An antibacterial membrane-penetrating polypeptide and its application

Abstract

This invention relates to an antibacterial membrane-penetrating peptide and its applications. The amino acid sequence of the antibacterial membrane-penetrating peptide includes the sequence shown in SEQ ID NO.1 or a sequence with more than 70% homology to the sequence shown in SEQ ID NO.1. This invention screened and obtained a short peptide that specifically binds to the bacterial Hsp70s protein. It was found that this peptide binds to the Hsp70s protein through a traditional substrate-binding pocket, exhibiting an unusually high affinity for the protein in the DnaK-ATP state. The short peptide can inhibit Hsp70s protein activity to a certain extent, preventing it from aiding in the renaturation of denatured luciferase. Furthermore, while maintaining the integrity of the bacterial cell membrane, this short peptide has a superior ability to cross the bacterial cell membrane. The invention further discusses its application potential in the preparation of antibacterial drugs and antibiotic adjuvants, providing new ideas for solving current problems such as bacterial resistance and the difficulty in discovering new antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an antibacterial membrane-penetrating polypeptide and its applications. Background Technology

[0002] Antibiotics play a crucial role in addressing bacterial infections during organ transplantation, cancer treatment, and other major disease management. However, the improper use of antibiotics has led to the emergence of drug-resistant bacteria. The prospect of discovering superior, safe, and clinically applicable "stronger antibiotics" in the short term is far from optimistic. This is due to several factors: a significant decrease in easily identifiable new antibacterial targets and lead antibiotics; and numerous difficulties in identifying and screening potential drugs. Currently, less than 20% of antibiotics are in clinical development, with the market largely dominated by generics. While emerging technologies such as phage infection, CRISPR gene editing, and AI-based antibiotic screening offer new approaches to addressing antibiotic resistance, they also present varying degrees of technological barriers, hindering rapid widespread adoption.

[0003] The mechanisms by which different types of antibiotics develop resistance share commonalities, which can be summarized as follows: (1) antibiotics cannot penetrate the interior of bacteria; (2) if they do penetrate, they cannot accumulate or remain stable within 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 development cycle is relatively short, providing a possibility for quickly solving the problem of drug-resistant bacteria. Currently, there are two main types of antibiotic adjuvants. The first type has a single function, mainly inhibiting the activity of a key protein that leads to antibiotic resistance (such as membrane porin, efflux pump protein, etc.) through its blocking function, but it does not have antibacterial activity itself and cannot guarantee the complete restoration of antibiotic activity. The second type is synergistic, which, in addition to having the above-mentioned antibiotic adjuvant functions, 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 most successful example of a synergistic antibiotic adjuvant is the short-peptide Host Defense Peptide (HDP). It first helps antibiotics enter the bacteria and exert their function by disrupting the bacterial cell membrane structure; simultaneously, it enhances the activity of host immune cells, thereby promoting the host's immune system to further clear the bacteria, demonstrating its synergistic antibiotic adjuvant performance. However, significant differences exist in the immune systems of individual hosts during application, leading to uncertainty in the synergistic effect of HDP-like short-peptides, thus limiting their clinical translation.

[0004] Hsp70s (70kDa heat shock proteins) are a class of heat shock molecular chaperone proteins that influence vital cellular activities by regulating intracellular protein homeostasis. Hsp70s are highly conserved proteins in both structure and function, primarily composed of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). Their function involves ATP-driven conformational coupling between these two domains, accompanied by the binding and release of substrate peptides. This facilitates substrate protein folding, assembly, and transport. Any molecule that hinders ATP binding to the NBD, substrate peptide binding to the SBD, or directly impedes Hsp70 conformational coupling can severely affect the protein's activity. While some reports indicate that small molecule compounds affecting Hsp70 conformational coupling can effectively inhibit the growth of mycobacteria, the specific mechanisms and applications remain largely unreported.

[0005] In conclusion, the development of novel antibiotic adjuvants is of great significance to the application of antibiotics. Summary of the Invention

[0006] In response to the shortcomings of existing technologies and practical needs, this invention provides an antibacterial membrane-penetrating polypeptide and its application, aiming to solve the current problems of bacterial resistance and the difficulty in discovering new antibiotics.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[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 includes the sequence shown in SEQ ID NO.1 or a sequence having more than 70% homology with the sequence shown in SEQ ID NO.1.

[0009] This invention identifies and screens short peptides that specifically bind to the bacterial Hsp70s protein. It discovers that these peptides bind to the Hsp70s protein via a traditional substrate-binding pocket, but exhibit an unusually high affinity for the protein in the DnaK-ATP state. These short peptides can inhibit Hsp70s protein activity to a certain extent, preventing it from aiding in the renaturation of denatured luciferase. Furthermore, while maintaining the integrity of the bacterial cell membrane, these short peptides possess superior ability to cross the bacterial cell membrane.

[0010] SEQ ID NO. 1: RLRSLAPRKLVIISK.

