Oligopeptide and application thereof in resisting escherichia coli multi-drug resistance

Abstract

The invention relates to a biotechnology, in particular to oligopeptide and application thereof in resisting multiple drug resistance of escherichia coli. The amino acid sequence of the oligopeptide (SZP) is shown as SEQ ID NO.1, and the homology of the oligopeptide (SZP) is more than 80%. The oligopeptide is an oligopeptide of a target AcrAB-TolC, and the amino acid sequence of the oligopeptide is SEQ ID NO. 1X1YX2RX3FVVX4X5; wherein X1 is selected from R, Y, H or K; x2 is selected from R, K or H; x3 is selected from G, F, Y, R, K or H; x4 is selected from G, A, W, F, D, E, K or H; and X5 is selected from G, D, E, R, K or H. The active peptide SZP disclosed by the invention can be used for remarkably enhancing the antibacterial activity of various antibiotics to multidrug-resistant escherichia coli through an efflux pump for generating multidrug resistance by targeting dominant bacteria, enhancing the permeability of cell membranes and a multidimensional mechanism for influencing the proton gradient of inner membranes of the bacteria, and can be obtained through a solid-phase synthesis technology, so that the preparation cost is relatively low.

Description

Technical Field

[0001] The present invention relates to biotechnology, and specifically to an oligopeptide and its application in combating multidrug-resistant Escherichia coli. Background Art

[0002] The emergence of multidrug-resistant (i.e., resistant to at least three classes of antimicrobial drugs) bacteria (multidrug-resistance organism, MDRO) has sharply reduced the success rate of modern medicine in treating infections and also posed a more severe challenge to the medical economic expenditures of various countries. According to statistics, approximately 700,000 people die from MDRO infections globally each year, with cumulative economic losses reaching up to $1 million. Escherichia coli is the most common in clinical infections, one of the 12 priority-resistant bacteria announced by the WHO, and also the one with the highest lethality rate among the globally highly concerned multidrug-resistant bacteria ESKAPE, posing a great threat to human health. According to the "National Bacterial Resistance Detection Report", the detection rate of clinical drug-resistant Escherichia coli strains in China has always ranked first, and in drug sensitivity tests, more than 80% show resistance to at least three antimicrobial drugs, mainly including antibiotics such as penicillins, cephalosporins, quinolones, aminoglycosides, and carbapenems. Therefore, there is an urgent need for effective treatment methods to solve its multidrug resistance problem.

[0003] An antibacterial sensitizer refers to a drug molecule that can weaken or even block the antibiotic resistance of bacteria. Different from the combined use strategy of antibiotics, it has weak or no antibacterial activity itself, so it is not easy to induce bacteria to develop new resistance mechanisms and is expected to become a sustainable strategy for combating multidrug resistance problems. Currently, common clinical antibacterial sensitizers mainly target bacterial resistance enzymes, efflux pumps, metabolic pathways, or host defense mechanisms.

[0004] The Resistance-Nodulation-Division (RND) family is one of the main drug efflux pumps in Gram-negative bacteria. It can penetrate the inner and outer membrane structures of Gram-negative bacteria, can remove a large number of various drug molecules in bacteria, and is closely related to its physiological functions such as colonization, virulence, and biofilm secretion in the host. Therefore, it plays a significant role in mediating multidrug resistance. AcrAB-TolC is the most main RND efflux pump in Escherichia coli. Inhibitors targeting it can reverse the drug resistance level of Escherichia coli by preventing the efflux of antimicrobial drugs and are a good source for developing broad-spectrum antibacterial sensitizers. However, currently, the development of related drugs is mainly based on small molecule compounds, and there are generally problems such as low efficacy, large side effects, and narrow antibacterial spectrum. Peptide drugs have many advantages such as high biological activity, low systemic toxicity, and low immunogenicity, and are a potential way to solve the above problems. Summary of the Invention

[0005] The object of the present invention is to provide an oligopeptide and its application in the treatment of Escherichia coli multidrug resistance.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] An oligopeptide, the oligopeptide (SZP) has an amino acid sequence as shown in SEQ ID NO.1 and sequences with a homology greater than 80%.

[0008] The oligopeptide is an oligopeptide targeting AcrAB-TolC, and its amino acid sequence is SEQ ID NO.1X 1 YX 2 RX 3 FVVX 4 X 5 ; wherein, X 1 is selected from R, Y, H or K; X 2 is selected from R, K or H; X 3 is selected from G, F, Y, R, K or H; X 4 is selected from G, A, W, F, D, E, K or H; X 5 is selected from G, D, E, R, K or H.

