Bacteriostatic transmembrane polypeptide and application thereof

Abstract

The invention relates to an antibacterial transmembrane polypeptide and application thereof. The amino acid sequence of the antibacterial transmembrane polypeptide comprises a sequence as shown in SEQ ID NO.1 or a sequence of which the homology with the sequence as shown in SEQ ID NO.1 is 70% or above. The specific binding oligopeptide of the bacterial Hsp70s protein is obtained through screening, it is found that the specific binding oligopeptide is bound with the Hsp70s protein through a traditional substrate binding pocket, the abnormally high affinity of binding with the protein is shown in the DnaK-ATP state, the oligopeptide can inhibit the activity of the Hsp70s protein to a certain degree and prevent the Hsp70s protein from helping denatured luciferase renaturation, and in addition, the specific binding oligopeptide has a good application prospect. On the basis of ensuring the completeness of the bacterial cell membrane, the oligopeptide has excellent capability of penetrating through the bacterial cell membrane. The application potential of the compound in preparation of drugs for inhibiting bacteria and preparation of antibiotic adjuvants is further discussed, and a new thought is provided for solving the problems that at present, bacteria are resistant to drugs, and new antibiotics are difficult to excavate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an antibacterial membrane-penetrating polypeptide and an application thereof. Background Art

[0002] Antibiotics play a key role in responding to and solving bacterial infections that occur during organ transplantation, tumors, or other major diseases. However, the irregular use of antibiotics has led to the emergence of drug-resistant bacteria. The situation of discovering "stronger antibiotics" with superior performance and safe clinical application in the short term is not optimistic. The reasons are: the number of new antibacterial targets and lead antibiotics that are easy to find has decreased significantly; the mining and screening of potential drugs are difficult. So far, the proportion of new antibiotics under clinical research is less than 20%, and the R&D market is mostly dominated by imitations. The emergence of some new technologies: phage infection technology, CRISPR gene editing technology, and screening of new antibiotics based on artificial intelligence AI technology, although it provides new ideas for solving the problem of antibiotic resistance, there are also technical barriers to varying degrees, which is not conducive to rapid promotion and application.

[0003] The mechanisms of resistance to different types of antibiotics are common and can be summarized as follows: (1) Antibiotics cannot enter the bacteria; (2) If they enter, they cannot accumulate or stably exist inside the bacteria. Antibiotic adjuvants restore antibiotic activity by specifically inhibiting the occurrence of antibiotic resistance mechanisms. Their research and development goals are clear and their research and development cycles are relatively short, which provides the possibility of quickly solving the problem of drug-resistant bacteria. There are currently two main types of antibiotic adjuvants. The first type has a single function and mainly uses its blocking function to specifically inhibit the activity of a key protein that causes antibiotic resistance (such as membrane pore proteins, efflux pump proteins, etc.). It does not have antibacterial activity itself and cannot guarantee complete restoration of antibiotic activity. The second type is synergistic. On the basis of having the above-mentioned antibiotic adjuvant functions, it can also synergistically inhibit bacterial growth through other pathways, thereby preventing the further development of bacterial resistance, and has significant advantages in clinical application. The current successful representative of synergistic antibiotic adjuvants is the short peptide Host Defense Peptide (HDP), which first helps antibiotics enter the bacteria to function by destroying the bacterial cell membrane structure; at the same time, it can also increase the activity of host immune cells, thereby promoting the host immune system to further eliminate bacteria, demonstrating its performance as a synergistic antibiotic adjuvant. However, during the application process, there are large differences in the individual immune systems of the host, which will lead to uncertainty in the synergistic effect of HDP short peptides, and therefore will limit its clinical transformation.

[0004] Hsp70s (70kDa heat shock proteins) are a class of heat shock chaperone proteins that affect important cellular life activities by regulating protein homeostasis inside cells. Hsp70s is a highly conservative protein in structure and function. It is mainly composed of a nucleotide binding domain (NBD) and a substrate binding domain (SBD). Its working principle is the occurrence of ATP-driven conformational coupling of the two domains, accompanied by the binding and release of substrate peptides, thereby helping the substrate protein folding, assembly, and transport. Any molecule that hinders ATP binding to NBD, hinders substrate peptide binding to SBD, and directly hinders the occurrence of Hsp70s conformational coupling can seriously affect the activity of the protein. It has been reported that small molecule compounds that affect the conformational coupling of Hsp70 proteins can effectively inhibit the growth of mycobacteria, but the specific mechanism and application are still rarely reported.

[0005] In summary, the development of new antibiotic adjuvants is of great significance to the field of antibiotic application. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides an antibacterial membrane-penetrating polypeptide and its application, in order to solve the current problems of bacterial resistance and difficulty in discovering new antibiotics.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an antibacterial membrane-penetrating polypeptide, wherein the amino acid sequence of the antibacterial membrane-penetrating polypeptide comprises the sequence shown in SEQ ID NO.1 or a sequence having a homology of more than 70% with the sequence shown in SEQ ID NO.1.