[0011] It is understood that any short peptides with similar functions obtained by conserved mutations based on the amino acid sequence of the short peptides of this invention should be within the scope of protection of this 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 the application of the antibacterial membrane-penetrating peptide described in the first aspect in the preparation of a delivery carrier.

[0017] The present invention has discovered that the antibacterial membrane-penetrating polypeptide has excellent ability to cross cell membranes without damaging cell structure. Therefore, it can be further applied to the preparation of delivery vectors that can carry macromolecules such as nucleic acids, proteins, and drugs into cells, and has broad application prospects in drug delivery, gene delivery, bioimaging and other fields.

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

[0019] In this invention, it was discovered that the antibacterial membrane-penetrating polypeptide can effectively penetrate bacteria, act on the Hsp70s protein, prevent it from helping denatured luciferase to renature, disrupt bacterial metabolism, and inhibit its growth.

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

[0021] Fourthly, the present invention provides the application of the antibacterial membrane-penetrating peptide described in the first aspect in the preparation of antibiotic adjuvants.

[0022] In this invention, different types of antibiotics are used at low concentrations to interfere with the growth of *E. coli* to simulate antibiotic resistance scenarios (where antibiotics cannot effectively penetrate the bacteria or accumulate inside them after penetration). After adding different concentrations of short peptides, the peptides can synergistically inhibit the growth of *E. coli* with different types of low-concentration antibiotics, with the synergistic antibacterial working concentration as low as 40 μM. Furthermore, under pressure conditions, the short peptides also exhibit strong inhibitory ability against the growth of multidrug-resistant *E. coli*, further demonstrating the potential of short peptides as novel synergistic antibiotic adjuvants for drug-resistant *E. coli* and other bacteria.

[0023] Fifthly, the present invention provides a bacterial inhibitor, the bacterial inhibitor comprising an antibiotic and the antimicrobial membrane-penetrating polypeptide described in the first aspect.

[0024] In this invention, bacterial inhibitors are further developed, utilizing antimicrobial membrane-penetrating peptides and antibiotics in synergy to break bacterial resistance mechanisms and achieve highly efficient antibacterial activity.

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

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

[0027] Preferably, the antibiotic includes 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] This invention delves deeper, screening for and obtaining a short peptide that specifically binds to the bacterial Hsp70s protein. It was discovered that this peptide binds to the Hsp70s protein through a traditional substrate-binding pocket, but exhibits an unusually high affinity for the protein in the DnaK-ATP state. The short peptide can inhibit Hsp70s protein activity to a certain extent, preventing it from aiding in the renaturation of denatured luciferase. Furthermore, while maintaining the integrity of the bacterial cell membrane, this short peptide possesses superior ability to cross the bacterial cell membrane. Its application potential in the preparation of delivery vectors, the development of antibacterial drugs, and the preparation of antibiotic adjuvants is further discussed, providing new ideas for solving current problems such as bacterial resistance and the difficulty in discovering new antibiotics. Attached Figure Description

[0030] Figure 1A Affinity curves of NR with DnaK and mutant proteins;

[0031] Figure 1B Affinity curves of VP5 with DnaK and mutant proteins;

[0032] Figure 2A Affinity curves of NR with DnaK-ATP and PP;

[0033] Figure 2B Affinity curves of VP5 with DnaK-ATP and PP;

[0034] Figure 2C The diagram shows the binding kinetics of NR, VP5 with DnaK-ATP and PP.

[0035] Figure 3A The image shows the results of VP5 inhibiting the renaturation of denatured luciferase.

[0036] Figure 3B This is a diagram showing the results of VP5's ability to penetrate the cell membrane of E. coli;

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

[0038] Figure 4B The figure shows the comparison of the average fluorescence intensity of the amino acid sequences used to analyze the membrane-penetrating properties of VP5.

[0039] Figure 5A Fluorescent micrograph showing VP5's ability to penetrate the cell membrane of Gram-positive bacteria;

[0040] Figure 5B Fluorescence intensity diagram of VP5 penetrating the membrane and entering Gram-positive bacteria;

[0041] Figure 6 The result shows that VP5 transmembrane penetration does not affect cell membrane integrity.

[0042] Figure 7 The figure shows the results of VP5 synergistic heat shock inhibition of Escherichia coli growth;

[0043] Figure 8 The results of the potential test of VP5 as a synergistic antibiotic adjuvant for Escherichia coli are shown in Figure A, which shows the inhibition of E. coli growth by different concentrations of kanamycin; Figure B shows the inhibition of E. coli growth by VP5 in combination with low concentrations of kanamycin; Figure C shows the inhibition of E. coli growth by different concentrations of gentamicin; Figure D shows the inhibition of E. coli growth by VP5 in combination with low concentrations of gentamicin; Figure E shows the inhibition of E. coli growth by different concentrations of azithromycin; Figure F shows the inhibition of E. coli growth by VP5 in combination with low concentrations of azithromycin; Figure G shows the inhibition of E. coli growth by different concentrations of tetracycline; and Figure H shows the inhibition of E. coli growth by VP5 in combination with low concentrations of tetracycline.