[0009] The X 1 , X 2 , X 3 or X 5 can be the same or different and are selected from R, H or K, and X 4 is selected from G.

[0010] An application of the oligopeptide as described above, the oligopeptide is used as an antibacterial sensitizer.

[0011] The oligopeptide is used as an antibacterial sensitizer targeting AcrAB-TolC.

[0012] An antibacterial sensitizer containing the oligopeptide as described above.

[0013] An application of the antibacterial sensitizer as described above, the application of the antibacterial sensitizer in the treatment of bacterial infections.

[0014] A combination of drugs for combined use, the combination of drugs for combined use contains the oligopeptide.

[0015] The composition is an oligopeptide and an antibacterial drug;

[0016] The antibacterial drug is one or more of chloramphenicol, ciprofloxacin, ampicillin, ceftazidime, imipenem, erythromycin.

[0017] Among them, the mass ratio of chloramphenicol to oligopeptide is 1:2.5 to 1:100, the mass ratio of ceftazidime to oligopeptide is 1:15 to 1:200, and the mass ratio of imipenem to oligopeptide is 1:60 to 1:800.

[0018] Use of a described combined medication combination, the use of the combined medication combination in the preparation of a drug for drug-resistant bacterial infections.

[0019] Preferably, the drug-resistant bacteria are Escherichia coli.

[0020] The bacteria are Escherichia coli MG1655 overexpressing AcrAB-TolC.

[0021] The combined medication combination can significantly improve the antibacterial activity of antibiotics against Escherichia coli MG1655 expressing AcrAB-TolC.

[0022] Use of the combined medication combination in the antibacterial sensitization activity against Escherichia coli overexpressing AcrAB-TolC.

[0023] Advantages of the present invention

[0024] The active peptide SZP of the present invention can significantly enhance the antibacterial activity of various antibiotics against multidrug-resistant Escherichia coli through a multi-dimensional mechanism of targeting the efflux pump that dominates the production of multidrug resistance in bacteria, enhancing cell membrane permeability, and affecting the proton gradient across the bacterial inner membrane, and can be obtained through solid-phase synthesis technology with a relatively low preparation cost. Therefore, it is expected to provide an effective way to solve the problems of low efficacy, strong in vivo toxicity, and narrow antibacterial spectrum of similar drugs, and provide an alternative solution for the treatment of clinically multidrug-resistant bacteria. Description of the drawings

[0025] Figure 1 This is for evaluating the inhibitory effect of SZP on the efflux function of the efflux pump of Escherichia coli MG1655 by Nile red efflux experiment provided in the examples of the present invention. Glu represents glucose;

[0026] Figure 2 This is the effect of SZP provided in the examples of the present invention on the cell membrane integrity of Escherichia coli MG1655.

[0027] Figure 3 This is the effect of SZP provided in the examples of the present invention on the proton gradient across the inner membrane of Escherichia coli MG1688 cells.

[0028] Figure 4 This is the detection of the hemolysis rate of rat red blood cells by SZP provided in the examples of the present invention. Detailed implementation manners

[0029] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and shall be included within the protection scope of the present invention.

[0030] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art.

[0031] Example 1 Obtaining of SZP

[0032] Nanjing Genscript Biotech Co., Ltd. was entrusted to prepare the SZP described in the present invention by solid-phase synthesis method. The oligopeptide of its target AcrAB-TolC has an amino acid sequence of SEQ ID NO.1X 1 YX 2 RX 3 FVVX 4 X 5 ; wherein, X 1 is selected from R, Y, H or K; X 2 is selected from R, K or H; X 3 is selected from G, F, Y, R, K or H; X 4 is selected from G, A, W, F, D, E, K or H; X 5 is selected from G, D, E, R, K or H. Purify to a purity of ≥95% by high-performance liquid chromatography; that is, obtain each oligopeptide. The following lists some oligopeptides:

[0033] SZP-1: RYRRRFVVGR

[0034] SZP-2: FYRRRFVVGR

[0035] SZP-3: RYRRRFVVRR

[0036] Example 2

[0037] The minimum inhibitory concentration (MIC) of SZP against Escherichia coli was detected by the microbroth dilution method to evaluate its inherent antibacterial activity. 100 μL of MH broth medium was added to the 1st - 9th wells of each row in a 96 - well plate. 200 μL of MH broth medium was added to the 10th well. 100 μL of the sample to be tested was taken and added to the 1st well of each row. Then, 100 μL of the solution was taken from the 1st well and added to the 2nd well, mixed well by pipetting. This operation was repeated until the 8th well, and 100 μL of the solution aspirated from the 8th well was discarded. 100 μL of the bacterial suspension to be tested was added to the 1st - 9th wells of each row. At this time, the final concentrations of SZP were 300 μM, 150 μM, 75 μM, 37.5 μM, 18.75 μM, 9.37 μM, 4.68 μM, and 2.34 μM respectively. The 9th well of each row was the positive control without the sample to be tested, and the 10th well was the negative control with only the medium. Subsequently, the 96 - well plate was incubated overnight in an incubator at 37 °C and then observed. The absence of visible bacterial growth in the bacterial suspension in each concentration well was recorded as negative, and the turbidity of the bacterial suspension was recorded as positive. The MIC of the drug against the bacteria was the lowest drug concentration at which no visible bacterial growth was observed in the bacterial suspension in the well. The determination of the drug sensitivity results was based on the Clinical and Laboratory Standards Institute (CLSI) guidelines. The quality control bacterium was Escherichia coli ATCC25922. The results showed that the MIC of each obtained oligopeptide was ≥ 300 μg / mL.

[0038] Meanwhile, the checkerboard micro - dilution method was used to detect the combined application effect of SZP with various antibiotics to evaluate its antibacterial sensitization effect. A single bacterial colony was picked and inoculated into fresh LB medium. After overnight incubation with constant shaking, it was diluted with MH liquid medium to a concentration of about 1 - 2×10 5CFU / mL. According to CLSI M100-ED34 and combined with the commonly used clinical doses of antibiotics, 10 concentration gradients were set for ampicillin, gentamicin sulfate, ciprofloxacin, chloramphenicol, tetracycline, ceftazidime, erythromycin, and imipenem, respectively, and they were combined with SZP at 90 μg / mL, 60 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL, respectively. The culture plates were incubated in an incubator at 37 °C, and the MIC determination criteria were the same as above. According to the literature, when the MIC of combined use of antibacterial drugs is ≥2 times higher than that of single use, it can be considered that the combined drugs can improve the sensitivity of antibacterial drugs, and ≥4 times indicates a significant improvement in the tolerance of bacteria to antibacterial drugs (Med Res Rev. 2019 39(6):2460-2504. doi:10.1002 / med.21591; Pharmacol Rep. 2021 73(1):1-16. doi:10.1007 / s43440-020-00160-9.); and LYRRRFVVGR (original oligopeptide) was used as a control oligopeptide (see Table 1).

[0039] Table 1 Combined antibacterial effects of SZP series oligopeptides and control oligopeptides with various antibiotics

[0040]

[0041] As can be seen from the above table, it can be seen from Table 1 that SZP-1 has a significant improvement effect on chloramphenicol, ceftazidime, and imipenem. Compared with single use, the MIC of combined use decreased by 8 times, 8 times, and 4 times, respectively. At the same time, compared with single use, the MIC of SZP series oligopeptides decreased by 2 times when combined with erythromycin, and the MIC of the drug also decreased by 2 times when SZP-3 was combined with ciprofloxacin.

[0042] Furthermore, some of the above drug combinations were further combined, and the FICI was calculated based on the MIC to evaluate the sensitive effect of the combination of two drugs (see Table 2). FICI is the fractional inhibitory concentration index, which refers to the sum of the ratios of the minimum inhibitory concentration of the combined use of various drugs to the MIC of single use when multiple drugs are used simultaneously. Among them, FICI can be divided into four situations: synergistic, additive, irrelevant, and antagonistic. Interpretation criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI < 1, additive effect; FICI ≥ 1, irrelevant effect; FICI ≥ 2, antagonistic effect.

[0043] Table 2 Combined antibacterial effects of SZP-1 oligopeptide and control oligopeptide with various antibiotics

[0044]

[0045] As can be seen from Table 2, the original polypeptide only showed an additive effect on chloramphenicol, which did not meet the definition of an antibacterial sensitizer. Compared with the original oligopeptide, SZP-1 had a significant sensitizing effect on three kinds of drug-resistant bacteria, not only meeting the requirements of a sensitizer but also having broad-spectrum properties.

