Application of FloA gene as target spot in reducing drug resistance of methicillin-resistant staphylococcus aureus to erythromycin

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a FloA gene as a target spot to reduction of drug resistance of methicillin-resistant staphylococcus aureus to erythromycin. The invention discovers that after the FloA gene of the methicillin-resistant staphylococcus aureus is knocked out by adopting a recombinant vector, the minimum inhibitory concentration of erythromycin to the methicillin-resistant staphylococcus aureus can be obviously reduced. Therefore, the FloA gene as a target spot can be used for screening drugs for reducing the drug resistance of the methicillin-resistant staphylococcus aureus to the erythromycin, and the FloA gene is combined with the erythromycin to realize better activity of resisting the methicillin-resistant staphylococcus aureus; meanwhile, by constructing the FloA gene knockout methicillin-resistant staphylococcus aureus strain, a foundation can be laid for researching the erythromycin drug resistance mechanism of the methicillin-resistant staphylococcus aureus.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of the FloA gene as a target in reducing the erythromycin resistance of methicillin-resistant Staphylococcus aureus. Background Art

[0002] Staphylococcus aureus is a Gram-positive bacterium with strong pathogenicity. It can produce toxins such as hemolysin, leukocidin, enterotoxin and enzymes, and can cause serious infections in poultry, livestock and humans such as bacteremia, necrotizing pneumonia, skin and soft tissue infections, osteomyelitis, infective endocarditis, etc., seriously threatening the safety of animal-derived food, livestock production safety and public health safety. Antibiotics play a crucial role in the treatment of Staphylococcus aureus infections. However, the unreasonable use of antibiotics has led to an increasingly serious situation of Staphylococcus aureus drug resistance, and the "superbug" methicillin-resistant Staphylococcus aureus (MRSA) has been frequently detected in the community and the livestock industry. MRSA is resistant to all β-lactam antibiotics and shows a strong degree of resistance to other classes of antibiotics such as macrolides and quinolones. At present, due to the lag in the research and development process of new antibiotics behind the evolution speed of bacterial drug resistance, we are gradually entering the "post-antibiotic era", and the continuous increase in bacterial drug resistance poses an unprecedented challenge to human health.

[0003] Functional membrane microdomains (FMMs) are microdomains rich in acyclic polyisoprenoids and the scaffold protein FloA on the bacterial cell membrane. This domain is similar to the "lipid raft" on the eukaryotic cell membrane and is involved in cell signal transduction, protein transport, and the localization and aggregation of membrane proteins. The synthesis of acyclic polyisoprenoids that make up FMMs depends on the mevalonate pathway (García-Fernández E, et al. Cell. 2017, 171:1354-1367.). Studies have confirmed that the resistance of MRSA strains to β-lactam antibiotics depends on the penicillin-binding protein (PBP2a) located on the bacterial membrane encoded by the exogenous methicillin resistance determinant mecA gene. PBP2a oligomerizes in FMMs, causing its active site to be embedded in a narrow and extended crack, making it difficult for β-lactam antibiotics to approach due to steric hindrance. Therefore, MRSA is resistant to β-lactam antibiotics (Peacock SJ and Paterson GK. Annu. Rev. Biochem. 2015, 84:577-601; Jiao F, et al. Antimicrob. Agents Chemother. 2023, 67:e0089523.).

[0004] The resistance mechanism of macrolide antibiotics is completely different from that of β-lactam antibiotics. The resistance of MRSA to erythromycin depends on the modification of 23S rRNA of the ribosomal 50S subunit mediated by the erm (erythromycin resistance methylase) gene. In addition, efflux pumps also play an important role in the process of erythromycin resistance. Efflux pumps are transmembrane proteins that can actively transport harmful molecules such as antibiotics out of the cell, thereby reducing the effective concentration of the drug inside the cell and causing the antibiotic to lose its effect on the bacteria. Currently, there is no research report on the effect of FMMs deletion in MRSA strains on erythromycin sensitivity, and it is not clear whether the proteins encoded by the erythromycin efflux pump protein genes such as LmrS and MsrA in MRSA strains are associated with FMMs. Summary of the Invention

