Clostridium difficile engineering strain with low MLSB antibiotic resistance as well as construction and application of clostridium difficile engineering strain

Abstract

The invention firstly provides a method for regulating and controlling the expression of the ermB gene by knocking out a target gene or reducing the activity / expression of a target gene sigmaV or anti-sigmaV gene. The target gene is a sigma V gene or an anti-sigma V gene. Wherein the sigma V has a nucleotide sequence as shown in SEQ ID NO: 1; the anti-sigma V has a nucleotide sequence as shown in SEQ ID NO: 2. On the basis, the invention provides a clostridium difficile engineering strain with low MLSB antibiotic resistance, and the engineering strain is constructed by knocking out a target gene sigma V gene or an anti-sigma V gene. The MLSB antibiotics comprise Macrolides (macrolides), Lcosamides (lincosamides) and Streptogramin B (streptozotocin type B), and the MLSB antibiotics comprise Macrolides (macrolides), Lcosamides (lincosamides) and Streptogramin B (streptozotocin type B). Compared with a wild strain, the clostridium difficile engineering strain reduces erm gene expression by knocking out sigma V or anti-sigma V genes, so that the drug resistance of MLSB antibiotics is remarkably reduced, the drug resistance problem of clostridium difficile is fundamentally solved, and the clostridium difficile engineering strain is expected to be applied to preparation of CDI treatment drugs, achieves a good treatment effect under the condition of low dosage of antibiotics, and has broad application prospects. The method has important social significance and wide market application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to an antibiotic-resistant engineering strain, in particular to an engineering strain of Clostridioes difficile with low MLSB antibiotic resistance and construction and application thereof. BACKGROUND

[0002] Clostridioes difficile is a gram-positive, obligate anaerobic bacillus that can produce spores and parasitizes in the normal human intestinal tract, and is an opportunistic pathogen. Under the use of a large dose of broad-spectrum antibiotics, immunosuppressants or chemotherapy drugs, the proportion of intestinal flora of patients is often out of balance, Clostridioes difficile takes the opportunity to multiply and secrete toxins, thereby causing Clostridium diffcile infection (CDI). CDI occurs mainly in children and the elderly with relatively low immunity, and the main clinical symptoms are diarrhea, abdominal pain, fever, etc. Severe cases can cause pseudomembranous colitis, and can further develop into toxic megacolon, intestinal perforation, septic shock, and even death. CDI patients have a longer hospital stay, a higher recurrence rate and a higher mortality rate, and are the primary factor of hospital-acquired bacterial infection and gastrointestinal inflammation-related death, which seriously threatens the health of hospitalized patients.

[0003] At the same time, due to the use of a large amount of antibiotics in the treatment of CDI, the extensive drug resistance of Clostridioes difficile is becoming more and more serious. At present, Clostridioes difficile shows resistance to many antibiotics including MLSB antibiotics, such as lincomycin and clindamycin, aminoglycosides, tetracyclines, macrolides, cephalosporins, penicillins and fluoroquinolones, etc. More seriously, some antimicrobial drugs, such as cephalosporins, clindamycin, carbapenems, trimethoprim, sulfonamides, erythromycin, broad-spectrum β-lactams, ampicillin, amoxicillin and new generation fluoroquinolones, are more likely to cause CDI than other types of antibacterial drugs. Therefore, the current situation of antibiotic resistance of Clostridioes difficile is very serious, and a high drug resistance rate has been reported worldwide. How to solve the drug resistance of Clostridioes difficile is a technical problem that needs to be solved for the treatment of CDI. The inventors' team has conducted research on the drug resistance of Clostridioes difficile. Among them, the invention patent CN115786380B discloses "Use of CD630_27900 gene in reducing tolerance of Clostridioes difficile". In this patent, the inventors reduce the tolerance of Clostridioes difficile to acidic environment and antibiotic drugs (ampicillin, metronidazole, amoxicillin, vancomycin, norfloxacin, cefoxitin, clindamycin and kanamycin) by knocking out the CD630_27900 gene, and reduce its cytotoxicity.

[0004] Research reports indicate that resistance to MLSB antibiotics in *C. diffusa* is primarily mediated by the *erm* gene. The mechanism involves an enzyme encoded by the *erm* gene methylating bacterial ribosomal target sites, leading to drug inactivation. Theoretically, reducing the expression of the *ermB* gene could fundamentally solve the drug resistance problem in *C. diffusa*. However, methods for reducing *ermB* gene expression have not yet been reported. Summary of the Invention

[0005] To address the problem of widespread drug resistance in Clostridium difficile in existing technologies, this invention first provides a method to address this by knocking out or reducing the σ value of the target gene. V Or anti-σ V This application describes a method for regulating ermB gene expression, specifically focusing on gene activity / expression. Based on this, it provides an engineered strain of *Clostridium difficile* with low resistance to MLSB antibiotics, achieved by knocking out the target gene σ. V Genes or anti-σ V The gene was successfully constructed. Compared with the wild strain, the engineered *Clostridium difficile* strain showed a significant decrease in resistance to MLSB antibiotics, fundamentally solving the drug resistance problem of *Clostridium difficile*, which has important social significance and broad market application prospects.

[0006] The technical solution of the present invention:

[0007] This application first provides the application of a target gene in regulating ermB gene expression, wherein the target gene is σ. V Or anti-σ V Gene. Wherein, the σ V Possessing the nucleotide sequence shown in SEQ ID NO:1; the anti-σ V It possesses the nucleotide sequence shown in SEQ ID NO:2. V The factor is a member of the Sigma 70 family of extracellular functional sigma factors (ECFs). This family of members is related to bacterial stress response and can regulate gene expression when needed; they participate in various bacterial stress responses, including responses to environmental stresses such as antibiotics, temperature, osmotic pressure, and heavy metal ions. Under normal circumstances, anti-sigma factors bind to sigma factors. The inventors unexpectedly discovered in their research that: (1) knocking out sigma factors separately V and anti-σ V After gene therapy, the strain's sensitivity to clindamycin decreased, and the sensitivity was partially restored in the replacement strain. (2) Anti-σ in wild-type strains under clindamycin stress V A high expression trend was observed, and σ was demonstrated through GST-pull-down experiments.V and Anti-σ V have direct interaction, knocking out anti-σ V post-σ V expression is up-regulated; knocking out anti-σ V The expression of the ermB gene is significantly down-regulated. (3) After overexpressing the ermB gene in the σ V and anti-σ V mutant strain, the strain's sensitivity to clindamycin is partially restored, and it is proved that σ V can bind to the promoter sequence of the ermB gene.

[0008] Based on the foregoing findings, the inventors first proposed that clindamycin stress Anti-σ V post-σ V promotes the expression of the clindamycin-resistant target gene ermB gene, thereby affecting the clindamycin resistance of C. difficile—namely, σ V and Anti-σ V by regulating the expression of the ermB gene, thereby affecting the clindamycin resistance. As mentioned above, it is well known to those skilled in the art that the resistance of C. difficile to MLSB antibiotics is mainly mediated by the erm gene, and reducing the expression of the erm gene can solve the problem of resistance. Based on this, the application of the target gene in regulating the expression of the ermB gene described in the present application provides theoretical support for the treatment of CDI, and has important significance.

[0009] On this basis, the present application provides a method for regulating the expression of the ermB gene, knocking out the target gene or reducing the activity / expression of the target gene in a cell or organism to down-regulate the expression of the ermB gene; the target gene is σ V or anti-σ V gene. Wherein, the method for knocking out the target gene is preferably CRISPR-Cas9 / Cpf1 / Cas12b / Cas12j / Cas-CLOVER, CRISPRi, Clostron, Thermotargetron (TMT), ACE and INTEGRATE system-mediated gene knockout.