[0009] The present invention mines and screens short peptides that specifically bind to bacterial Hsp70s proteins, and finds that they bind to Hsp70s proteins through traditional substrate binding pockets, but show abnormally high affinity for binding to proteins in the DnaK-ATP state. The short peptides can inhibit the activity of Hsp70s proteins to a certain extent, preventing them from helping denatured luciferase to renature. In addition, on the basis of ensuring the integrity of the bacterial cell membrane, the short peptides have superior ability to cross the bacterial cell membrane.

[0010] SEQ ID NO. 1: RLRSLAPRKLVIISK.

[0011] It is understood that short peptides with similar functions obtained by conservative mutation based on the amino acid sequence of the short peptide of the present invention should be within the protection scope of the present invention.

[0012] Preferably, the amino acid sequence of the antibacterial membrane-penetrating polypeptide includes the sequence shown in SEQ ID NO.2.

[0013] Preferably, the amino acid sequence of the antibacterial membrane-penetrating polypeptide includes the sequence shown in SEQ ID NO.3.

[0014] SEQ ID NO. 2: ALRSLAPRKLVIISK.

[0015] SEQ ID NO. 3: RLASLAPRKLVIISK.

[0016] In a second aspect, the present invention provides use of the antibacterial membrane-penetrating polypeptide described in the first aspect in preparing a delivery vector.

[0017] The present invention finds that the antibacterial membrane-penetrating polypeptide has an excellent ability to cross the cell membrane without destroying the cell structure. Therefore, it can be further used to prepare a delivery vector that can carry macromolecules such as nucleic acids, proteins, and drugs into cells, and has broad application prospects in the fields of drug delivery, gene delivery, and bioimaging.

[0018] In a third aspect, the present invention provides use of the antibacterial membrane-penetrating polypeptide described in the first aspect in the preparation of a drug for inhibiting bacteria.

[0019] In the present invention, it is found that the antibacterial membrane-penetrating polypeptide can effectively enter bacteria and act on Hsp70s protein, preventing it from helping denatured luciferase to renature, disrupting bacterial metabolism and inhibiting its growth.

[0020] Preferably, the bacteria comprises Escherichia coli or Staphylococcus aureus.

[0021] In a fourth aspect, the present invention provides use of the antibacterial membrane-penetrating polypeptide described in the first aspect in the preparation of an antibiotic adjuvant.

[0022] In the present invention, different types of antibiotics are used to interfere with the growth of Escherichia coli at low concentrations to simulate the occurrence of antibiotic resistance (the situation where antibiotics cannot effectively enter the bacteria and cannot accumulate inside the bacteria after entering): after adding short peptides at different concentrations, the short peptides can synergize with different types of low-concentration antibiotics to effectively inhibit the growth of Escherichia coli, and the synergistic antibacterial working concentration can be as low as 40μM; in addition, under stress conditions, the short peptide also shows a strong inhibitory ability on the growth of multi-drug resistant Escherichia coli, further demonstrating the application potential of short peptides as novel synergistic antibiotic adjuvants for resistant Escherichia coli and the like.

[0023] In a fifth aspect, the present invention provides a bacterial inhibitor, which comprises an antibiotic and the antibacterial membrane-penetrating polypeptide described in the first aspect.

[0024] In the present invention, bacterial inhibitors are further developed, and the antibacterial membrane-penetrating polypeptides and antibiotics are used in synergy to break the bacterial resistance mechanism and achieve efficient antibacterial effect.

[0025] Preferably, the bacteriostatic agent is in the form of a compound preparation or a combination of two separate preparations.

[0026] Preferably, the antibiotics include at least one of tetracycline antibiotics, macrolide antibiotics or aminoglycoside antibiotics.

[0027] Preferably, the antibiotic comprises at least one of kanamycin, gentamicin, azithromycin, tetracycline or spectinomycin.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] The present invention conducts in-depth excavation and screens to obtain a short peptide that specifically binds to the bacterial Hsp70s protein. It is found that it binds to the Hsp70s protein through the traditional substrate binding pocket, but shows an abnormally high affinity for binding to the protein in the DnaK-ATP state. The short peptide can inhibit the activity of the Hsp70s protein to a certain extent, preventing it from helping the denatured luciferase to renature. In addition, on the basis of ensuring the integrity of the bacterial cell membrane, the short peptide has a superior ability to cross the bacterial cell membrane, and further discusses its application potential in the preparation of delivery vectors, the preparation of drugs that inhibit bacteria, and the preparation of antibiotic adjuvants, providing new ideas for solving the current problems of bacterial resistance and the difficulty in exploring new antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A is the affinity curve of NR with DnaK and mutant protein;

[0031] Figure 1B is the affinity curve of VP5, DnaK and mutant protein;

[0032] Figure 2A is the affinity curve of NR with DnaK-ATP and PP;