[0005] To address the above technical problems, the present invention unexpectedly discovers that knocking out the FloA gene of methicillin-resistant Staphylococcus aureus can significantly reduce the resistance of methicillin-resistant Staphylococcus aureus to erythromycin, providing a new idea for studying the resistance mechanism of MRSA to erythromycin and also providing a new strategy for improving the sensitivity of MRSA to erythromycin. Specifically, it includes the following content:

[0006] In a first aspect, the present invention provides the use of the FloA gene as a target in screening for drugs that reduce the resistance of methicillin-resistant Staphylococcus aureus (MRSA) to erythromycin, wherein the drugs inhibit the expression of the FloA gene using the FloA gene as a target.

[0007] Preferably, the drugs are selected from small molecule compounds that inhibit the expression of the FloA gene, interfering RNAs targeting the FloA gene, FloA gene knockout vectors, or primers.

[0008] Preferably, the method for constructing the FloA gene knockout vector is as follows: using the genomic DNA of methicillin-resistant Staphylococcus aureus as a template, designing and synthesizing a fusion fragment of the upstream and downstream homologous arms of the FloA gene; ligating the fusion fragment with the knockout vector to construct the FloA gene knockout vector.

[0009] Preferably, the nucleotide sequence of the fusion fragment is shown as SEQ ID No.1.

[0010] Preferably, the knockout vector is pBT2.

[0011] In a second aspect, the present invention provides a pharmaceutical composition for treating methicillin-resistant Staphylococcus aureus infection, the pharmaceutical composition comprising erythromycin and at least one of the following:

[0012] (i) small molecule compounds that inhibit the expression of the FloA gene;

[0013] (ii) interfering RNAs targeting the FloA gene;

[0014] (iii) FloA gene knockout vectors or primers.

[0015] Preferably, the method for constructing the FloA gene knockout vector is as follows: using the genomic DNA of methicillin-resistant Staphylococcus aureus as a template, designing and synthesizing a fusion fragment of the upstream and downstream homologous arms of the FloA gene; ligating the fusion fragment with the knockout vector to construct the FloA gene knockout vector.

[0016] Preferably, the nucleotide sequence of the fusion fragment is shown as SEQ ID No.1.

[0017] Preferably, the knockout vector is pBT2.

[0018] In a third aspect, the present invention provides the use of the pharmaceutical composition described in the second aspect above in the preparation of a drug for treating methicillin-resistant Staphylococcus aureus infection.

[0019] In a fourth aspect, the present invention provides a method for increasing the sensitivity of methicillin-resistant Staphylococcus aureus to erythromycin, the method being: inhibiting or knocking out the FloA gene of methicillin-resistant Staphylococcus aureus.

[0020] In a fifth aspect, the present invention provides the use of a methicillin-resistant Staphylococcus aureus FloA gene knockout strain in studying the erythromycin resistance mechanism of methicillin-resistant Staphylococcus aureus or in increasing the sensitivity of methicillin-resistant Staphylococcus aureus to erythromycin.

[0021] Preferably, the method for constructing the methicillin-resistant Staphylococcus aureus FloA gene knockout strain comprises the following steps:

[0022] (1) Using the genomic DNA of methicillin-resistant Staphylococcus aureus strain N315 as a template, designing and synthesizing a fusion fragment of the upstream and downstream homologous arms of the FloA gene;

[0023] (2) Connecting the fusion fragment with a knockout vector to construct a recombinant knockout vector;

[0024] (3) Electrotransforming the recombinant knockout vector into Staphylococcus aureus strain RN4220, screening positive monoclonal colonies through a resistance plate, and verifying by double digestion with restriction endonucleases;

[0025] (4) Electrotransforming the recombinant knockout vector modified by RN4220 into methicillin-resistant Staphylococcus aureus strain N315, screening positive monoclonal colonies through a resistance medium, and verifying by PCR and sequencing to obtain the methicillin-resistant Staphylococcus aureus FloA gene knockout strain.

[0026] Preferably, the nucleotide sequence of the fusion fragment in step (1) is shown as SEQ ID No.1.