[0010] The method as described above is applied in reducing the MLSB antibiotic resistance of Clostridium difficile. The MLSB antibiotic includes Macrolides (macrolides), Lincosamides (lincomycin) and Streptogramin B (streptogramin B). The macrolide antibiotic includes 14-membered ring antibiotics erythromycin, clarithromycin, roxithromycin, flurithromycin and dirithromycin, 15-membered ring antibiotic azithromycin and 16-membered ring antibiotic josamycin, midecamycin, acetyl midecamycin, spiramycin, rokitamycin, tylosin and oleandomycin; the lincomycin antibiotic is clindamycin and lincomycin; the streptogramin B antibiotic is quinupristin and dalfopristin. Based on the fact that the resistance of Clostridium difficile to MLSB antibiotics is mainly mediated by erm gene, the inventors further study the influence of knocking out σ V or anti-σ V gene on the resistance of Clostridium difficile to clindamycin as a representative of lincomycin, erythromycin as a representative of macrolide and quinupristin as a representative of streptogramin B. The results show that the Clostridium difficile with knocked out σ V or anti-σ V gene has significantly reduced resistance to the three antibiotics; this also confirms that reducing the expression of erm gene is an effective way to solve the drug resistance of Clostridium difficile.

[0011] The application also provides a Clostridium difficile engineering strain with low MLSB antibiotic resistance. The engineering strain is a Clostridium difficile mutant strain with knocked out σ V or anti-σ V gene as described above. The Clostridium difficile engineering strain reduces the expression of erm gene by knocking out σ V or anti-σ V gene, thereby significantly reducing the MLSB antibiotic resistance.

[0012] The application of the Clostridium difficile engineering strain as described above in preparing CDI treatment drugs. Traditional treatment of CDI usually uses more antibiotics (such as vancomycin, fidaxomicin), not only has a very high recurrence rate (about 20-30%), but also forms a vicious cycle. In contrast, the drug using the Clostridium difficile engineering strain works in the opposite direction, fundamentally reduces the antibiotic resistance of Clostridium difficile, so that a low dose of antibiotic can achieve good therapeutic effect.

[0013] The application also provides a pharmaceutical composition comprising an agent capable of reducing the activity of the target gene or inhibiting the expression of the target gene as described above, and a pharmaceutically acceptable carrier.

[0014] Use of the pharmaceutical composition described above in the preparation of a medicament for the treatment or prevention of ermB gene-mediated antibiotic-resistant bacterial infections.

[0015] The beneficial effects of this invention are:

[0016] (1) This application innovatively provides the target gene σ V Or anti-σ V Application of the gene in regulating ermB gene expression; based on the application, this application achieves downregulation of ermB gene expression by knocking out the target gene or reducing the activity / expression of the target gene, overcoming the difficulties of the prior art, providing theoretical support for the treatment of CDI, and is of great significance.

[0017] (2) This application provides a Clostridium difficile engineered strain with low MLSB antibiotic resistance. The Clostridium difficile engineered strain is obtained by knocking out σ... V Or anti-σ V The gene reduced ermB gene expression, thereby significantly reducing resistance to MLSB antibiotics and fundamentally solving the problem of widespread drug resistance in Clostridium difficile.

[0018] (3) The Clostridium difficile engineered strain with low resistance to MLSB antibiotics described in this application is expected to be used in the preparation of CDI treatment drugs, achieving good therapeutic effects under low doses of antibiotics, which has important social significance and broad market application prospects. Attached Figure Description

[0019] Appendix Figure 1 For σ V Gene knockout vector construction and knockout process; among which, (A)σ V (B) Gene location of the gene, (C) Construction of the target vector pCC1-S, (D) σ V Construction of gene knockout vector pCC1, (D) Assembly of gene editing effect complex, (E) Construction of ∆σ V The process of mutants.

[0020] Appendix Figure 2 For σ V The process of constructing gene complementation vectors and complementation lines; among which, (A)σ V Replenishment carrier construction, (B)σ V The process of constructing replacement strains.

[0021] Appendix Figure 3 For σ V Screening and verification of mutant strains and complemented strains; among which, (A) PCR verification σ V Gene deletion, (B) gene sequencing verification σ VGene deletion, (C)RT-qPCR verification successfully constructed σ V Mutant strains and complemented strains.

[0022] Appendix Figure 4 For anti-σ V Gene knockout vector construction and knockout process; among which, (A) anti-σ V (A) Gene location of the gene, (B) Construction of the target vector pCC3-S, (C) anti-σ V Construction of gene knockout vector pCC3, (D) Assembly of gene editing effect complex, (E) Construction of ∆anti-σ V The process of mutants.

[0023] Appendix Figure 5 For anti-σ V Gene complementation vector and complementation strain construction process; among which, (A) anti-σ V Complementary vector construction, (B) anti-σ V The process of constructing replacement strains.

[0024] Appendix Figure 6 For anti-σ V Screening and verification of mutant strains and complemented strains; among which, (A) PCR verification of anti-σ V Gene deletion, (B) gene sequencing verification of anti-σ V Gene deletion, (C)RT-qPCR anti-σ V Gene deletion verification successfully constructed anti-σ V Mutant strains and replacement strains.

[0025] Appendix Figure 7 Wild type (WT), ∆σ V mutant strain, ∆anti-σ V Mutant strains, replacement strains (::σ) V ) and replenishment strain (::anti-σ V The growth curve of ).

[0026] Appendix Figure 8 For ∆σ V mutant strains and ∆anti-σ V The MSLB antibiotic resistance phenotype of mutant strains on brucellosis plates; among which, (A) wild-type (WT), ∆σ V mutant strain, ∆anti-σ V Mutant strains, replacement strains (::σ) V ) and replenishment strain (::anti-σ V (A) Clindamycin resistance; (B) Wild-type (WT), ∆σ VMutant, Δanti-σ V Mutant, complemented (::σ V ) and complemented (::anti-σ V ) erythromycin resistant phenotype; (C) Wild type (WT), Δσ V mutant, Δanti-σ V mutant, complemented (::σ V ) and complemented (::anti-σ V ) quinupristin resistant phenotype.

[0027] Figure 4 Figure 9 Erythromycin resistant phenotype of Δσ V mutant and Δanti-σ V mutant in liquid BHIS liquid medium; Wherein (A) OD600 values of wild type (WT), Δσ V mutant, Δanti-σ V mutant, complemented (::σ V ) and complemented (::Δanti-σ V ) under different antibiotic concentrations, (B) Clindamycin sub-inhibitory concentration tolerance curves of wild type (WT), Δσ V mutant, Δanti-σ V mutant, complemented (::σ V ) and complemented (::anti-σ V ), (C) Growth status of wild type (WT), Δσ V mutant, Δanti-σ V mutant, complemented (::σ V ) and complemented (::anti-σ V ) under clindamycin concentration of 0.5 (μg / mL).

[0028] Figure 5 Figure 10 Anti-σ V binding with clindamycin and increased expression under stress; Wherein, (A) Binding pocket display of Anti-σ V interacting with clindamycin, (B) Interaction details of Anti-σ V with clindamycin, (C) Equilibrium dissociation constant curve of Anti-σ V under different clindamycin concentrations, (D) Real-time sensing curve of Anti-σ V interacting with different concentrations of clindamycin surface plasmon resonance, (E) RT-qPCR detection of wild type (WT) anti-σ VExpression of genes, (F) RT-qPCR detection of σ V Expression of genes.

[0029] Figure 8 Figure 11 Figure 9 V Figure 10 V Figure 11 V Figure 12 V Figure 13 V Figure 14 V Figure 15 V Figure 16 V Figure 17 V Figure 18 V Figure 19 V Figure 20 V Figure 21 V Figure 22 V Figure 23 V Figure 24 V Figure 25 V Figure 26 V Figure 27

[0030] Figure 28 Figure 12 Figure 29 V Figure 30 V Figure 31 V Figure 32 V Figure 33 V Figure 34 V Figure 35 V Figure 36 V Figure 37 V::ermB) and overexpressing strains (∆anti-σ) V Erythromycin tolerance of ::ermB), wild-type (WT), ∆σ was determined by (E)BHIS plate assay. V mutant strain, ∆anti-σ V Mutant strains, overexpression strains (∆σ) V ::ermB) and overexpressing strains (∆anti-σ) V Quinuprudin tolerance (::ermB)

[0031] Appendix Figure 13 For σ V The expression of ermB is regulated by direct binding to the ermB promoter; (A) the genomic location of the ermB gene in Clostridium difficile strain 630, and (B) the σ predicted by the MEME online tool. V Based on consensus motifs, (C)EMSA determined σ V Binding to the promoter binding region of the ermB gene.

