HF using the Cloning assembly kit. After obtaining the recombinant system, the NEBExpress competent cells were transformed. Colonies were formed on the screening plate. Colony PCR was performed on LB solid plates containing 10 μg / mL chloramphenicol to screen the transformants. The positive transformants were transferred to 10 μg / mL LB liquid culture medium for bacterial growth. Finally, the plasmid was extracted using a plasmid extraction kit to obtain the CD630_27900 gene knockout plasmid pYJY1.

[0039] 1.3 Construction of pyrF and CD630_27900 gene complementation plasmids

[0040] The primer pairs HW733 / HW734 and HW818 / HW819 were used to amplify the pyrF and CD630_27900 genes from Clostridium difficile CD630, respectively. The pMTL82151 vector was linearized with Hind III and then connected to the pyrF gene, and then transformed into competent cells. After screening, the recombinant pYJY2-S was obtained. The pYJY2-S vector was digested with restriction endonuclease Xho I, and the CD630_27900 gene was connected to it. After transformation, the pyrF and CD630_27900 double complementation vector pYJY2 was obtained by screening.

[0041] 2 Results

[0042] Using the CD630 genome of Clostridium difficile as a template, PCR amplified the homology arms, and obtained the upstream and downstream homology arms with band sizes of 500 bp and 1300 bp, respectively. After overlapping PCR, a complete homology arm fragment of 1800 bp was obtained. The homology arm fragment was connected to the pMTL-BY vector linearized with Not I-HF, transformed into competent cells, and colony PCR screening was performed on chloramphenicol resistance plates. When the molecular weight was 1973 bp, it indicated that the plasmid was successfully constructed, and the CD630_27900 gene knockout plasmid pYJY1 was obtained. Figure 2 As shown in A. After primer pairs HW733 / HW734 and HW818 / HW819 were used to amplify the promoter-containing pyrF and CD630_27900 gene fragments from Clostridium difficile CD630, pyrF was connected to the HindIII linearized pMTL82151 vector, and the recombinant plasmid pYJY2-S ( Figure 2 B), the molecular size after successful connection of the pyrF fragment was 2316bp. The CD630_27900 gene fragment was then connected to the linearized pYJY2 plasmid to obtain the CD630_27900 complementation vector pYJY2 ( Figure 2 C), the detected molecular weight is 1997 bp.

[0043] Example 2: Obtaining CD630_27900 gene knockout strains and complemented strains

[0044] 1 Method

[0045] 1.1 Conjugation of targeting plasmid and complementing plasmid to transform C. difficile ΔpyrF

[0046] Construction of the ΔpyrF chassis strain of Clostridium difficile Using the CD630 genome as a template, primers HW731 / HW732 were used to amplify the 798 bp upstream homology arm of the CD630_pyrF gene, and primers HW733 / HW734 were used to amplify the 798 bp downstream homology arm of the CD630_pyrF gene. The upstream and downstream fragments of the CD630_pyrF gene were used as templates to perform overlapping PCR to obtain the complete homology arm fragments. The PCR product of the CD630_pyrF gene deletion was ligated with the HindIII linearized pMTL82151 plasmid using the ClonExpress MultiS One Step Cloning Kit homologous recombination reagent to construct the pyrF knockout plasmid pMTL-BJ1. The method of transforming Clostridium difficile by targeting plasmid conjugation was referred to the article published by Hong et al. (Hong W, Zhang J, Cui G, et al. Multiplexed CRISPR-Cpf1-Mediated Genome Editing in Clostridium difficile toward the Understanding of Pathogenesis of C. difficile Infection [J]. ACS synthetic biology, 2018, 7 (6): 1588–1600.). The pyrF knockout plasmid pMTL-BJ1 was transformed into Escherichia coli CA434, and the transformants were transferred to LB liquid medium for culture. After the strain grew, it was mixed with Clostridium difficile liquid and spotted on a BHIS solid plate. After 18 hours, the bacteria were scraped off and spread on a BHIS medium containing cefoxitin, D-cycloserine, and thiamphenicol for transformant screening. The above transformants were cultured on a BHIS plate supplemented with 400 μg / mL 5-FOA for 48 hours. When colonies were found on the plate, primers HW735 / HW736 were used to identify whether the pyrF gene was knocked out. The CD630_27900 targeting plasmid pYJY1 and the complementing plasmid pYJY2 were used to transform Clostridium difficile ΔpyrF in the same manner as above.