[0027] Preferably, the knockout vector in step (2) is pBT2.

[0028] Preferably, the restriction endonucleases in step (3) are EcoRⅠ and KpnⅠ.

[0029] Preferably, the method for screening positive monoclonal in step (4) is as follows: inoculate the N315 strain carrying the recombinant knockout vector at a ratio of 1:1000 into 2 mL of TSB (chloramphenicol, 20 μg / mL) medium, and culture overnight at 30 °C and 200 rpm; the next day, transfer it at a ratio of 1:100 to fresh 2 mL of TSB medium, culture at 42 °C and 200 rpm, transfer once every 24 h, for a total of 5 times; streak on a TSA plate (chloramphenicol, 20 μg / mL), and culture overnight at 42 °C; pick monoclonal and transfer it to fresh 2 mL of TSB medium, culture at 30 °C and 200 rpm, transfer once every 24 h, for a total of 3 times; streak on a TSA plate, and culture overnight at 30 °C; pick monoclonal and transfer it to a 96-well plate containing 200 μL of TSB medium, and culture at 30 °C until turbid; transfer the culture solution in the 96-well plate at a ratio of 1:100 to another 96-well plate of TSB medium (chloramphenicol, 20 μg / mL), and culture overnight at 30 °C. Compare the two groups of 96-well plates, and select the clones that grow in the blank TSB medium but do not grow in the chloramphenicol TSB medium.

[0030] Preferably, the primers for PCR verification in step (4) are:

[0031] FloA-up-F: GACTCCCTCAACACGAA;

[0032] FloA-down-R: GGATGACAACATCGAAAC.

[0033] The beneficial effects of the present invention are as follows:

[0034] The present invention obtained a methicillin-resistant Staphylococcus aureus FloA gene knockout strain through homologous recombination. It was unexpectedly found that knocking out the FloA gene of methicillin-resistant Staphylococcus aureus can significantly reduce the resistance of methicillin-resistant Staphylococcus aureus to erythromycin. Compared with the minimum inhibitory concentration of erythromycin against wild methicillin-resistant Staphylococcus aureus (>256 μg / mL), after the FloA gene was inhibited, the minimum inhibitory concentration of erythromycin against methicillin-resistant Staphylococcus aureus was significantly reduced (0.5 μg / mL), and methicillin-resistant Staphylococcus aureus was sensitive to erythromycin; at the same time, there were no significant differences in the growth and colony morphology between the FloA gene knockout strain and the wild strain, which provided convenience for comparing and studying the FloA gene knockout strain and the wild strain at the phenotypic level; therefore, the FloA gene can be used as a target to screen drugs that reduce the resistance of methicillin-resistant Staphylococcus aureus to erythromycin, and combine with erythromycin to achieve better anti-methicillin-resistant Staphylococcus aureus activity. The present invention provides a new perspective for studying the resistance mechanism of methicillin-resistant Staphylococcus aureus to erythromycin, and also provides a new strategy for improving the sensitivity of methicillin-resistant Staphylococcus aureus to erythromycin. Description of the Drawings

[0035] Figure 1 Double digestion verification diagram of the pBT2-FloA vector extracted for DH5α transformation: Lane M is DL10000 DNA Marker; Lane 1 is pBT2-FloA;

[0036] Figure 2 Double digestion verification diagram of the pBT2-FloA vector extracted for RN4220 transformation: Lane M is DL5000 DNA Marker, and Lane 1 is pBT2-FloA;

[0037] Figure 3 Double digestion verification diagram of the pBT2-FloA vector extracted for N315 transformation: Lane M is DL5000 DNA Marker; Lane 1 is pBT2-FloA;

[0038] Figure 4 PCR identification diagram of N315ΔFloA strain, Lane M is DL5000 DNA Marker; Lane 2 is N315 wild strain; Lane 3 is N315ΔFloA strain;

[0039] Figure 5 Forward sequencing result diagram of N315ΔFloA strain;

[0040] Figure 6 Reverse sequencing result diagram of N315ΔFloA strain;

[0041] Figure 7 Comparison diagram of growth and colony morphology between wild strain N315 WT and knockout strain N315ΔFloA;

[0042] Figure 8 Detection diagram of the minimum inhibitory concentration of erythromycin for wild strain N315 WT and knockout strain N315ΔFloA. Detailed implementation manners

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments, and those skilled in the art can make similar improvements without departing from the connotation of the present invention.