[0033] Figure 2B is the affinity curve of VP5 with DnaK-ATP and PP;

[0034] Figure 2C This is the result diagram of the binding kinetics of NR, VP5, DnaK-ATP and PP;

[0035] Figure 3A This is a graph showing the result of VP5 inhibiting the refolding of denatured luciferase;

[0036] Figure 3B This is a result diagram showing that VP5 has the property of penetrating the cell membrane of E. coli;

[0037] Figure 4A To analyze the fluorescence distribution of the amino acid sequence of VP5 transmembrane properties;

[0038] Figure 4B This is a comparison of the mean fluorescence intensity of the amino acid sequence of VP5 transmembrane properties;

[0039] Figure 5A This is a fluorescence micrograph showing that VP5 has the ability to penetrate the cell membrane of Gram-positive bacteria;

[0040] Figure 5B This is the fluorescence intensity diagram of VP5 penetrating the membrane into Gram-positive bacteria;

[0041] Figure 6 This is the result diagram showing that VP5 penetrates the membrane without affecting the integrity of the cell membrane;

[0042] Figure 7 This is a graph showing the results of VP5 synergizing with heat shock conditions to inhibit the growth of E. coli;

[0043] Figure 8 Figure 5 is a graph showing the potential of VP5 as a synergistic antibiotic adjuvant for Escherichia coli, wherein Figure A is a graph showing the inhibition of Escherichia coli growth by different concentrations of kanamycin, Figure B is a graph showing the inhibition of Escherichia coli growth by VP5 in cooperation with a low concentration of kanamycin, Figure C is a graph showing the inhibition of Escherichia coli growth by different concentrations of gentamicin, Figure D is a graph showing the inhibition of Escherichia coli growth by VP5 in cooperation with a low concentration of gentamicin, Figure E is a graph showing the inhibition of Escherichia coli growth by different concentrations of azithromycin, Figure F is a graph showing the inhibition of Escherichia coli growth by VP5 in cooperation with a low concentration of azithromycin, Figure G is a graph showing the inhibition of Escherichia coli growth by different concentrations of tetracycline, and Figure H is a graph showing the inhibition of Escherichia coli growth by VP5 in cooperation with a low concentration of tetracycline;

[0044] Fig. 9A This is a graph showing the results of VP5 synergistically inhibiting the growth of drug-resistant Escherichia coli under heat shock conditions;

[0045] Fig. 9B This is the result of VP5 cooperating with antibiotics to inhibit the growth of drug-resistant Escherichia coli;

[0046] Fig. 9C This is the result of VP5 synergizing with low concentration of tetracycline to inhibit the growth of drug-resistant Escherichia coli;

[0047] Fig.10 This is a graph showing the effect of VP5 on the growth of eukaryotic cells HEK293. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0049] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0050] Example 1

[0051] This example targets the Escherichia coli Hsp70s protein (DnaK) for mining.

[0052] A short peptide VP5 that targets and binds to DnaK was screened out through a peptide chip.

[0053] Screening of short peptides was performed based on the indirect ELISA method. First, a peptide chip was made using iPDMS nanomaterials and the selected short peptide sequence. Then, the peptide chip was incubated with Hsp70 protein containing a histidine fusion tag to capture the protein-bound short peptides coupled to the chip. After the incubation, the excess Hsp70 protein was washed, and the positive peptide signal point was detected and imaged using a His tag-specific monoclonal antibody (HPR marker).

[0054] A short peptide (named VP5) that targets and binds to DnaK was obtained, and the amino acid sequence was RLRSLAPRKLVIISK (SEQID NO.1).

[0055] Example 2

[0056] This example analyzes the binding force and binding site of the short peptide VP5 and DnaK (in the non-ATP state).

[0057] The binding force and binding site of the short peptide VP5 and DnaK (in the non-ATP state) were detected by fluorescence polarization experiment in liquid solution, and the binding situation was compared with that of the model short peptide NR and DnaK (in the non-ATP state).

[0058] Fluorescence polarization experimental materials:

[0059] a) Reaction buffer 1: containing 1 M Hepes-KOH (pH 7.5), 3 M KCl, 1 M Mg(OAc)2, 1 mM DTT, 10% glycerol and 1 mg / mL BSA (bovine serum albumin);

[0060] b) FITC-labeled short peptide: FITC was labeled at the N-terminus of the short peptide, synthesized at Nanjing GenScript Biotechnology Co., Ltd., with a purity greater than 95%.

[0061] DnaK and other proteins were diluted 2-fold in series starting from 20 μM concentration using reaction buffer 1 until the final concentration was about 0.0006 μM. Then, the different concentrations of proteins in each tube were evenly mixed with the FITC-labeled short peptide to be tested (the final concentration of the short peptide was 10 nM), transferred to a 384-well plate (Corning, 3575, USA) and incubated at room temperature in the dark for a certain period of time until the short peptide and protein reaction reached equilibrium. Finally, the static fluorescence polarization signal in each well was detected by Cytation 3 (BioTek) multifunctional microplate reader (Benteng Instrument Co., Ltd., China), and the obtained signal value was analyzed by GraphPad PRISM 7.0.