[0032] Appendix Figure 14 For σ V The ermB promoter positively regulates ermB gene expression; among which, (A)σ V A schematic diagram showing the regulation of gusA reporter gene expression by binding to the ermB gene promoter sequence. (B) Qualitative detection of GUS enzyme activity. (C) Changes in fluorescence value in solution over time from 0 to 100 min.

[0033] Appendix Figure 15 For anti-σ V With σ V A diagram illustrating the mechanism by which the regulation of the ermB gene leads to clindamycin resistance in Clostridium difficile. Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments.

[0035] Example 1: σ V Amino acid sequence alignment and phylogenetic tree construction

[0036] σ VAmino acid sequence alignment: (1) The target gene (CD630_15580) was retrieved by searching the National Center for Biotechnology Information NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the amino acid sequence was extracted; (2) ProteinBLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to select the refseq_select database, filter typical strain sequences (such as Clostridium difficile homologs), and download all FASTA files; (3) The integrated sequence was submitted to CLUSTALW (https: / / www.genome.jp / tools-bin / clustalw) to generate *.aln files; (4) The protein sequence was analyzed using the AlphaFold Protein Structure Database (https: / / www.alphafold.ebi.ac.uk / ). The ESPript3.0 (https: / / espript.ibcp.fr / ESPript / ESPript / index.php) program was used for visual output drawing, and the sigma V Amino acid multiple sequence alignment and secondary structure prediction. The sigma V Protein of Clostridium bolteae was aligned with Clostridioides difficile 630, Clostridium uliginosum, Metaclostridioides mangenotii, Clostridium butyricum, and Sedimentibacter acidaminivorans homologous sequences. After alignment, it was found that the amino acid sequence of the target gene (CD630_15580) was highly conserved in multiple regions with sigma70 factors of multiple Clostridium species, proving that the target gene may be a sigma70 family member with common evolutionary origin and core functional structure.

[0037] Phylogenetic tree construction: (1) The target gene (CD630_15580) was retrieved from the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ) to extract the amino acid sequence; (2) ProteinBLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to select the refseq_select database, filter typical strain sequences (such as C. difficile homologs), and download all FASTA files; (3) Then use MEGA 11 software to construct the phylogenetic tree. According to the phylogenetic tree, the sigma70 factors of different strains of C. difficile were clustered into one branch, indicating that the target gene encodes the sigma70 factor in the C. difficile 630 strain. V V Protein.

[0038] Example 2: Anti-sigma V Amino acid sequence alignment and phylogenetic tree construction

[0039] Anti-sigma V Amino acid sequence alignment: the method is the same as example 1, except that the target gene retrieved in step (1) is (CD630_15590). The ESPript3.0 (https: / / espript.ibcp.fr / ESPript / ESPript / index.php) program was used for visual output drawing to obtain the Anti-sigma V Amino acid sequence alignment and secondary structure prediction. The Anti-sigma V Protein of C. difficile was aligned with the homologous sequences of C. difficile 630 strain (Clostridioides difficile 630), unclassified Clostridioides, Romboutsia maritimum, Metaclostridioides mangenotii and Caproiciproducens galactitovorans, respectively. After alignment, it was found that the amino acid sequence of the target gene (CD630_15590) was conserved in the Anti-sigma RsiV factor structure in C. difficile and its close relatives, indicating that the target gene may encode the Anti-sigma factor RsiV.

[0040] Phylogenetic tree construction: the method is the same as example 1, except that the target gene retrieved in step (1) is (CD630_15590). Figure 2 ​Phylogenetic analysis of Anti-σV homologous proteins in Clostridium difficile and related strains revealed that the anti-σ factor RsiV encoded by the target gene is highly conserved in Clostridium difficile and its closely related genera, demonstrating that the function of this σ factor RsiV is relatively stable during evolution.

[0041] Example 3: σ V Construction of gene knockout plasmids and σ V Construction of knockout strains

[0042] Figure 1 A is σ V The gene's location. For example... Figure 1 As shown in A, σ V The gene is located between CD630_15570 and CD630_15590. To obtain σ V For gene-deficient strains, the knockout plasmid is first constructed. The construction of this plasmid involves two steps: first, obtaining the target vector pCC1-S; and second, inserting the homologous arm σ into the pCC1-S target vector. V -The arm fragment is used to obtain the knockout vector pCC1. Figure 1 B is the construction of the target vector pCC1-S. Using plasmid pWH55 as a template, σ was amplified using primer pair YW3105 / HW1712. V The -PsRNA::crRNA fragment was used to ligate the amplified target fragment with the BtgZI linearized pWH34 plasmid via homologous recombination. After screening with primer pair HW1094 / HW1095, the recombinant plasmid pCC1-S was obtained. Figure 1 C is σ V Construction of the gene knockout vector pCC1. Using the genome of Clostridium difficile strain 630 (purchased from ATCC) as a template, σ gene knockout vectors were amplified using primer pairs HW1714 / HW1715 and HW1716 / HW1717, respectively. V Upstream and downstream homologous arms of the gene were obtained by overlapping extension PCR using primer pair HW1714 / HW1718 to obtain the complete σ gene. V -arm fragment, then amplified σ through homologous recombination V The -arm fragment was ligated with the XhoI linearized pCC1-S plasmid, and the knockout plasmid pCC1 was obtained after screening with primer pair HW1539 / HW1095.

[0043] After constructing σ V After the plasmid is knocked out, σ is performed. V Construction of knockout strains includes conjugation transformation and screening. Figure 1 D represents the assembly of the gene editing effect complex and plasmid loss. Figure 1 E is used to construct ∆σ V The process of creating mutants. (The text abruptly shifts to a seemingly unrelated topic about σ.)V The pCC1 plasmid was knocked out and transformed into *E. coli* CA434. Transformants were obtained and transferred to LB broth for culture. After growth, the strain was mixed with *Clostridium difficile* culture and spotted onto BHIS solid plates. After 18 hours, the bacterial cells were scraped off and spread onto BHIS medium containing cefoxitin, D-cycloserine, and thiamphenicol. Once transformants appeared on the plates, they were picked and transferred to BHI broth containing thiamphenicol for further culture. The culture was then streaked onto BHI solid medium containing lactose and thiamphenicol to induce double crossover. After single colonies grew on the selection plates, single colonies were picked and HW1718 / HW1719 were selected by PCR using primers. Finally, σ... V The strain was knocked out. Finally, the plasmid was lost through continuous passage.

[0044] Example 4: σ V Construction of gene complementation plasmid and σ V Construction of replacement strains

[0045] The promoter-carrying σ gene was amplified from *Clostridium difficile* using primer pair HW2008 / HW2009. V Gene fragment. BamHI linearization of PMTL82151 vector followed by σ with promoter. V Gene fragment ligation, transformation of competent cells, and selection yielded the pCC2 complement plasmid, such as... Figure 2 As shown in Figure A. Construct σ. V After replenishing the carrier, σ is performed. V Construction of replacement strains includes conjugation transformation and screening, such as Figure 2 As shown in B. Specifically: σ V The pCC2 plasmid was reintroduced into *E. coli* CA434. Transformants were obtained and transferred to LB broth for culture. After the strain grew, it was mixed with *Clostridium difficile* culture and spotted onto BHIS agar plates. After 18 hours, the bacterial cells were scraped off and spread onto BHIS medium containing cefoxitin, D-cyclic serine, and thiamphenicol. Once transformants appeared on the plates, single colonies were picked and screened by PCR using primer pair HW2060 / 2061 to obtain σ... V Replenishment of the plant.