[0044] The reagents used in the embodiments of the present invention, unless otherwise specified, are all purchased from biochemical reagent suppliers, and the experimental techniques used, unless otherwise specified, are all conventional techniques.

[0045] In the following embodiments, the nucleotide sequences are shown as follows:

[0046] The homologous arm fusion fragment sequence of the FloA gene is shown in SEQ ID No.1:

[0047]

[0048] Example 1

[0049] This example provides a method for constructing a methicillin-resistant Staphylococcus aureus FloA gene knockout strain and its application, including the following content:

[0050] 1. Construction of the homologous recombination vector pBT2-FloA

[0051] (1) Using the genomic DNA of Staphylococcus aureus strain N315 (GenBank: BA000018.3) as a template, a fusion fragment pUC57-FloA (shown in SEQ ID No. 1) of the upstream and downstream homologous arms of the FloA gene was designed and synthesized. After the pUC57-FloA fusion fragment and the pBT2 knockout vector were double-digested with the restriction endonucleases EcoRⅠ and KpnⅠ and linearized fragments were obtained, the fusion fragment and the knockout vector were recovered from the gel using an agarose gel recovery kit.

[0052] The double-digestion reaction was carried out using the restriction endonucleases of Takara Company. The reaction system (50 μL) was as follows:

[0053]

[0054] Reaction conditions: 37°C, 30 min. The size of the enzyme digestion product was detected by 1% agarose gel electrophoresis.

[0055] (2) The fusion fragment recovered from the gel was ligated to the pBT2 vector using a ligase and transformed into DH5α competent cells. The cells were spread on an LB plate (Amp, 100 μg / mL) and cultured overnight at 37°C; the next day, positive monoclonal colonies were picked and plasmids were extracted for double-digestion verification with EcoRⅠ and KpnⅠ. The ligation reaction was carried out using the ligase of Takara Company. The reaction system (10 μL) was as follows:

[0056] solutionⅠ 2.5 μL

[0057] pBT2 vector 2.5 μL

[0058] Fusion fragment 5 μL

[0059] Reaction conditions: 16°C, 40 min.

[0060] The results were as Figure 1 shown. The double-digestion electrophoresis result in lane 1 was in line with the expectation of about 2029 bp, and the recombinant knockout vector pBT2-FloA was obtained.

[0061] 2. Electrotransformation of the recombinant knockout vector pBT2-FloA into Staphylococcus aureus RN4220

[0062] Prepare competent Staphylococcus aureus RN4220; Take 7 μL of the successfully constructed pBT2-FloA recombinant plasmid and add it to 100 μL of RN4220 competent cells, then incubate on ice for 30 min; After electroporation (parameters: voltage 2.5 kV, capacitance 50 μF, resistance 200 Ω), quickly place it on ice and let it stand for 10 min; Add 1 mL of BHI culture medium, and culture it with shaking at 30 °C and 150 rpm for 1 h, then spread it on a TSA plate (chloramphenicol, 20 μg / mL) and culture it overnight at 30 °C; The next day, pick positive clones and extract plasmids for double digestion identification with EcoRⅠ and KpnⅠ.

[0063] The results are as Figure 2 shown. The double digestion electrophoresis result in lane 1 is in line with the expectation of about 2029 bp.

[0064] 3. Electrotransform the recombinant knockout vector pBT2-FloA modified by RN4220 into Staphylococcus aureus N315

[0065] Prepare competent Staphylococcus aureus N315; Electrotransfer the recombinant knockout vector pBT2-FloA modified by RN4220 into N315 competent cells; Electrotransformation conditions: voltage 2.5 kV, capacitance 50 μF, resistance 200 Ω; Spread the electrotransformed bacterial solution on a TSA plate (chloramphenicol, 20 μg / mL); The next day, pick positive clones and extract plasmids for double digestion identification with EcoRⅠ and KpnⅠ.