[0047] 1.2 Screening of C. difficile CD630_27900 mutant strains

[0048] The C. difficile transformants carrying the pYJY1 targeting plasmid were streaked onto a defined minimal medium for C. difficile growth (CDMM) (McAllister KN, Bouillaut L, Kahn JN, Self WT, Sorg JA. Using CRISPR-Cas9-mediated genome editing to generate C. difficile mutants defective in selenoproteins synthesis [J]. SciRep. 2017, 7 (1): 14672.), and single-exchange strains were screened by colony PCR using primer pair HW681 / HW739. The single-exchange strains were then plated on BHIS medium containing 2 mg / mL 5-fluoroorotic acid (5-Fluoroorotic, 5-FOA) to screen for double-exchange strains. After colonies grew on the screening plate, single clones were picked and screened by colony PCR using primer pair HW738 / HW739. Finally, the ΔCD630_27900 gene knockout strain was obtained, and the plasmid was lost by continuous passage.

[0049] 2 Results

[0050] ACE is a gene editing method for Clostridium difficile published by Heap et al. (Heap JT, Ehsaan M, Cooksley CM, Ng YK, Cartman ST, Winzer K, Minton NP. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker [J]. Nucleic Acids Res. 2012, 40 (8): e59.) in 2012. This method is based on the uracil-deficient strain ΔpyrF and ACE. By adjusting the length of the homologous arms and controlling the order of homologous arm exchange, the purpose of gene knockout is achieved. The principle is: if the pyrF gene is missing, the bacteria will die due to the inability to synthesize uracil. When this gene exists, it can convert 5-FOA into toxic 5-fluorouracil (5-fluorouracil, 5-FU) and participate in bacterial genome synthesis, ultimately leading to bacterial death. The process of using this method to seamlessly edit Clostridium difficile and obtain mutant strains is as follows. Figure 3 shown.

[0051] During the experiment, the probability of a single double exchange of homologous arms in the strain is low. Using Clostridium difficile ΔpyrF as the chassis strain to obtain a new mutant strain usually requires two screenings. First, pure single-exchange strains are screened on a chloramphenicol plate, and then the single-exchange strains are placed under 5-FOA pressure to screen for double exchanges of homologous arms to obtain mutant strains. When screening pure homologous arm single-exchange strains on CDMM culture medium, the primer pairs HW681 / HW739 (to detect single-exchange strains) and HW738 / HW739 (to detect wild-type strains) are used to simultaneously amplify the same single clone by colony PCR. When the single-exchange strain amplifies a band (2681bp) and the wild type (1513bp) does not amplify a band, the colony can be judged as a pure single-exchange strain. 24 colonies were randomly picked, and the results of screening single-exchange strains are as follows Figure 4 As shown in A, the results of screening wild type Figure 4 As shown in B. Figure 4 A The results showed that except for pores 19, 21, and 22, all other strains underwent single exchange. Figure 4 Judging from the results of B, 4 pure single exchange strains were screened ( Figure 4 B pores 4, 5, 6, 7). Figure 4 As shown in channels 12 and 20 in B, a mutant strain (805 bp) was screened during the screening of pure single-exchange strains, and both mutant strains were mixed with single-exchange ( Figure 4 A) in the holes 12, 20). Figure 4 After the colonies corresponding to lane 20 in A and 4B were purified on 5-FOA medium, 24 colonies were randomly selected for mutation verification. The results showed that all 24 colonies were mutant strains. Figure 4 C. After sequencing results comparison, CD630_27900 gene (708bp, sequence as shown in SEQ ID NO.1) was successfully deleted. Figure 4 D. The pyrF gene complementing plasmid pYJY2 and the pyrF and CD630_27900 gene complementing plasmid pYJY3 were transformed into the ΔCD630_27900ΔpyrF double mutant strain to obtain the single mutant strain ΔCD630_27900 and the complementing strain ::CD630_27900.