[0046] Example 5: σ V Screening and validation of mutant strains and complemented strains

[0047] Plasmid σ is lost through continuous passage. V After knocking out the vector, a stable σ was obtained. V Mutant strain. Then, PCR detection was performed using primers for HW1718 / HW1719, σ VThe PCR product of the mutant strain was 1286 bp, while the PCR product of the wild-type strain was 1522 bp. The PCR products were sent to Shanghai Sangon Biotech for sequencing to verify the σ mutant. V Gene successfully deleted, σ V The mutant strain was successfully constructed, such as Figure 3 As shown in Figure A. Take WT and ∆σ during the logarithmic growth phase. V mutant strain, ::σ V The reintroduced strains were inoculated into fresh BHIS medium and cultured anaerobically at 37°C until the OD600 reached 0.6. The cells were collected by centrifugation at 12000 r / min for 3 min. Using a bacterial total RNA extraction kit (TIANGEN, Beijing), WT and Δσ values ​​were separately extracted. V 、::σ V Total RNA was collected. The total RNA was reverse transcribed into cDNA using the FasKinggDNA Dispelling RT SuperMi (TIANGEN Beijing) kit. The 16S ribosomal RNA expression gene (rrs) was used as an internal control for the σ expression of each strain. V Gene expression levels were analyzed. The final data were statistically analyzed using Prism 10 software (GraphPad Software, Inc.). See details for the results. Figure 3 B and 5C. Among them, such as... Figure 3 As shown in B, gene sequencing confirmed the presence of σ in the Clostridium difficile 630 genome. V A 236 bp DNA sequence within the gene has been deleted; for example... Figure 3 As shown in C, RT-qPCR detection showed ∆σ V σ in mutant strain V Gene expression is completely absent, while in the complemented strain (:: σ V Its expression was restored in )

[0048] Example 6: anti-σ V Gene knockout plasmid construction and anti-σ V Knockout strain construction process

[0049] Figure 4 A is anti-σ V The gene's location. For example... Figure 4 As shown in A, anti-σ V The gene is located between CD630_15580 and CD630_15600. To obtain anti-σ... VFor gene-deficient strains, the knockout plasmid was first constructed. The construction of this plasmid involved two steps: first, obtaining the target vector pCC3-S; second, inserting the homologous arm anti-σ into the pCC3-S target vector. V -The ARM fragment was used to obtain the knockout vector pCC3. Figure 4 B represents the construction of the target vector pCC3-S. The construction process is the same as in Example 3, except that the target fragment is anti-σ. V The primers for PsRNA::crRNA amplification are YW3105 / HW1833. Figure 4 C represents anti-σ V Construction of the gene knockout vector pCC3. The construction process is the same as in Example 3, except that the target fragment is anti-σ. V -arm amplification primers are HW1834 / HW1835 and HW1836 / HW1837.

[0050] After constructing anti-σ V After the vector was knocked out, anti-σ was performed. V Construction of knockout strains includes conjugation transformation and screening. Figure 4 D represents the assembly of the gene editing effect complex and plasmid loss. Figure 4 E is for constructing ∆anti-σ V The mutant screening process is the same as in Example 3, except that the screening primers are HW1838 / HW1839.

[0051] Example 7: anti-σ V Gene complementation plasmid construction and anti-σ V Replenishment strain construction process

[0052] Anti-σ promoters were amplified from Clostridium difficile using primer pair HW2068 / HW2069. V Gene fragment. BamHI linearization of PMTL82151 vector followed by anti-σ with promoter. V Gene fragment ligation, transformation into competent cells, and selection yielded the pCC4 complement plasmid, such as... Figure 5 As shown in Figure A. Construct anti-σ V After plasmid replenishment, anti-σ was performed. V Construction of replacement strains includes conjugation transformation and screening, such as Figure 5 As shown in B. The specific construction process is the same as in Example 4, except that the replenishment plasmid is pCC4.

[0053] Example 8: anti-σ V Screening and validation of mutant strains and complemented strains

[0054] Plasmid anti-σ is lost through continuous passage. V After knocking out the vector, stable anti-σ was obtained. V Mutant strain. Following this, PCR detection was performed on HW1838 / HW1839 using primers, and anti-σ... V The PCR product of the mutant strain was 1364 bp in size, while the PCR product of the wild-type strain was 1864 bp. The PCR products were sent to Shanghai Sangon Biotech for sequencing to verify the anti-σ antibody. V Gene successfully deleted, anti-σ V The mutant strain was successfully constructed, such as Figure 6 As shown in A. WT and ∆anti-σ were extracted during the logarithmic growth phase according to the method in Example 5. V mutant strain, ::anti-σ V Total RNA from the replacement strain was reverse transcribed into cDNA. The 16S ribosomal RNA expression gene (rrs) was used as an internal reference for anti-σ assays of each strain. V Gene expression levels were analyzed. The final data were statistically analyzed using Prism 10 software (GraphPad Software, Inc.). See details for the results. Figure 6 B and Figure 6 C. Among them, such as Figure 6 As shown in B, gene sequencing confirmed the presence of anti-σ in the Clostridium difficile 630 genome. V A 500 bp DNA sequence within the gene has been deleted; for example Figure 6 As shown in C, RT-qPCR detection showed ∆anti-σ V anti-σ mutant strain V Gene expression is completely absent, while in the complemented strain (::anti-σ) V Its expression was restored in )

[0055] Example 9: ∆σ V mutant strains and ∆anti-σ V Growth curve determination of mutant strains

[0056] Clostridium difficile 630 (WT) and mutant strain ∆σ were used. V , mutant strain ∆anti-σ V 、Replenishment plant::σ V 、Replenishment strain::anti-σ VThe strains were streaked onto solid plates, and single colonies were picked from the plates and inoculated into BHIS liquid medium. When the strains reached the logarithmic growth phase and the OD600 value was 0.5, they were inoculated into fresh medium at a 1% inoculum, with three replicates per group, and cultured anaerobically at 37 °C. The OD600 value was measured every 3 hours using a cell density meter (Ultrospec, USA). A growth curve was plotted with time on the x-axis and OD600 value on the y-axis. See details. Figure 7 The x-axis represents culture time (hours), and the y-axis represents cell turbidity at OD600. For example... Figure 7 As shown, there was no significant difference in the growth of the five strains from 0 to 21 hours; after 21 hours, CD630 and the mutant strain ∆anti-σ showed significant differences. V 、Replenishment plant::σ V 、Replenishment strain::anti-σ V Autolysis, while mutant ∆σ V It is in a plateau phase. Therefore, it can be concluded that σ is missing in *Clostridium difficile*. V The autolysis rate of the gene-derived strain was significantly slowed down, which may directly lead to the continuous release of toxins by Clostridium difficile and enhanced biofilm formation, ultimately resulting in increased pathogenicity, more stubborn infection and higher recurrence rate.

[0057] Example 10: ∆σ V mutant strains and ∆anti-σ V Tests on the resistance of mutant strains to MLSB antibiotics

[0058] This embodiment uses the solid agar plate dilution method to determine ∆σ. V mutant strains and ∆anti-σ VDetermination of resistance to MLSB antibiotics in mutant strains. In accordance with the "Methods for Testing the Drug Susceptibility of Anaerobic Bacteria" (M11Ed9E) (2018) recommended by the Clinical Laboratory Standards Committee (CSLI), Brucella agar plates containing different types and concentrations of the above antibiotics were prepared and MIC tests were performed on the strains. The specific process is as follows: (1) Weigh 45.0 g of Brucella culture medium and add it to 1 L of distilled water. Add 5 mg / mL of heme chloride and 1 mg / mL of vitamin K1 solution at a volume ratio of 1,000:1. Autoclave at 121 °C for 15 min. After cooling to about 60 °C, add 5% defibrinated sheep blood. Set the concentration gradients of clindamycin, erythromycin and quinupristin as 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, respectively. (2) Each strain was cultured to a bacterial suspension of 0.5 McFarland turbidity units, with a concentration of approximately 1 × 10^8 CFU / mL. Each strain was then spotted onto an antibiotic plate and incubated at 37 ℃ for 48 h in an anaerobic workstation. A control group without any antibiotics was also included. Each strain was replicated in triplicate. See details for the results. Figure 8 A- Figure 8 C.