[0066] The results are as Figure 3 shown. The double digestion electrophoresis result in lane 1 is in line with the expectation of about 2029 bp.

[0067] 4. Screening and identification of N315ΔFloA strain

[0068] (1) Inoculate the N315 strain carrying the recombinant knockout vector at a ratio of 1:1000 into 2 mL of TSB (chloramphenicol, 20 μg / mL) medium and culture it overnight at 30 °C and 200 rpm; The next day, transfer it at a ratio of 1:100 to fresh 2 mL of TSB medium and culture it at 42 °C and 200 rpm to induce homologous recombination of the plasmid. Transfer it once every 24 h for a total of 5 times.

[0069] (2) Dip a small amount of the bacterial solution cultured at 42 °C for 5 generations with an inoculation loop and streak it on a TSA plate (chloramphenicol, 20 μg / mL), and culture it overnight at 42 °C; The next day, pick monoclonal colonies and transfer them to fresh 2 mL of TSB medium and culture them at 30 °C and 200 rpm to induce homologous recombination of the plasmid again. Transfer it once every 24 h for a total of 3 times.

[0070] (3) Dip an inoculation loop into a small amount of the bacterial liquid cultured at 30 °C for 3 generations and streak it on a TSA plate, then culture it overnight at 30 °C; pick a monoclonal colony and transfer it to a 96-well plate containing 200 μL of TSB medium, and culture it at 30 °C until it becomes turbid; transfer the culture solution in the 96-well plate to another 96-well plate containing TSB medium (chloramphenicol, 20 μg / mL) at a ratio of 1:100, and culture it overnight at 30 °C.

[0071] (4) Compare the two 96-well plates, select the clones that grow on the blank TSB medium but not on the chloramphenicol TSB medium, and use PCR to identify whether the FloA gene has been knocked out. The PCR primers are:

[0072] FloA-up-F: GACTCCCTCAACACGAA;

[0073] FloA-down-R: GGATGACAACATCGAAAC.

[0074] Use Taq DNA Polymerase from Takara to perform a conventional PCR reaction. The reaction system (50 μL) is as follows:

[0075]

[0076] Reaction conditions: Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 1 min; annealing at 53 °C for 45 s; extension at 72 °C for 3 min 10 s; 33 cycles; final extension at 72 °C for 8 min. Detect the band size and specificity of the PCR products by 1% agarose gel electrophoresis.

[0077] The PCR identification result diagram of the N315ΔFloA strain is as Figure 4 shown. Lane 2 is the amplification product of the wild strain N315WT, 3009 bp; lane 3 is the amplification product of the knockout strain N315ΔFloA, 2019 bp. The results are as expected, confirming that the FloA gene has been knocked out.

[0078] (5) Send the amplification product in Figure 4 lane 3 to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0079] The sequencing results are as Figure 5 and 6 shown. The sequence is as expected, confirming the successful construction of the N315ΔFloA strain.

[0080] 5. Comparison of the growth and colony morphology between the wild strain N315 WT and the knockout strain N315ΔFloA

[0081] Two 2 mL aliquots of sterile TSB were cultured overnight at 37°C and 200 rpm for the wild-type strain N315 WT and the knockout strain N315ΔFloA. The next day, sheep blood plates were streaked and cultured overnight at 37°C. The monoclonal bacterial suspension was resuspended in physiological saline and the concentration of the bacterial suspension was adjusted to 0.5 × 10 using a McFarland turbidimeter. 8 , spread on sheep blood plates, and observe the growth and colony morphology after overnight culture.

[0082] The results are as follows Figure 7 As shown, there was no significant difference in growth and colony morphology between the wild strain N315 WT and the knockout strain N315ΔFloA.