[0062] The results showed that, similar to the model short peptide NR, in the non-ATP state, VP5 also binds to the DnaK protein through the SBD traditional binding pocket (L 1,2 and L 3,4 Composition) combined with protein, such as Figure 1A , Figure 1B As shown: The binding curve of VP5 with the full-length DnaK protein (WT) is very similar to that with SBD. 1,2 Two important amino acid mutations related to substrate binding (T403GM404G) and L 3,4 The deletion mutations all showed that they had no binding to the short peptide NR and VP5. In addition, the second potential substrate binding region L 5,6 The mutation also showed that the mutant protein had a weaker ability to bind to NR and VP5 ( Figure 1A , Figure 1B ). Furthermore, through the non-fluorescent (Native) short peptide competition experiment, that is, introducing 400μM of the other non-fluorescent short peptide before adding the FITC-labeled short peptide (FITC-short peptide), it was found that both short peptides can effectively shield the binding of the other FITC-short peptide with the protein ( Figure 1A , Figure 1B ). The above results fully demonstrate that in the non-ATP state, VP5 and NR short peptides bind to proteins in a similar manner, and the way VP5 binds to DnaK protein is conservative. The relevant affinity parameters of the above results are shown in Table 1.

[0063] Table 1

[0064]

[0065] Example 3

[0066] This example analyzes the affinity of the short peptide VP5 to DnaK.

[0067] The affinity between the short peptide and DnaK in the ATP state was detected by fluorescence polarization experiment. Detection of the binding kinetics of short peptides and proteins in the non-ATP state: The detection method refers to Example 1, except that after the protein is diluted with a concentration gradient, ATP (2mM, Solebao, A8270, China) needs to be added to the protein dilutions of different concentrations in advance, and incubated at room temperature for 2 minutes before performing subsequent experimental steps. Detection of the binding kinetics of short peptides and proteins in the ATP state: Different from the above-mentioned detection method of the binding kinetics of short peptides and proteins in the non-ATP state, it is necessary to add ATP (2mM) and incubate with the protein for 2 minutes before adding FITC-short peptides, and then perform subsequent tests.

[0068] As expected, the binding affinity of the short peptide NR to DnaK-ATP decreased by at least 2 orders of magnitude compared to the protein in the non-ATP state ( Figure 2A , Table 2); and the affinity of NR to the DnaK mutant protein PP (which locks the protein conformation in the ATP state) is also consistent with expectations ( Figure 2A , Table 2). In contrast, in the ATP state, the short peptide VP5 still maintains a high affinity state with DnaK-ATP ( Figure 2B , Table 2), indicating that in the ATP state, the binding of the short peptide VP5 to DnaK-ATP is special. Based on the abnormally high affinity of the short peptide VP5 to DnaK-ATP, we believe that the non-fluorescent VP5 in the ATP state will strongly antagonize the binding of FITC-NR to DnaK. To verify this hypothesis, we further detected the mutual antagonism between the two short peptides in the ATP state through competitive fluorescence polarization experiments. The results showed that 200μM non-fluorescent VP5 can completely antagonize the binding of FITC-NR to PP protein, and even 400μM non-fluorescent NR can only reduce the binding of FITC-VP5 to PP protein to a certain extent, and the antagonistic effect is far less than that of VP5 ( Figure 2A , 2B ). The above experimental results once again fully prove that, unlike the model short peptide NR, the short peptide VP5 can bind to DnaK with high affinity under ATP state.

[0069] Table 2

[0070]

[0071] Next, we further analyzed the binding characteristics of the short peptide VP5 with DnaK-ATP and PP mutations from a kinetic perspective. Figure 2CAs shown in the figure, through the fluorescence polarization kinetics model, we found that: under the ATP state, the model short peptide NR binds to DnaK-ATP relatively quickly, and the instrument cannot capture the dynamic changes in the signal value; on the contrary, the binding kinetics of the short peptide VP5 to the above two proteins is relatively slow, and the dynamic change trend of the signal value is also relatively obvious. In addition, the end point fluorescence polarization value of the kinetic detection result of the short peptide is also consistent with Figure 2A , Figure 2B The results are consistent. The above experiments also show from the perspective of binding kinetics that the binding of VP5 to DnaK-ATP is special.

[0072] Example 4

[0073] This example analyzes whether the abnormally high affinity binding of VP5 to DnaK-ATP will affect the refolding system activity of DnaK.

[0074] The effect of short peptide VP5 on DnaK activity was tested by in vitro denatured luciferase renaturation experiment.

[0075] Experimental materials for detecting the refolding of denatured luciferase: 1) Buffer A: containing 25mM Hepes-KOH (pH7.5), 100mM KCl, 10mM Mg(OAC)2 and 2mM DTT; 2) Refolding buffer: 3mM ATP was added to Buffer A.