[0059] like Figure 8 As shown in A, (1) wild type (WT), ∆σ V mutant strain, ∆anti-σ V Mutant strains, replacement strains (::σ) V ) and replenishment strain (::anti-σ V (1) It can grow normally on solid blood plates with clindamycin concentration of 0 μg / mL; (2) On solid blood plates with clindamycin concentrations of 0.5 μg / mL and 64 μg / mL, ∆σ V mutant strains and ∆anti-σ V The mutant strain does not grow, while the wild-type (WT) replacement strain (::σ) V ) and replenishment strain (::anti-σ V (3) All of them can grow normally; when the concentration of clindamycin reaches 128 μg / mL, they cannot grow. Figure 8 As shown in Figure B, on solid blood agar plates with erythromycin concentrations of 0.5 μg / mL and 128 μg / mL, ∆σ V mutant strains and ∆anti-σ V The mutant strain does not grow, while the wild-type (WT) replacement strain (::σ) V ) and replenishment strain (::anti-σ VBoth showed normal growth. Similarly, on solid blood agar plates with quinupristin concentrations of 0.5 μg / mL and 32 μg / mL, ∆σ V mutant strains and ∆anti-σ V The mutant strain does not grow, while the wild-type (WT) replacement strain (::σ) V ) and replenishment strain (::anti-σ V All of them can grow normally. Therefore, it can be concluded that the absence of σ in *Clostridium difficile* indicates a deficiency in this bacteria. V or anti-σ V The strains that underwent gene modification showed a significant reduction in resistance to MLSB antibiotics, such as clindamycin, erythromycin, and quinupristin.

[0060] To demonstrate whether the experimental trend of significantly reduced resistance to MLSB antibiotics in the mutant strain is consistent in liquid culture medium, and to further investigate the minimum inhibitory concentration of the mutant strain against MLSB antibiotics, this example further employs a liquid gradient microdilution method to adjust ∆σ. V mutant strains and ∆anti-σ V The tolerance of mutant strains to MLSB antibiotics (represented by clindamycin) was determined using the following steps: Antibiotic concentrations of 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 in 5 mL of liquid BHIS medium. Each test tube was inoculated with 1% WT and Δσ (1% OD600 = 0.6). V ∆anti-σ V 、::σ V and ::anti-σ V Each bacterial strain was used in triplicate. All strains were cultured in an anaerobic environment at 37 °C for 24 h, and the OD600 values ​​were read using a microplate reader. Data are expressed as mean ± standard error (SEM). Statistical comparisons were performed using two-way ANOVA and Tukey's post-hoc test. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, and **** indicates P ≤ 0.0001. See details in [link to results]. Figure 9 A- Figure 9 C.

[0061] Figure 9 In Figure A, the vertical axis represents the OD600 value, and the horizontal axis represents the concentration (μg / mL) of different antibiotics. Yellow, gray, purple, pink, and blue bars represent the wild-type (WT), Δσ, and Δσ values, respectively. V mutant strain, ∆anti-σ VMutant strains, replacement strains (::σ) V ) and replenishment strain (::anti-σ V OD600 values ​​at different antibiotic concentrations. Figure 9 As shown in Figure A, all five strains grew normally at a clindamycin concentration of 0 μg / mL, but stopped growing at a concentration of 128 μg / mL. The mutant strains stopped growing at clindamycin concentrations ranging from 0.5 to 64 μg / mL, and the replacement strains recovered to wild-type levels. Therefore, compared to the wild-type (WT), ∆σ V mutant strains and ∆anti-σ V The mutant strains all showed significantly reduced sensitivity to clindamycin, while the complement strains (::σ) V ) and replenishment strain (::anti-σ V The sensitivity to clindamycin was partially restored to wild-type levels.

[0062] Figure 9 B represents wild type (WT), ∆σ V mutant strain, ∆anti-σ V Mutant strains, replacement strains (::σ) V ) and replenishment strain (::anti-σ V The tolerance curves for subinhibitory concentrations of clindamycin were plotted; the x-axis represents the concentration (μg / mL) of different antibiotics, and the y-axis represents the turbidity of cells at OD600. Figure 9 As shown in Figure B, when the antibiotic concentration is 0.3 μg / mL, ∆anti-σ V The mutant strain did not grow, and Δσ was observed when the antibiotic concentration was 0.5 μg / mL. V The mutant strain does not grow. Therefore, compared to the wild type (WT), ∆σ V mutant strains and ∆anti-σ V The mutant strains showed significantly reduced sensitivity to clindamycin, ∆anti-σ V The mutant strain exhibited a suppressive inhibitory concentration of 0.3 μg / mL for clindamycin, while Δσ V The mutant strain exhibited a suppressive inhibitory concentration of 0.5 μg / mL against clindamycin, while the complement strain (::σ) V ) and replenishment strain (::anti-σ V The sensitivity to clindamycin was restored to the wild-type level.

[0063] Figure 9 C represents the wild-type (WT) and ∆σ at a clindamycin concentration of 0.5 (μg / mL). V mutant strain, ∆anti-σ V Mutant strains, replacement strains (::σ) V ) and replenishment strain (::anti-σ V The growth state of ( ). For example,Figure 9 As shown in Figure C, when the clindamycin concentration was 0.5 μg / mL, the mutant strains did not grow in glass test tubes containing liquid culture medium. Therefore, it can be concluded that the mutant strains cannot grow at a clindamycin concentration of 0.5 μg / mL.

[0064] Example 11: Anti-σ V Prediction of protein-to-clindamycin molecular docking

[0065] The structure file of clindamycin was obtained by searching the PubChem website (https: / / pubchem.ncbi.nlm.nih.gov / ), and the SDF file was converted to a PDB file using Open Babel 2.3.2 software. The protein structure was predicted using Alphafold3, and the structure of Anti-σ was determined using AutoDockTools software. V After hydrogenation and charge balancing modifications, the receptor protein and ligand small molecules were converted into PDBQT format. Molecular docking of the receptor protein and ligand small molecules was performed using AutoDock Vina 1.1.2, and the docking results were analyzed using PLIP. The docking results were visualized using PyMOL. (See attached image for details.) Figure 10 A and Figure 10 B. Figure 10 A is Anti-σ V A demonstration of the binding pocket to clindamycin. (By...) Figure 10 As shown in A, Anti-σ V There is a binding pocket, and clindamycin small molecules can enter the pocket. Therefore, Anti-σ... V Proteins can bind to small molecules of clindamycin. Figure 10 B represents Anti-σ V Details of its interaction with clindamycin (including hydrogen bonding and hydrophobic interactions). (By...) Figure 10 Anti-σ shown in B V Proteins 19ILE, 21LYS, 23LEU, and 183VAL form hydrophobic interactions with small molecules, while proteins 250GLN, 251ASP, and 265ASP form hydrogen bonds with small molecules (Anti-σ). V The binding energy with the small molecule clindamycin is -6.4 kcal / mol. Therefore, Anti-σ... V It has a strong binding affinity to small molecule clindamycin.

[0066] Example 12: Surface Plasmon Resonance Measurement of Anti-σ V Protein affinity for clindamycin, a small molecule.

[0067] Surface plasmon resonance (SPR) experiments were performed on a Biacore 8K instrument (Cytiva) using a CM5 sensor chip. The chip surface was activated for 420 s by injecting a freshly prepared solution of 400 mM N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) and 100 mM N-hydroxysuccinimide (NHS) in a 1:1 volume ratio at a flow rate of 10 μL / min. Subsequently, Anti-σ... V After dilution to 20 μg / mL (immobilization buffer), the sample channel (Fc2) was immobilized at a flow rate of 10 μL / min, with an immobilization level of approximately 12,600 response units (RU). The reference channel (Fc1) remained untreated. After immobilization, residual active ester groups were blocked by injecting 1 M ethanolamine hydrochloride (pH 8.5) at a flow rate of 10 μL / min for 420 s. For kinetic analysis, clindamycin was diluted to eight different concentrations (0.08–5 μM) using running buffer (PBS-T, i.e., phosphate-buffered saline solution containing 0.05% (v / v) Tween-20). Each concentration was sequentially injected into two channels (Fc1–Fc2) at a flow rate of 20 μL / min, with the binding phase lasting for 100 s and the dissociation phase lasting for 180 s. A total of eight ascending concentration cycles were performed, and the chip surface was regenerated after each cycle to restore the baseline state. Experimental data were processed using Biacore Insight Evaluation software (Cytiva) and fitted using a 1:1 Langmuir model. See details for the results. Figure 10 C-10D.