[0052] Example 3: Growth rate determination of wild-type, knockout and complemented strains of Clostridium difficile CD630

[0053] 1 Method

[0054] The strains CD630, ΔCD630_27900, and ::CD630_27900 were streaked on solid plates, and single clones were picked from the plates and inoculated into BHIS liquid medium. The strains were cultured to the logarithmic growth phase, and the OD 600When the value was 0.5, the cells were inoculated into new culture medium at a 1% inoculum, with three replicates per group, and cultured anaerobically at 37°C. During this period, the OD was measured every 3 hours using a cell density meter (Ultrospec, USA). 600 Value. Taking time as the horizontal axis, OD 600 The value is the vertical axis and the growth curve is drawn.

[0055] 2 Results

[0056] The growth curves of WT, ΔCD630_27900, and ::CD630_27900 are shown in Figure 5 As shown. There was no significant difference in the growth rate of WT, ΔCD630_27900, and ::CD630_27900 strains during the logarithmic phase (0-12h). The autolysis rate of the ΔCD630_27900 mutant strain was significantly lower than that of the wild-type CD630 strain (24-60h) during the death phase (12-72h), and this difference was statistically significant. The autolysis rate of the ::CD630_27900 complement strain was significantly increased compared with both CD630 and ΔCD630_27900.

[0057] Example 4: Antibiotic sensitivity analysis of wild-type, knockout and complemented strains of Clostridium difficile CD630

[0058] 1 Method

[0059] The microdilution method was used to detect the differences in drug resistance of CD630, ΔCD630_27900, and ::CD630_27900 strains under different antibiotics. First, 150 μL of BHIS liquid culture medium was added to the wells of a 96-well plate, and then 256 μg / mL concentrations of metronidazole, vancomycin, amoxicillin, kanamycin, cefoxitin, norfloxacin, D-cycloserine, chloramphenicol, ampicillin, streptomycin, clindamycin, erythromycin and other antibiotics were added to the first column of different wells. After 11 consecutive two-fold dilutions, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL were obtained. Finally, 10 μL of CD630, ΔCD630_27900, and ::CD630_27900 strains in the logarithmic growth phase were inoculated into each well plate. Three replicates were set for each strain, and blank controls were set for all antibiotics. After anaerobic culture at 37°C for 18 hours, the OD was measured. 600 value.

[0060] 2 Results

[0061] The results of drug resistance test of WT, ΔCD630_27900, and ::CD630_27900 strains to commonly used antibiotics are shown in Figure 6 As shown. Compared with WT, ΔCD630_27900 strain is more sensitive to ampicillin (A), metronidazole (B), amoxicillin (C), vancomycin (D), norfloxacin (E), cefoxitin (F), clindamycin (G) and kanamycin (H), and the drug resistance is reduced. After complementation of ΔCD630_27900 strain, the drug resistance of ::CD630_27900 strain is restored or partially restored to the wild-type state. According to statistical analysis, there are statistically significant differences in the sensitivity of ΔCD630_27900 and WT, ΔCD630_27900 and ::CD630_27900 strains in the above antibiotics.

[0062] The MIC test results of CD630, ΔCD630_27900, and ::CD630_27900 strains against commonly used antibiotics are shown in Table 2. Compared with CD630, the MIC of ΔCD630_27900 to ampicillin, metronidazole, amoxicillin, vancomycin, norfloxacin, cefoxitin, and kanamycin was reduced, and the resistance of ::CD630_27900 was restored. According to the MIC interpretation standards provided by CSLI, the resistance of the ΔCD630_27900 mutant to vancomycin, metronidazole, norfloxacin, and amoxicillin changed from intermediate resistance or resistance to sensitivity. This shows that the deletion of the CD630_27900 gene reduces the resistance of Clostridium difficile to the above antibiotics.

[0063] Table 2 Analysis of minimum inhibitory concentration of Clostridium difficile

[0064]

[0065] Example 5: pH sensitivity analysis of wild-type, knockout and complemented strains of Clostridium difficile CD630

[0066] 1 Method

[0067] BHIS liquid medium with pH 1-12 was prepared with hydrochloric acid (HCl) and sodium hydroxide (NaOH), and the OD values ​​of wild-type CD630, mutant strain ΔCD630_27900, and complemented strain ::CD630_27900 were measured. 600 The value was cultured to 0.5, 1 mL of bacterial solution was taken from each tube of strain, centrifuged at 6000 r / min for 3 minutes, the supernatant was removed, and then 500 μL of culture medium with different pH values ​​was added. After culturing for 3 hours, 1 μL was spotted on a BHIS solid plate and cultured for 24 to 36 hours to observe the growth results of the strain.