[0076] Protocol for detecting denatured luciferase refolding assay:

[0077] a) Preparation of protein stock solution: dilute DnaK, DnaJ, and GrpE to appropriate concentrations with Refolding buffer; and dilute with Buffer A to prepare luciferase protein stock solution (1-5 μM is sufficient);

[0078] b) Prepare luciferase for experiment:

[0079] i. Prepare denatured luciferase (for negative control and experimental groups): Heat in Refolding buffer at 42°C (recommended for 10-20 minutes) to denature the luciferase protein. After denaturation, place at room temperature;

[0080] ii. Prepare undenatured luciferase (for positive control): dilute the luciferin stock solution 10-fold with Refolding buffer and place at room temperature;

[0081] c) Prepare the reaction system

[0082] i. Positive control: Take 3 μL of the above undenatured luciferase and add it to 30 μL Refolding buffer and test immediately;

[0083] ii. Negative control: Take 3 μL of the above denatured luciferase and add it to 30 μL Refolding buffer and test immediately;

[0084] iii. Experimental group: 3 μL of the above denatured luciferase was mixed with the refolding system to promote the refolding of the luciferase protein. The refolding system consisted of DnaK, DnaJ, and GrpE. The reaction was carried out at room temperature for 30 minutes before detection.

[0085] iv. Interference group (short peptide added): Compared with the experimental group, short peptide was added to the renaturation system as an experimental factor; we added different concentrations of VP5 short peptide (20μM, 40μM, 80μM, 160μM) or different concentrations of NR short peptide (400μM, 1000μM, 2000μM, 4000μM) after adding DnaK, reacted with DnaK for 2 minutes in advance, and then added other components of the renaturation system, reacted at room temperature for 30 minutes before detection;

[0086] d) Detection system

[0087] 5 μL of sample was aspirated and reacted with luciferase detection system protein substrate (Promega, E1500) in a 96-well plate (Thermo, 176037, Denmark), and the cold luminescence reading was measured by the luminescence mode of the Cytation 3 multifunctional microplate reader.

[0088] The results show that Figure 3A ), the short peptide VP5 can strongly inhibit the activity of the protein folding system (refolding system) of DnaK and destroy its effect on the renaturation of the denatured substrate luciferase. 80μM VP5 can significantly inhibit the renaturation of denatured luciferase by about 70%, and when the working concentration of VP5 is 160μM, it completely inhibits the renaturation of denatured luciferase; the inhibition rate of 4000μM NR on the renaturation of denatured luciferase is about 50%, which is far lower than the interference ability of VP5 on the refolding system of DnaK.

[0089] We further tested whether VP5 also had the ability to penetrate the cell membrane of E. coli.

[0090] Inoculate a single colony of wild-type Escherichia coli (E. coli ATCC 25922) into LB liquid medium and culture overnight at 37°C, 200 rpm. The next day, inoculate fresh bacterial culture at a ratio of 100× and culture again until OD 600= about 0.55. Take 1mL of bacterial solution and divide it into 1.5mL centrifuge tubes, centrifuge at 1500×g, 4℃, 5min, and discard the supernatant. Wash the cells with 1×PBS, centrifuge at 1500g, 4℃, 5min, and discard the supernatant. Then resuspend the cells with solution A (Takara, the main component is CaCl2), place on ice for 10-20min, centrifuge at 1500×g, 4℃, 5min, discard the supernatant, resuspend the bacteria with LB and dilute for use. Add FITC-short peptide to the above bacterial solution to a working concentration of 30μM and incubate in the dark for 30min. After the reaction is completed, centrifuge at 8000rpm at room temperature for 3min, remove the supernatant, and then wash the bacteria thoroughly with PBS, and finally dissolve in a suitable volume of PBS solution for fluorescence microscopy. When observing with a fluorescence microscope, use the blue channel (470 / 490) with a magnification of 600 times.

[0091] The results show that Figure 3B ), unlike the model short peptide NR, VP5 can penetrate the membrane and enter the interior of E. coli: after E. coli incubated with FITC-NR and washed with PBS, no fluorescently labeled E. coli cells could be observed under a fluorescence microscope; on the contrary, a large number of fluorescently labeled E. coli cells remained in the FITC-VP5 group. Based on this experiment, we preliminarily concluded that VP5 has the ability to penetrate the cell membrane of E. coli.

[0092] Through fluorescence microscopy, we preliminarily determined that the short peptide VP5 has a good ability to penetrate the E. coli cell membrane. In order to reveal the essence of the reason why the short peptide VP5 can penetrate the membrane, we performed a series of mutations on the amino acid sequence of VP5 (Table 3) to specifically analyze the key amino acid sites that affect VP5 membrane penetration.