[0046] Example 3 Evaluation of SZP as an antibacterial sensitizer for efflux inhibitory activity

[0047] Nile red is one of the substrates of bacterial efflux pumps, and it shows fluorescence inside the cell membrane but basically has no fluorescence in aqueous solution. The Escherichia coli cultured overnight was inoculated into LB medium and cultured overnight with shaking. The cells were collected by centrifugation at room temperature and resuspended and washed with PBS buffer containing magnesium chloride. After centrifuging and resuspending the cells again, the cell concentration was adjusted to an appropriate level. After standing still, 2 mL of each liquid was transferred to a centrifuge tube, and the uncoupler CCCP was added to adjust the bacterial solution to an appropriate concentration to cut off the energy source of the efflux pump. After 15 min, except for one tube set as a negative control, the appropriate concentration of PAβN (positive control) and SZP (taking the above SZP-1 as an example) were added to the others respectively. After another 15 min, Nile red was added, and then the mixture was incubated in a constant temperature shaker at 37 °C for 3 h. After standing at room temperature and centrifuging, the supernatant was removed, and the precipitate was resuspended in the buffer. Immediately, the cell suspension was taken and added to a quartz cuvette. The fluorescence spectrophotometer was set with a slit width of 10 nm, an excitation wavelength of 552 nm, and an emission wavelength of 636 nm. After tracking the fluorescence of the cell suspension for 100 s, 100 μL of glucose (1 M) was added to restore the energy supply of the efflux pump to rapidly trigger the efflux of Nile red, and the fluorescence intensity change was monitored again for 200 s. The inhibitory efficacy of SZP on the efflux pump function was evaluated by the change in fluorescence value (see Figure 1 ).

[0048] The results are shown in Figure 1 . Compared with Escherichia coli MG1655 treated with the negative control (PBS), SZP at a high concentration significantly inhibited the efflux of Nile red, but the effect was inferior to that of the positive control (PAβN).

[0049] Example 4 Evaluation of bacterial outer membrane integrity

[0050] PI is a nucleic acid dye that cannot penetrate the intact cell membrane. When the integrity of the bacterial cell membrane is lost, PI enters the cell and binds to DNA / RNA, emitting red fluorescence. Bacteria in the mid-logarithmic phase were collected and added to 50 ml of LB medium at a ratio of bacterial solution:LB liquid medium = 1:100, and cultured in a shaker at 37 °C and 220 rpm until the OD 600 reached 0.6 - 0.8. The cells were collected by centrifugation for min; the cells were washed 3 times with PBS and resuspended in PBS until the OD 600was about 0.5, then PI was added to a final concentration of 10 uM, and incubated at 37 °C in the dark for 30 min. Then, a certain concentration of SZP (taking SZP-1 above as an example), PAβN (positive control), or PBS (negative control) was added. After mixing, it was incubated at 37 °C for 1 h. The fluorescence intensity of the sample excited by the excitation light at 535 nm and emitting light at a wavelength of 615 nm was detected using a multifunctional microplate reader (see Figure 2 ).

[0051] The results are shown in Figure 2 . The disruption of bacterial outer membrane integrity by SZP showed a dose-dependent manner. The destructive effect at low concentration was weaker than that of the positive control (PAβN), and was comparable to it at high concentration.

[0052] Example 5 Evaluation of the influence on the proton gradient of bacterial inner membrane

[0053] The lipophilic fluorescent dye DiOC 2 (3) can accumulate on the hyperpolarized membrane and increase the fluorescence intensity, and is often used as a membrane potential probe. The energy-consuming inhibitor destroys the membrane potential and depolarizes it, resulting in a decrease in fluorescence intensity. It can accumulate on the hyperpolarized membrane and enhance its fluorescence intensity. Therefore, when DiOC 2 (3) is added to Escherichia coli with intact inner membrane and normal membrane potential, the fluorescence intensity will increase significantly. When the energy-consuming inhibitor is added, it will destroy the membrane potential and depolarize it, then the fluorescence intensity will decrease. The bacteria cultured overnight were inoculated on LB medium and shaken in a constant temperature shaker at 37 °C until they grew to the mid-logarithmic phase and then centrifuged for collection. The bacterial liquid was transferred to 50 ml of LB medium at a ratio of bacterial liquid:LB medium = 1:100 and shaken for 3 h. It was resuspended and washed with PBS buffer. The obtained precipitate was resuspended with PBS buffer containing potassium ethylenediaminetetraacetate (EDTA-K) and incubated at 37 °C for 20 minutes. Then it was centrifuged and the precipitate was resuspended with buffer until OD 600 was 1. DiOC 2 (3) was added to the bacterial liquid to a final concentration of 30 uM. The SZP (taking SZP-1 above as an example), PAβN (positive control), or PBS (negative control) was added to the black 96-well plate substrate respectively, and then 200 uL of the bacterial liquid was added. The fluorescence was measured using a microplate reader. The excitation wavelength was adjusted to 450 nm, the emission wavelength was 670 nm, and the excitation and emission slit widths were 10 nm and 5 nm respectively. After tracking the fluorescence of the cell suspension for 60 seconds, Dioc2(3) was added to an appropriate concentration. After the fluorescence intensity was stable, glucose was added to maintain the inner membrane potential energy to keep the fluorescence intensity at a high level. Then CCCP (control group) and EPI were added, and the fluorescence intensity was detected until it was stable (see Figure 3 ).