[0068] 2 Results

[0069] The pH sensitivity results of WT, ΔCD630_27900, and ::CD630_27900 strains are shown in Figure 7 After treating the strains with culture media of different pH values ​​for 3 hours, it was observed that when pH = 12, all strains did not grow, and when pH was 5-11, all strains could grow on BHIS solid plates. When pH = 4, WT could generate colonies on the plate, but ΔCD630_27900 could not grow. After complementation of the mutant strain, the ::CD630_27900 strain recovered its growth ability at pH = 4. It can be seen that knocking out the CD630_27900 gene will reduce the tolerance of Clostridium difficile to acidic environments.

[0070] Example 6: Cytotoxicity detection of wild-type and knockout strains of Clostridium difficile CD630

[0071] 1 Method

[0072] CD630 and ΔCD630_27900 strains were cultured to obtain supernatant toxins, which were diluted 10-fold and used to infect African green monkey kidney cells (Vero cells). No supernatant toxins were added to the blank control, and the Vero cell morphology was observed under an optical microscope at 200 times magnification.

[0073] 2 Results

[0074] like Figure 8 As shown, the cell morphology of the control group without the addition of toxin was spindle-shaped ( Figure 8 Undiluted CD630 ( Figure 8 A2), ΔCD630_27900( Figure 8 The supernatant toxins in B2 can cause the Vero cells to become round and suspended (indicated by the arrows). 1 ~10 6 times (the dilution step is 10 times) and then infected Vero cells ( Figure 8 The maximum dilution factor of CD630 that caused morphological changes in Vero cells was 1×10 5 times (such as Figure 8 The CD630 toxin supernatant was diluted to 1×10 6 The cell morphology returned to normal when the toxin dilution factor of ΔCD630_27900 was 1×10 1 ~1×10 3 times, the cell rounding ratio and suspension degree gradually decreased with the increase of dilution times ( Figure 8 B3-B5), as the dilution factor continues to increase, the cell morphology returns to normal ( Figure 8The results showed that the cytotoxicity of ΔCD630_27900 was significantly reduced compared with CD630.

[0075] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0076] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The use of knocking out the CD630_27900 gene in reducing the cytotoxicity of Clostridium difficile and its tolerance to acidic environments and antibiotics, characterized in that: The sequence of the CD630_27900 gene is shown in SEQ ID NO.1, the pH of the acidic environment is 4, and the antibiotic drugs are ampicillin, metronidazole, amoxicillin, vancomycin, norfloxacin, cefoxitin, clindamycin and kanamycin.

2. The use according to claim 1, characterized in that The step of knocking out the CD630_27900 gene comprises: S1. Connect the CD630_27900-arm fragment to the pMTL-BY vector linearized by NotI-HF to obtain a knockout plasmid. The sequence of the CD630_27900-arm fragment is shown in SEQ ID NO.

2. S2, transforming the knockout plasmid into Escherichia coli to obtain recombinant Escherichia coli; S3, co-culturing the recombinant Escherichia coli and Clostridium difficile to perform homologous recombination; S4. Screening of Clostridium difficile CD630_27900 gene knockout transformants.

3. The use according to claim 2, characterized in that: The Escherichia coli is Escherichia coli CA434.

4. The use according to claim 1, characterized in that A preparation for targeted knockout of the CD630_27900 gene is used to prepare a therapeutic drug for diseases caused by Clostridium difficile infection.

5. The use according to claim 4, characterized in that The preparation can reduce the cytotoxicity of Clostridium difficile and its tolerance to an acidic environment and antibiotic drugs, the pH of the acidic environment is 4, and the antibiotic drugs are ampicillin, metronidazole, amoxicillin, vancomycin, norfloxacin, cefoxitin, clindamycin and kanamycin.

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