[0093] Table 3

[0094] Short peptide name sequence VP5 RLRSLAPRKLVIISK VP5-5 RLRSLAPRKAAAASK VP5-6 ALRSLAPRKLVIISK VP5-7 RLASLAPRKLVIISK VP5-8 ALASLAPAKLVIISK VP5-9 RLRSLAPRDLVIISD VP5-10 RLRSLAPRKSVSSSK VP5-11 RSRSSASRKLVIISK VP5-R RKLVIISKRLRSLAP

[0095] All short peptides (including mutant short peptides) were synthesized at Nanjing GenScript Biotechnology Co., Ltd. with a purity greater than 95%. The preliminary sample preparation process is consistent with the sample preparation process of the above-mentioned method for detecting the ability to penetrate the cell membrane of Escherichia coli. The difference is that after the sample is finally dissolved with a suitable volume of PBS solution, the efficiency of short peptide penetration is quantitatively analyzed by flow cytometry. When detecting by flow cytometry, the FITC channel is selected and the voltage is set to 407V.

[0096] The results are as follows Figure 4A and 4B As shown, it can be seen that mutation of VP5 can affect its membrane penetration ability, among which, mutation of the basic amino acid at the N-terminus of the short peptide (VP5-6) alone can improve the membrane penetration ability to a certain extent, indicating that VP5-6 is a short peptide with development potential.

[0097] We further tested whether VP5 could penetrate the cell membrane of Gram-positive bacteria with higher peptidoglycan content. Similar to the above method, we used Staphylococcus aureus (BMZ137680) as the research object. The results of fluorescence microscopy showed that ( Figure 5A ), most of the Staphylococcus aureus cells incubated with FITC-VP5 for a period of time were able to emit fluorescence, indicating that the ability of VP5 to penetrate the membrane is not limited to Gram-negative bacteria. Further, quantitative analysis by flow cytometry showed that the rate of short peptide VP5 penetrating Staphylococcus aureus was about 75% (e.g. Figure 5B In summary, our experiments have once again proved that VP5 is a short peptide with excellent membrane-penetrating ability. Unlike small molecules such as antibiotics that are passively transported into bacteria, VP5 once again shows its application potential of entering bacteria across the membrane to exert its function.

[0098] Example 5

[0099] This example further analyzes whether VP5 will damage the integrity of the bacterial cell membrane after penetrating the membrane into the bacteria.

[0100] Propidium iodide fluorescent dye (Biyuntian, catalog number: ST511) was used for verification. The preliminary bacterial treatment refers to the above-mentioned membrane penetration ability detection experimental scheme. 1) Treatment method of propidium iodide staining positive control group: add 70% isopropanol solution to treat the above bacterial solution, incubate at 39°C for 30 minutes, centrifuge the treated sample, wash and resuspend with 1×PBS, and then incubate in the dark with 10μL propidium iodide (working concentration is 50μg / mL) for 15 minutes in the dark, then wash and resuspend with 1×PBS, take 5μL onto the slide, and observe using the fluorescence microscope Texa Red (595 / 613) channel. 2) Treatment method of VP5 group: add short peptides with working concentrations of 30μM and 120μM, and incubate in a biochemical incubator at 37°C for 60 minutes. Then perform the labeling, washing and observation operations of propidium iodide (working concentration is 50μg / mL) as the above positive group.

[0101] Propidium iodide can penetrate cells with damaged membranes and bind to nucleic acid substances (DNA and RNA) in the cells, emitting red fluorescence under a fluorescence microscope ( Figure 6), microscopic observation results showed that when different concentrations (30μM, 120μM) of FITC-VP5 were incubated with E. coli cells at 37°C for a certain period of time, the green fluorescence channel could detect fluorescently labeled E. coli cells, but the red fluorescence channel could not capture obvious fluorescently labeled E. coli cells. The above experimental results show that VP5 can ensure the integrity of the cell membrane of E. coli without destroying it, which is different from the mechanism of action of most antibacterial peptides and has its own uniqueness.

[0102] Example 6

[0103] This example tests the antibacterial ability of VP5.

[0104] Inoculate a single colony of wild-type E. coli (E. coli ATCC 25922) into LB liquid medium: 37°C, 200 rpm overnight culture. The next day, inoculate fresh bacterial culture at a ratio of 100× and culture again until OD 600 = about 0.55. Dilute the bacterial solution with LB to OD 600 =0.2 as the stock solution, and diluted 10 times to the working concentration. Then, different working concentrations of VP5 (40μM, 80μM, 160μM, 320μM) were added to the 96-well plate with bacterial solution, and the group without VP5 was used as the positive control. After that, the 96-well plate bacterial solution was statically cultured at 37℃ and 42℃ for 16 to 20 hours, and then the absorbance (OD) of the bacterial solution in the 96-well plate was measured by HTX multifunctional microplate reader (SynergyHTX). 600 ).

[0105] The results are as follows Figure 7 As shown, VP5 also has a concentration-dependent inhibition on the growth of Escherichia coli, and under heat shock conditions, the antibacterial effect of VP5 is more significant.