[0054] The results are shown in Figure 3。SZP significantly reduces the proton gradient across the bacterial inner membrane at high doses, but the effect is weaker than that of CCCP; it has no obvious effect at low doses.

[0055] Example 6 Detection of erythrocyte hemolysis rate

[0056] Collect blood samples from the periorbital plexus of healthy rats and place them in anticoagulant tubes. After centrifugation, discard the upper layer of liquid. Add 0.9% normal saline for washing, and discard again after centrifugation. Repeat this step until the upper layer of liquid is clear. Subsequently, add 0.9% normal saline to make a 2% erythrocyte suspension. Add SZP (taking SZP-1 above as an example) and the erythrocyte suspension to an EP tube at a ratio of 1:1, and incubate at 37 °C for 2 h. Add 200 μL of erythrocyte suspension and equal amounts of SZP at different concentrations (taking SZP-1 above as an example), 1.0% Triton X-100 (positive control), and 0.9% saline buffer (negative control) to a 96-well plate. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at 540 nm, and calculate according to the following formula: Hemolysis rate (%) = (absorbance of sample - absorbance of negative control group) / (absorbance of positive control group - absorbance of negative control group) × 100%. The results were judged according to the Pharmacopoeia of the People's Republic of China (2020 Edition). A hemolysis rate < 5% indicates no risk of erythrocyte hemolysis (see Figure 4 ).

[0057] The results are shown in Figure 4 。SZP did not produce hemolytic activity at concentrations below 360 μg / ml.

[0058] In summary, as shown in the above examples, SZP of the present invention can significantly improve the antibacterial activities of multidrug-resistant Escherichia coli against chloramphenicol, ceftazidime, imipenem, and erythromycin. The best reduction in the minimum inhibitory concentration (MIC) of the first three drugs can reach 4 - 8 times. At the same time, it has no erythrocyte hemolytic property. Multidimensional experiments show that SZP achieves the above effects mainly by directly inhibiting the function of the RND efflux pump, disrupting the proton concentration gradient across the bacterial inner membrane to interfere with the energy transfer of the efflux pump, and enhancing cell permeability. Therefore, SZP of the present invention is a broad-spectrum antibacterial sensitizing active peptide with high efficiency and low toxicity, and can be used to treat infectious diseases caused by multidrug-resistant bacteria represented by Escherichia coli.

Claims

1. An oligopeptide, characterized in that: The oligopeptide is an amino acid sequence as shown in SEQ ID NO.1 and a sequence with a homology greater than 80%.

2. The oligopeptide according to claim 1, characterized in that: The oligopeptide is an oligopeptide targeting AcrAB-TolC, and its amino acid sequence is SEQ ID NO.1X1YX2RX3FVVX4X5; wherein X1 is selected from R, Y, H or K; X2 is selected from R, K or H; X3 is selected from G, F, Y, R, K or H; X4 is selected from G, A, W, F, D, E, K or H; X5 is selected from G, D, E, R, K or H.

3. The oligopeptide according to claim 2, characterized in that: Said X1, X2, X3 or X5 may be the same or different and selected from R, H or K, and X4 may be selected from G.

4. A use of the oligopeptide according to claim 1, characterized in that: The oligopeptides serve as antimicrobial sensitizers.

5. The use of oligopeptides according to claim 4, characterized in that: The oligopeptide serves as an antibacterial sensitizer targeting AcrAB-TolC.

6. An antimicrobial sensitizer, characterized in that: The antibacterial sensitizer contains the oligopeptide according to claim 1.

7. Use of the antimicrobial sensitizer according to claim 5, characterized in that: The antibacterial sensitizer is used as a drug for treating bacterial infection.

8. A combination drug, characterized in that: The combined pharmaceutical composition comprises the oligopeptide according to claim 1.

9. The combined drug composition according to claim 7, characterized in that: The antibacterial drug is one or more of chloramphenicol, ciprofloxacin, ampicillin, ceftazidime, imipenem and erythromycin.

10. A use of the combined drug combination according to claim 7, characterized in that: The application of the combined drug combination in the preparation of drugs for drug-resistant bacterial infections.

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