[0068] By analyzing Anti-σ V Calculate the equilibrium dissociation constant (KD) for the equilibrium binding reaction at different clindamycin concentrations. Figure 10 As shown in Figure C, the concentration-response curve was fitted based on the steady-state affinity model, yielding a KD value of 1.7 μM. Therefore, Anti-σ... V It exhibits specific binding with moderate affinity to clindamycin. For example... Figure 10 As shown in D, the SPR real-time sensing curve displays Anti-σ V The binding kinetics of clindamycin at seven different concentrations (78.1 nM to 5000 nM) were studied. The curves clearly show the two stages of association and dissociation, with the response signal (RU) increasing with increasing concentration. This indicates that Anti-σ... VThe interaction with clindamycin is typically concentration-dependent and reversible.

[0069] Example 13: RT-qPCR detection of anti-σ gene in wild-type Clostridium difficile under clindamycin stress V σ V and ermB expression

[0070] WT strains in the logarithmic growth phase were inoculated into fresh BHIS medium containing 0.2 μg / mL clindamycin and clindamycin-free medium, respectively, and cultured until OD600nm reached 0.6. Total RNA was extracted using a bacterial total RNA extraction kit (TIANGEN, Beijing). The total RNA was then reverse transcribed into cDNA using a FasKing gDNA Dispelling RT SuperMix kit (TIANGEN, Beijing). The 16S ribosomal RNA expression gene (rrs) was used as an internal control for anti-σ assays of the strain. V σ V The expression levels of the ermB gene were analyzed. For detailed RT-qPCR results, please refer to [link to RT-qPCR analysis]. Figure 10 E, Figure 10 F and Figure 12 B. Data are expressed as mean ± standard error (SEM). One-way ANOVA was used for statistical testing, and Tukey's method was used for multiple comparisons after the fact. * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001.

[0071] Depend on Figure 10 E indicates that under 0.2 μg / ml clindamycin stress, wild-type (WT) anti-σ V Gene expression increased significantly. Figure 10 F indicates that under 0.2 μg / ml clindamycin stress, wild-type (WT) σ V Gene expression increased significantly. Figure 12 B indicates that under 0.2 μg / ml clindamycin stress, the expression of the wild-type (WT) ermB gene significantly increased. In summary, anti-σ... V σ V The ermB gene is closely associated with clindamycin resistance in Clostridium difficile.

[0072] Example 14: Anti-σ V Protein and σ V Protein-binding molecular docking prediction

[0073] Protein interactions were predicted using AlphaFold3; results are detailed below.Figure 11 A and Figure 11 B. Among them Figure 11 A is the σ predicted by alphafold3. V Protein tertiary structure model; Figure 11 B is the Anti-σ predicted by alphafold3. V Protein tertiary structure model. Pymol v2.3.4 was used to analyze the predicted structures, identifying amino acid pairs that can form hydrogen bonds between proteins. PRODIGY was used to analyze the predicted multimeric structures, obtaining a binding energy of -20.7 kcal / mol between proteins. See Figure 12C for details. Figure 11 From C, we know that Anti-σ V Protein and σ V Proteins interact with each other (cyan represents protein Anti-σ). V Purple represents protein σ. V (interaction), where anti-σ V S94, E98, Y148, N145, E33, S178, K39, S177, G272, Y273, Y267, S226, M222, K235, D24, I17, H15, Y14, E13, K11, K9, S3 and σ V Hydrogen bonds are formed between K54, K3, Y58, D137, Y48, K65, R69, D73, K77, Y81, N83, R23, K18, E125, F124, D126, F123, R105, D101, and K157.

[0074] Example 14: Anti-σ V Protein and σ V Protein-bound GST-pulldown

[0075] This embodiment uses the GST pull-down experiment to verify protein σ. V With protein Anti-σ V The interaction between them. The protein σ V The coding sequence was cloned into the pGEX-4T-1 vector to construct the GST fusion protein, which was then transformed into E. coli BL21(DE3). After overnight expression induction with 0.05 mM IPTG at 16°C, bacterial cells were collected and lysed by sonication in PBS containing protease inhibitors. The protein was then purified to obtain σ. V -GST fusion protein. The protein Anti-σ... V The coding sequence was cloned into the pET-28a vector to construct Anti-σ VA fusion protein with a his tag was formed and transformed into E. coli BL21 (DE3). After overnight expression induction with 0.5 mMIPTG at 16°C, bacterial cells were collected; the protein was purified by sonication in PBS containing protease inhibitors to obtain Anti-σ. V -HIS fusion protein. GST-σ was measured using the BCA protein assay kit. V (bait protein), Anti-σ V The concentrations of three proteins—his (prey protein), GST (negative control), and GST—determine the sample loading volume. Add 100 μL of gel suspension to two separate 1.5 ml centrifuge tubes, centrifuge at 1000 g for 1 min to remove the stock solution, add 100 g of lysis buffer to thoroughly resuspend, centrifuge at 1000 rpm for 1 min, and discard the supernatant. Repeat the above washing steps twice. Then add GST-σ... V (25µg) with Anti-σ V -HIS (10µg) protein mixture, and GST (25µg) with Anti-σ V Mix 10 µg of HIS with lysis buffer, then bring the volume to 1 ml. Incubate overnight at 4°C on a shaker. Centrifuge at 2000 rpm at 4°C, discard the supernatant, and wash 8 times (repeatedly inverted) with 100 µL of lysis buffer for 10 minutes each time. After the final wash, centrifuge and discard the supernatant, add 30 µL of elution buffer, boil at 98°C for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and run the supernatant on a gel. Separate by SDS-PAGE, and perform Western blot analysis using His antibody and GST antibody. See details below. Figure 10 D.

[0076] Depend on Figure 11 E indicates that σ, which integrates GST, V Proteins or GST proteins are immobilized on glutathione agarose beads, along with Anti-σ proteins containing HIS tags. V The proteins were co-incubated; the bound proteins were analyzed by SDS-PAGE electrophoresis and immunoblotting using anti-HIS and anti-GST antibodies. The results showed that Anti-σ... V Can be σ V Specific enrichment, but inability to bind with the GST control, indicates that σ V With Anti-σ V There is a direct interaction between them. Therefore, the GST pull-down experiment verifies that σ... V and Anti-σ V The interaction between them. Figure 11 D is the σ verified by the GST pull-down experiment. Vand Anti-σ V A schematic diagram of the interaction process.

[0077] Example 15: ∆σ V mutant strains and ∆anti-σ V Transcriptomic analysis of mutant strains

[0078] Sample preparation and sequencing: WT, ∆σ V ∆anti-σ V The strains were inoculated into BHIS medium and cultured until the OD600 value was 0.6. After centrifugation at 4000 r / min for 10 min, the supernatant was removed and the bacterial cells of each strain were collected. The cells were transported to Shanghai Meiji Gene Technology Co., Ltd. on dry ice for transcriptome analysis. First, RNA extraction and quality testing were performed. After the samples were qualified, sequencing was performed according to the following steps: (1) ribosomal RNA was removed, (2) mRNA was enriched and purified, (3) mRNA was fragmented, (4) sequencing libraries were constructed and library quality was tested, and (5) sequencing was performed on the Illumina HiSeq platform.

[0079] Data processing and bioinformatics analysis: (1) Raw data processing, filtering and quality assessment: After the sample is taken off the machine, image files are obtained. The sequencing platform software converts the data to generate raw data in 4 GB FASTQ format. The raw data is processed as follows: First, the base quality distribution, base content and other data of the sample taken off the machine need to be quality controlled; second, low quality reads need to be further filtered; next, the measured transcriptome data is compared with the reference genome GCF_000009205.2_ASM920v2_genomic.fna on NCBI; finally, the measured genome is quantitatively analyzed for expression; FPKM (Fragments Per Kilo basesper Millionfragments) is used to standardize the expression level. Generally, FPKM > 1 indicates that the gene is expressed. (2) Differential expression gene clustering analysis: used to determine the expression pattern of differentially expressed genes under different experimental conditions; unknown biological connections between genes can be found through expression level clustering. Bidirectional clustering analysis was performed on the union of differentially expressed genes and samples across all comparison groups using the Pheatmap package in R. Clustering was based on the expression levels of the same gene in different samples and the expression patterns of different genes within the same sample. The Euclidean method was used to calculate distances, and hierarchical clustering was performed using the Complete Linkage method. See details for the results. Figure 11F. (3) Differentially expressed gene analysis: Differentially expressed mRNAs were analyzed using DEseq software. mRNAs with a fold change > 2 and a p-value < 0.05 were considered differentially expressed. Volcano plots of the differentially expressed mRNAs were generated using the ggplots2 package in R. See details for the results. Figure 11 G.