[0106] Example 7

[0107] This example tests whether VP5 can function as a synergistic antibiotic adjuvant for antibiotics with specific drug resistance mechanisms (antibiotics cannot enter bacteria efficiently or cannot accumulate at high concentrations inside bacteria).

[0108] Based on the model Escherichia coli, the scenarios of several antibiotic resistance mechanisms were simulated, and the ability of synergistic antibacterial after the addition of VP5 was detected. For the preliminary bacterial culture and preparation, refer to Example 6, and the prepared bacteria were inoculated in a 96-well plate at 200 μL per well, and cultured in a 37°C biochemical incubator overnight. For the antibiotic group and the VP5 synergistic antibiotic group, short peptides and antibiotic solutions of corresponding working concentrations need to be added and mixed before continuing the culture. After mixing, the 96-well plate was transferred to a 37°C biochemical incubator and continued to be cultured for about 15 hours. After the culture was completed, the absorbance (OD 600 ).

[0109] The antibiotics tested included aminoglycosides, macrolides, and tetracyclines. Aminoglycosides are often unable to enter the bacteria due to mutations in bacterial membrane pore proteins (e.g. kanamycin). + , Gentamicin GM + ); while macrolide antibiotics (azithromycin AMZ + ) and tetracycline antibiotics (tetracycline TC + ) Often due to the overexpression of efflux pump proteins on the bacterial cell membrane, antibiotics are pumped out of the bacteria in large quantities, thus failing to accumulate inside the bacteria and causing antibiotic resistance. Figure 8 As shown in Figure 2, we first screened the concentrations of different antibiotics that could kill about half of E. coli (simulating the antibiotic resistance scenario) and found that in Kana + 15 μg / mL ( Figure 8 Figure A), GM + 2.5 μg / mL ( Figure 8 (C), AMZ + 10 μg / mL ( Figure 8 Middle E), TC + 1 μg / mL ( Figure 8 Second, after determining the inefficient working concentrations of different antibiotics, we added VP5 short peptides of different concentration gradients while the bacteria were growing to detect the antibacterial ability of VP5 in synergy with low-concentration antibiotics, such as Figure 8 As shown in Figures B, D, F, and H, the addition of different concentrations (as low as 40 μM) of VP5 can completely inhibit the growth of E. coli. Figure 7The results show that the minimum antibacterial activity rate of VP5 (40-120 μM) alone at 37°C is only about 45%, indicating that the antibacterial effect of VP5 alone is limited. After adding it to a low-efficiency antibiotic concentration environment (bacterial activity rate above 40%), the overall antibacterial effect is significantly improved, and the bacterial activity rate is as low as below 10%, indicating that VP5 not only has a certain antibacterial function itself, but can also break the drug resistance mechanism (antibiotics cannot efficiently enter bacteria or cannot accumulate at high concentrations inside bacteria) and restore the antibacterial effect of antibiotics, thereby proving the potential of this short peptide as a new synergistic antibiotic adjuvant for Escherichia coli. The experimental results also further reflect the reality of the short peptide VP5 exerting a synergistic antibacterial function by targeting DnaK, an important antibacterial target.

[0110] Example 8

[0111] This example further verifies the feasibility of using VP5 to solve the problem of bacterial resistance.

[0112] A strain of multidrug-resistant Escherichia coli (Shanghai Collection of Microorganisms, D24652, resistant to tetracycline, chloramphenicol, streptomycin, and spectinomycin) was used as the test object to detect the inhibitory effect of short peptide VP5 on its growth under heat shock conditions. The detection method is similar to that of Example 7, except that the strain cultured this time is multidrug-resistant Escherichia coli.

[0113] turn out( Fig. 9A ), at 37°C, 320μM VP5 could inhibit the growth of multidrug-resistant bacteria by half; under heat shock conditions, 160μM VP5 could almost completely inhibit the growth of resistant bacteria. + ), spectinomycin (STM + ), Streptomycin (SM + ), chloramphenicol (CAP + ) against the growth of multidrug-resistant Escherichia coli. 20 μM VP5 was used in combination with 50 μg / mL of each antibiotic. The results showed that ( Fig. 9B ), 20μM VP5 can cooperate with 50μg / mL tetracycline and spectinomycin to completely inhibit the growth of multi-drug resistant Escherichia coli, but due to the complexity of antibiotic resistance mechanism, the antibacterial effect of VP5 in cooperation with streptomycin and chloramphenicol is not obvious. In addition, we also found that ( Fig. 9C), 40μM VP5 synergized with 10μg / mL tetracycline (normal working concentration for non-resistant bacteria) can also achieve complete inhibition of the growth of multidrug-resistant Escherichia coli. In summary, due to the complex resistance mechanism of this multidrug-resistant bacteria, the effect of VP5 alone is not ideal, and a higher concentration of short peptides is required to achieve the antibacterial effect; but if synergized with heat shock conditions (bacterial growth under simulated stress conditions); or synergized with low concentrations of antibiotics (especially tetracycline and spectinomycin), VP5's inhibitory effect on multidrug-resistant Escherichia coli is significantly improved. The experimental results once again show the high fit and rationality of VP5 as a synergistic antibiotic adjuvant for Escherichia coli, and it is not a traditional antibacterial peptide that can perform efficient antibacterial functions alone.