[0083] 6. Determination of the minimum inhibitory concentration of erythromycin

[0084] The minimum inhibitory concentration of erythromycin was determined by the E-test strip method with reference to the Clinical and Laboratory Standards Institute (CLSI): two sterile 2 mL TSB aliquots were cultured overnight at 37°C and 200 rpm for WT wild-type strain N315 WT and knockout strain N315ΔFloA. The next day, sheep blood aliquots were streaked and cultured overnight at 37°C. The monoclonal bacterial suspension was resuspended in physiological saline and the concentration of the bacterial suspension was adjusted to 0.5 × 10 using a McFadden turbidimeter. 8 , dip a sterile cotton swab in the bacterial solution and evenly spread it on the MH agar plate, use tweezers to pick up the erythromycin E-test strip and place it on the plate, invert and culture overnight at 37℃, and observe the minimum inhibitory concentration of erythromycin the next day.

[0085] The results are as follows Figure 8 As shown, the minimum inhibitory concentration (MIC) of erythromycin against the wild-type strain N315 WT was greater than 256 μg / mL, while that against the FloA knockout strain N315ΔFloA was only 0.5 μg / mL. This indicates that knocking out or inhibiting FloA gene expression significantly increases the susceptibility of methicillin-resistant Staphylococcus aureus to erythromycin and reduces its resistance to erythromycin. Therefore, targeting the FloA gene can be used to screen for drugs that reduce erythromycin resistance in methicillin-resistant Staphylococcus aureus. These drugs, when combined with erythromycin, exhibit enhanced anti-MRSA activity, which is of great significance for inhibiting MRSA infection.

[0086] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. Use of the FloA gene as a target in screening drugs for reducing the resistance of methicillin-resistant Staphylococcus aureus to erythromycin, characterized in that, The drug targets the FloA gene to inhibit the expression of the FloA gene.

2. The application according to claim 1, wherein The drug is selected from small molecule compounds that inhibit the expression of the FloA gene, interfering RNAs targeting the FloA gene, FloA gene knockout vectors or primers.

3. The application according to claim 2, characterized in that, The method for constructing the FloA gene knockout vector is as follows: Using the genomic DNA of methicillin-resistant Staphylococcus aureus as a template, designing and synthesizing a fusion fragment of the upstream and downstream homologous arms of the FloA gene; ligating the fusion fragment with the knockout vector to construct the FloA gene knockout vector.

4. The application according to claim 3, wherein The nucleotide sequence of the fusion fragment is shown as SEQ ID No.

1.

5. The application according to claim 4, characterized in that, The knockout vector is pBT2.

6. A pharmaceutical composition for treating methicillin-resistant Staphylococcus aureus infection, characterized in that, The pharmaceutical composition comprises erythromycin and at least one of the following: (i) Small molecule compounds that inhibit the expression of the FloA gene; (ii) Interfering RNAs targeting the FloA gene; (iii) FloA gene knockout vectors or primers.

7. Use of the pharmaceutical composition according to claim 6 in the preparation of a drug for treating methicillin-resistant Staphylococcus aureus infection.

8. A method for increasing the sensitivity of methicillin-resistant Staphylococcus aureus to erythromycin, characterized in that The method is: inhibiting or knocking out the FloA gene of methicillin-resistant Staphylococcus aureus.

9. Use of a methicillin-resistant Staphylococcus aureus FloA gene knockout strain in studying the resistance mechanism of methicillin-resistant Staphylococcus aureus to erythromycin or in enhancing the sensitivity of methicillin-resistant Staphylococcus aureus to erythromycin.

10. The use according to claim 9, characterized in that, The method for constructing the methicillin-resistant Staphylococcus aureus FloA gene knockout strain comprises the following steps: (1) Using the genomic DNA of methicillin-resistant Staphylococcus aureus strain N315 as a template, designing and synthesizing a fusion fragment of the upstream and downstream homologous arms of the FloA gene; (2) Ligating the fusion fragment with the knockout vector to construct a recombinant knockout vector; (3) Electrotransforming the recombinant knockout vector into Staphylococcus aureus strain RN4220, screening positive monoclonal colonies through a resistance plate, and verifying by double digestion with restriction endonucleases; (4) Electrotransforming the recombinant knockout vector modified by RN4220 into Staphylococcus aureus strain N315, screening positive monoclonal colonies through a resistance medium, and verifying by PCR and sequencing to obtain the methicillin-resistant Staphylococcus aureus FloA gene knockout strain.

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