[0080] Figure 11 F represents the wild type (WT) and ∆anti-σ V Cluster analysis of differentially expressed genes in mutant strains; where the horizontal axis represents genes and the vertical axis represents samples, each column represents a sample, pink indicates highly expressed genes, blue indicates low-expressed genes, and genes with high correlation in expression levels are grouped together. Figure 11 From F, we know that ∆anti-σ V The mutant strain showed significant changes compared to the wild-type WT. Figure 11 G is a volcano plot showing differential gene expression between wild-type (WT) and ∆anti-σV mutants; where the horizontal axis represents the gene expression between wild-type (WT) and ∆anti-σV mutants. V Expression differences in mutant strains (log2 fold change, Log2FC), with the ordinate representing the significance level (-log10 Padi). Pink dots indicate significantly upregulated genes (312), blue dots indicate significantly downregulated genes (393), and gray dots indicate genes with no significant difference (3276). Figure 11 As shown in G, ∆anti-σ V σ in mutant strain V Genes are represented by pink dots, and the arrows in the diagram label σ. V Genes in ∆anti-σ V The mutant strain showed significantly upregulated expression.

[0081] Figure 12 A represents wild type (WT) and ∆anti-σ V MA plot of differentially expressed genes in mutant strains; where the horizontal axis represents the gene expression in wild type (WT) and ∆anti-σ. V The expression differences in mutant strains (log2 fold change, Log2FC) are shown on the y-axis, representing the significance level of the difference (log10 TMP). Red dots represent significantly upregulated genes (312), blue dots represent significantly downregulated genes (393), and gray dots represent genes with no significant change in expression (3276). Figure 12 As shown in A, Δanti-σ V In the mutant strain, the ermB gene is represented by a blue dot. The arrows in the figure indicate the ermB gene at ∆anti-σ. VThe mutant strain showed significantly downregulated expression. In summary, σ... V The ermB gene plays a key role in the regulation of clindamycin resistance.

[0082] Example 16: RT-qPCR detection of ∆anti-σ V σ in mutant strain V expression

[0083] Wild-type WT strains and ∆anti-σ were collected during the logarithmic growth phase. V The mutant strains were inoculated into fresh BHIS medium and cultured until the OD600nm value reached 0.6. Total RNA was extracted using a bacterial total RNA extraction kit (TIANGEN, Beijing). The total RNA was then reverse transcribed into cDNA using a FasKing gDNA Dispelling RT SuperMix kit (TIANGEN, Beijing). The 16S ribosomal RNA expression gene (rrs) was used as an internal control to measure the σ of the strains. V Gene expression levels were analyzed. Data are expressed as mean ± standard error (SEM). One-way ANOVA was used for statistical testing, and Tukey's method was used for multiple comparisons afterward. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, **** indicates P ≤ 0.0001. See details in [link to results]. Figure 11 H.

[0084] like Figure 11 As shown in H, ∆anti-σ V σ in mutant strain V The gene was significantly upregulated. Therefore, RT-qPCR results showed that, compared to the wild type, there was significant upregulation in ∆anti-σ. V σ in mutant strain V Gene expression was significantly upregulated, consistent with transcriptome sequencing results.

[0085] Example 17: ∆σ V ::ermB strain and ∆anti-σ V ::ermB strain MSLB antibiotic resistance assay

[0086] The method is the same as in Example 10. The difference is that BHII solid medium was used, and the test strains were wild-type (WT) and Δσ. V mutant strain, ∆anti-σ V Mutant strains and overexpression strains (∆σ) V ::ermB) and overexpressing strains (∆anti-σ) V ::ermB)

[0087] For detailed results, please see [link / reference]. Figure 11 D. Data are expressed as mean ± standard error (SEM). RT-qPCR was statistically analyzed using one-way ANOVA, followed by Tukey's test for multiple comparisons. Clindamycin tolerance curves were statistically compared using two-way ANOVA and Tukey's test. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, and **** indicates P ≤ 0.0001. See details for results. Figure 11 E.

[0088] Depend on Figure 12 C indicates that: (1) Wild type (WT), ∆σ V mutant strain, ∆anti-σ V Mutant strains, overexpression strains (∆σ) V ::ermB) and overexpressing strains (∆anti-σ) V ::ermB) It can grow normally on solid BHIS agar plates with clindamycin concentration of 0 μg / mL; (2) It can grow normally on solid BHIS agar plates with clindamycin concentrations of 0.5 μg / mL and 2.0 μg / mL. V mutant strains and ∆anti-σ V The mutant strain does not grow, as do the wild-type (WT) and overexpression strains (∆σ). V ::ermB) and overexpressing strains (∆anti-σ) V ::ermB) can grow normally; (3) when the concentration reaches 16 μg / mL, wild type (WT) and overexpression strain (∆anti-σ) can grow normally; V ::ermB) Normal growth while ∆σ V mutant strain, ∆anti-σ V Mutant strains and overexpression strains (∆σ) V ::ermB) does not grow. (By) Figure 12 D indicates that on solid BHIS agar plates with erythromycin concentrations of 0.5 μg / mL and 128 μg / mL, ∆σ V mutant strains and ∆anti-σ V The mutant strain does not grow, as do the wild-type (WT) and overexpression strains (∆σ). V ::ermB) and overexpressing strains (∆anti-σ) V Both ::ermB) can grow normally. (From) Figure 12 E indicates that on solid BHIS agar plates with quinupristine concentrations of 0.5 μg / mL and 8 μg / mL, ∆σ V mutant strains and ∆anti-σ VThe mutant strain does not grow, as do the wild-type (WT) and overexpression strains (∆σ). V ::ermB) and overexpressing strains (∆anti-σ) V ::ermB) can all grow normally. This indicates that σ V and Anti-σ V By directly or indirectly regulating the ermB gene, Clostridium difficile can develop resistance to MLSB antibiotics, such as clindamycin, erythromycin, and quinupristine.

[0089] Example 18: EMSA Experimental Verification of σ V Combining with the ermB promoter

[0090] Predict σ using the MEME online website V For details regarding the sequence, please refer to [link / reference]. Figure 13 B. Then, the samples were sent to Shanghai Sangon Biotech for oligonucleotide probe synthesis and 5'-biotin modification. The complementary strands were denatured at 95°C for 5 min in an equimolar ratio, followed by slow cooling to room temperature for annealing to obtain double-stranded probes. These probes were quantified and diluted to working concentrations (final working concentration of the end-labeled probes was 0.05–0.5 nM). The recombinantly expressed and affinity-purified σ... V Protein quantification was performed using BCA. The binding reaction was conducted in a 20 µL system containing: Beyotime EMSA binding buffer, sterile enzyme-free water, target protein, and probe. The system contained labeled probe (0.5 ng) and protein at different concentration gradients (4, 5, 6, 7 mg), and was incubated at 25°C for 30 min. To verify specificity, the following controls were set up: probe alone (free probe control), and 50 and 100 times the amount of unlabeled specific competitive oligonucleotides (specific competition), incubated for 30 min. A 6% polyacrylamide gel was prepared and pre-cooled with 0.5×TBE buffer. Electrophoresis was performed at 100 V at 4°C or room temperature until the free probe migrated to approximately 2 / 3 of the gel length. After electrophoresis, the gel was transferred or directly immobilized via membrane transfer: DNA was transferred from the gel to the membrane using electroporation, cross-linked with UV light, and immediately transferred to blocking buffer for 30 min. The membrane was then incubated at room temperature for 30 min with an incubation solution containing avidin-horseradish peroxidase. After thorough washing, chemiluminescent substrate was added, and the signal was captured using a chemiluminescent imaging system. Images were taken using linear exposure, and the original images were saved for quantitative analysis. See results below. Figure 13 C.