[0114] Example 9

[0115] This example detects the cytotoxicity of VP5.

[0116] Human embryonic kidney cells HEK293, which are commonly used in the laboratory, were used as the test object to detect the effects of different concentrations of VP5 short peptides on their growth. Take HEK293 cells in the logarithmic growth phase, count them by a blood cell counting plate to obtain the accurate cell density, and then dilute them with fresh DMEM culture medium (10% fetal bovine serum, FBS) to form a cell suspension of a certain density. The above cell suspension was inoculated in a 96-well plate (10,000 / well) and cultured overnight at 37°C, 5% CO2 until completely adherent. The next day, fresh DMEM culture medium (10% fetal bovine serum, FBS) was replaced, and different concentrations of puromycin (Biyuntian Biotechnology Company, ST551) or VP5 short peptides to be tested were added to the culture medium at the same time, and then cultured for 2 days at 37°C, 5% CO2, and the positive control (normal cultured cells, without the tested antibiotics and VP5) and background control wells (only cell culture medium and different test drugs were added, without cells) were set up. After the culture was completed, the cell culture medium in the 96-well plate was completely removed, and fresh cell culture medium (containing 10% CCK8 reaction solution by volume, purchased from Bio-Tech Biotech, C0037) was immediately added, and the culture was continued at 37°C, 5% CO2 for 1 hour. The absorbance (OD) in each well was then detected by HTX multifunctional microplate reader (Synergy HTX). 450 The cell growth inhibition curves affected by drugs were analyzed and plotted using GraphPad PRISM 7.0.

[0117] The results are as follows Fig.10As shown, compared with puromycin (PURO), which effectively inhibits the growth of HEK293 cells, VP5 has very little effect on the growth of the eukaryotic cells. 320μM VP5 can still keep the viability of HEK293 cells above 90%. This shows that the safety of VP5 short peptide as a synergistic antibiotic adjuvant to solve the problem of antibiotic resistance is guaranteed to a certain extent, which once again shows the good application prospects of this short peptide.

[0118] In summary, the present invention conducts in-depth excavation and screens to obtain a short peptide that specifically binds to the bacterial Hsp70s protein. It is found that it binds to the Hsp70s protein through the traditional substrate binding pocket, but exhibits an abnormally high affinity for binding to the protein in the DnaK-ATP state. The short peptide can inhibit the activity of the Hsp70s protein to a certain extent, preventing it from helping the denatured luciferase to renature. In addition, on the basis of ensuring the integrity of the bacterial cell membrane, the short peptide has a superior ability to cross the bacterial cell membrane, and further discusses its application potential in the preparation of delivery vectors, the preparation of drugs that inhibit bacteria, and the preparation of antibiotic adjuvants, providing new ideas for solving the current problems of bacterial resistance and the difficulty in exploring new antibiotics.

[0119] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An antibacterial membrane-penetrating polypeptide, characterized in that: The amino acid sequence of the antibacterial membrane-penetrating polypeptide includes the sequence shown in SEQ ID NO.1 or a sequence with a homology of more than 70% to the sequence shown in SEQ ID NO.

1.

2. The antibacterial membrane-penetrating polypeptide according to claim 1, characterized in that The amino acid sequence of the antibacterial membrane-penetrating polypeptide includes the sequence shown in SEQ ID NO.

2.

3. The antibacterial membrane-penetrating polypeptide according to claim 1, characterized in that The amino acid sequence of the antibacterial membrane-penetrating polypeptide includes the sequence shown in SEQ ID NO.

3.

4. Use of the antibacterial membrane-penetrating polypeptide according to any one of claims 1 to 3 in the preparation of a delivery vector.

5. Use of the antibacterial membrane-penetrating polypeptide according to any one of claims 1 to 3 in the preparation of a drug for inhibiting bacteria.

6. The use according to claim 5, characterized in that: The bacteria include Escherichia coli and / or Staphylococcus aureus.

7. Use of the antibacterial membrane-penetrating polypeptide according to any one of claims 1 to 3 in the preparation of an antibiotic adjuvant.

8. The use according to claim 7, characterized in that: The antibiotic adjuvant includes a synergistic antibiotic adjuvant.

9. A bacterial inhibitor, characterized in that The bacterial inhibitors include antibiotics and the antibacterial membrane-penetrating polypeptide according to any one of claims 1 to 3.

10. The bacterial inhibitor according to claim 9, characterized in that The bacterial inhibitor is in the form of a compound preparation or a combination of two separate preparations; Preferably, the antibiotics include at least one of tetracycline antibiotics, macrolide antibiotics or aminoglycoside antibiotics.

Images