[0091] Figure 13 A represents the genomic location of the ermB gene in Clostridium difficile strain 630; as shown in the figure. Figure 13 As shown in A, σ VConservative motifs are predicted by combining promoter regions. Figure 13 B indicates that the prediction σ obtained by the MEME online tool... V The consensus motif is 5'-caaatcacaacgtg-3'. For example... Figure 13 As shown in C, EMSA measurement of σ V The binding of σ to the promoter binding region of the ermB gene shows that, with a constant amount of biotin-labeled probe, the retardation band gradually deepens with increasing protein concentration and gradually fades with increasing cold-competitive probe concentration. Therefore, σ... V It regulates the expression of the ermB gene by directly binding to the promoter sequence of the ermB gene.

[0092] Example 19: Quantitative analysis of reporter gene gusA / quantitative experimental verification of σ V Binding direction with ermB promoter

[0093] Reporter gene strain construction: The ermB gene promoter fragment was amplified using the genome of Clostridium difficile strain 630 as a template. The amplified ermB gene promoter fragment was then ligated into the BtgZI linearized PMTL82151 vector carrying the gusA reporter gene, resulting in the PermB-gusA-82151 vector. V Construction of overexpression vector: σ was amplified using the genome of Clostridium difficile strain 630 as a template. V Gene fragments, amplified σ with promoter V The fragment was ligated into the HindIII linearized PMTL82151 vector to obtain σ V Overexpression vectors. Three vectors were transformed into Clostridium difficile strain 630 to obtain strains carrying the gusA reporter gene without a promoter, strains carrying the PermB promoter and the gusA reporter gene, and strains carrying the PermB promoter and the gusA reporter gene and overexpressing σ. V strains.

[0094] GUS enzyme qualitative assay: Take 2 mL of the bacterial culture grown after picking transformants, centrifuge at 10000 × g, 4 ℃ for 20 min, discard the supernatant, wash the precipitate twice with 1 mL of ice-cold 1×TE buffer, resuspend in 500 μL of PBS solution, and homogenize using a tissue homogenizer. Add 100 μL of the cell fragments to 100 μL of GUS staining solution and incubate at 37 ℃ in the dark. Observe whether the solution turns blue and record the results. See details below. Figure 14 B.

[0095] GUS enzyme activity assay: 100 μL of the supernatant from the lysate used for GUS qualitative analysis was added to 400 μL of 1×PBS buffer solution preheated to 37℃, followed by 500 μL of 4-methylumbelliferone-β-D-glucuronide (4-MUG) substrate. The mixture was incubated at 37℃ in the dark. At 0 min, 20 min, 40 min, 60 min, 80 min, and 100 min, 200 μL of the mixed reaction solution was added to 800 μL of the reaction stop solution. The mixture was stored at room temperature in the dark. After the assay, the sample was loaded into a black 96-well microplate. Fluorescence was measured using a microplate reader at an excitation wavelength of 365 nm and an emission wavelength of 455 nm. All experimental samples were performed in triplicate. One-way ANOVA was used to compare fluorescence intensity bars between groups. Two-way ANOVA and Tukey's post-hoc test were used for statistical comparison of fluorescence intensity curves. * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, and **** indicates P ≤ 0.0001. See results for details. Figure 14 C.

[0096] Figure 14 A is σ V The regulation involves binding to the ermB gene promoter sequence, thereby initiating gusA reporter gene expression. (See diagram.) Figure 14 As shown in A, without the addition of overexpression σ V When expressed in a vector, the genome within the bacteria is expressed normally, σ V The protein activates the expression of the gusA reporter gene by binding to the promoter of the ermB gene; adding overexpression of σ V After being expressed in the vector, gusA expression is overactivated, resulting in increased expression levels. For example... Figure 14 As shown in Figure B, Clostridium difficile strain 630 was transformed with plasmid without promoter (left image), with plasmid containing ermB gene promoter (middle image), and with plasmid containing ermB gene promoter and overexpression of σ, respectively. V Transcription regulatory factor plasmids (right image). Plasmids without promoter sequence showed no color change after staining with a GUS staining kit. Plasmids with promoter added and σ overexpression... V The protein stained more deeply with GUS than when the promoter was added alone. This further illustrates that σ... V It regulates the expression of the ermB gene by directly binding to the promoter sequence of the ermB gene.

[0097] Figure 14 C shows the strain without a promoter (vector), the strain with a promoter (PermB), and the strain with a promoter and overexpression of σ. V strain (WT::σ)V The fluorescence intensity of ( ) varies over time. Figure 14 C indicates that adding a promoter and overexpressing σ... V strain (WT::σ) V The fluorescence intensity of σ was stronger than that of the strain with only the promoter added (PermB), indicating that overexpression of σ... V The strain produced more GUS protein, confirming σ V The mechanism by which GUS protein is specifically bound to ermB.

[0098] Based on the above research findings, the applicant proposed Anti-σ V With σ V Regulation of the ermB gene leads to the mechanism of clindamycin resistance in Clostridium difficile, see appendix for details. Figure 15 Specifically, in the model construction process, Anti-σ... V Upon sensing external environmental stimuli, clindamycin releases σ V Transcriptional regulators, free σ V It combines with RNA polymerase and the core enzyme to form the RNA polymerase holoenzyme. The RNA polymerase holoenzyme binds to the promoter of the ermB gene, which encodes methyltransferase, to initiate the expression of the ermB gene. The ermB methyltransferase causes methylation of the clindamycin binding site A2058 on the 23S rRNA of the ribosomal subunit, thereby preventing clindamycin from binding. The proteins that maintain normal bacterial growth continue to be transcribed and translated, thus producing clindamycin resistance.

[0099] Based on this research, this application innovatively provides the target gene σ V Or anti-σ V Application of genes in regulating ermB gene expression; by knocking out the target gene or reducing the activity / expression of the target gene, the expression of the ermB gene is downregulated. Based on this, this application provides a Clostridium difficile engineered strain with low MLSB antibiotic resistance. The Clostridium difficile engineered strain, by knocking out σ... V Or anti-σ V The gene reduced ermB gene expression, resulting in a significant reduction in resistance to MLSB antibiotics. This technology holds promise for the development of CDI treatment drugs, enabling good therapeutic effects with low-dose antibiotics. It has significant social implications and broad market application prospects.

Claims

1. The application of the target gene in regulating ermB gene expression, characterized by: The target gene is σ. V Or anti-σ V Gene; the σ V Possessing the nucleotide sequence shown in SEQ ID NO:1; the anti-σ V It has a nucleotide sequence as shown in SEQ ID NO:

2.

2. A method for regulating ermB gene expression, characterized in that: The target gene is knocked out or its activity / expression is reduced in cells or organisms to downregulate the expression of the ermB gene; the target gene is σ. V Or anti-σ V Gene.

3. The method for regulating ermB gene expression according to claim 2, characterized in that: The knockout of target genes or reduction of target gene activity / expression is achieved through gene knockout mediated by CRISPR-Cas9 / Cpf1 / Cas12b / Cas12j / Cas-CLOVER, CRISPRi, Clostron, Thermotargetron (TMT), ACE, and INTEGRATE systems.

4. The application of the method as described in claim 2 or 3 in reducing resistance to MLSB antibiotics in Clostridium difficile.

5. The application according to claim 3, characterized in that: The MLSB class of antibiotics includes macrolides, lincosamides, and streptogramin B.

6. The application according to claim 3, characterized in that: The macrolide antibiotics include 14-membered ring antibiotics such as erythromycin, clarithromycin, roxithromycin, fluerythromycin, and tererythromycin; 15-membered ring antibiotics such as azithromycin; and 16-membered ring antibiotics such as josamycin, midecamycin, acetyl-midecamycin, spiramycin, rotamycin, tylosin, and tylosin; the lincosamide antibiotics are clindamycin and lincomycin; and the streptozotocin B antibiotics are quinupristin and dalfopristin.

7. A strain of Clostridium difficile with low resistance to MLSB antibiotics, characterized in that: The engineered strain is a gene that knocks out the target gene σ as described in claim 1. V Genes or anti-σ V A Clostridium difficile mutant strain.

8. The use of the engineered Clostridium difficile strain as described in claim 7 in the preparation of CDI therapeutic agents.

9. A pharmaceutical composition, characterized in that: It comprises: an agent capable of reducing the activity of the target gene as described in claim 1 or inhibiting the expression of the target gene, and a pharmaceutically acceptable carrier.

10. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for treating or preventing ermB gene-mediated antibiotic-resistant bacterial infections.

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