[0044] Figure 9A The figure shows the results of VP5 synergistic inhibition of the growth of drug-resistant Escherichia coli under heat shock conditions;

[0045] Figure 9B The figure shows the results of VP5 synergistically inhibiting the growth of drug-resistant Escherichia coli with antibiotics;

[0046] Figure 9C The figure shows the results of VP5 synergistically inhibiting the growth of drug-resistant Escherichia coli with low concentrations of tetracycline.

[0047] Figure 10 The figure shows the effect of VP5 on the growth of eukaryotic cells HEK293. Detailed Implementation

[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely 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 is determined by the claims.

[0049] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0050] Example 1

[0051] This embodiment focuses on the discovery of the Escherichia coli Hsp70s protein (DnaK).

[0052] A short peptide, VP5, that targets and binds to DnaK was identified through peptide microarray screening.

[0053] Short peptides were screened using an indirect ELISA method. First, a peptide chip was fabricated using iPDMS nanomaterials and selected short peptide sequences. Then, the peptide chip was incubated with Hsp70 protein containing a histidine fusion tag to capture protein-bound short peptides coupled to the chip. After incubation, excess Hsp70 protein was washed away, and positive peptide signal points were detected and imaged using a His tag-specific binding monoclonal antibody (HPR label).

[0054] A short peptide targeting DnaK (named VP5) was obtained, with the amino acid sequence RLRSLAPRKLVIISK (SEQ ID NO.1).

[0055] Example 2

[0056] This embodiment analyzes the binding affinity and binding site between the short peptide VP5 and DnaK (in the non-ATP state).

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

[0058] Materials for fluorescence polarization experiments:

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

[0060] b) FITC-labeled short peptides: FITC is labeled on the N-terminus of short peptides, synthesized by Nanjing Genscript Biotech Co., Ltd., with a purity greater than 95%.

[0061] DnaK and other proteins were serially diluted twofold from 20 μM to a final concentration of approximately 0.0006 μM using reaction buffer 1. Then, the different protein concentrations in each tube were uniformly mixed with the FITC-labeled short peptides (all short peptides had a final concentration of 10 nM), transferred to 384-well plates (Corning, 3575, USA), and incubated at room temperature in the dark until the short peptides and proteins reached equilibrium. Finally, the static fluorescence polarization signal in each well was detected using a Cytation 3 (BioTek) multi-mode microplate reader (BioTek Instruments Co., Ltd., China), and the obtained signal values ​​were analyzed using GraphPad PRISM 7.0.

[0062] The results showed that, similar to the model peptide NR, VP5 also binds to the SBD conventional binding pocket of DnaK proteins (L) in a non-ATP state. 1,2 and L 3,4 (Composition) binds to proteins, such as Figure 1A , Figure 1B As shown: the binding curves of VP5 with the full-length DnaK protein (WT) and with SBD are extremely similar, L 1,2 Two important amino acid mutations related to substrate binding (T403GM404G) and L 3,4 Deletion mutations all showed no binding to the short peptides NR and VP5. Furthermore, a second potential substrate-binding region, L... 5,6 The mutation also showed that the mutant protein had a weaker binding affinity to NR and VP5. Figure 1A , Figure 1B Furthermore, through a non-fluorescent (native) short peptide competition experiment, i.e., introducing a 400 μM non-fluorescent short peptide of the other before adding the FITC-labeled short peptide (FITC-short peptide), it was found that both short peptides could effectively shield the binding of the other FITC-short peptide to the protein. Figure 1A , Figure 1B The above results clearly demonstrate that, under non-ATP conditions, VP5 binds to NR short peptides in a similar manner, and the binding mode of VP5 to DnaK protein is conserved. The relevant affinity parameters for the above results are shown in Table 1.

[0063] Table 1

[0064]

[0065] Example 3

[0066] This embodiment analyzes the affinity of the short peptide VP5 for DnaK.

[0067] The affinity between short peptides and DnaK in the ATP state was detected using fluorescence polarization experiments. For detecting the binding kinetics of short peptides to proteins in the non-ATP state: the detection method was the same as in Example 1, except that after serially diluting the protein, ATP (2 mM, Solarbio, A8270, China) was added to the protein dilutions at different concentrations before incubation at room temperature for 2 min before subsequent experimental steps. For detecting the binding kinetics of short peptides to proteins in the ATP state: unlike the method described above for detecting the binding kinetics of short peptides to proteins in the non-ATP state, ATP (2 mM) was added before adding the FITC-short peptide and incubated with the protein for 2 min before subsequent testing.

[0068] As expected, the binding affinity of the short peptide NR to DnaK-ATP decreases by at least two orders of magnitude compared to the protein in its non-ATP state. Figure 2A (Table 2); and the affinity of NR for the DnaK mutant protein PP (which locks the protein conformation in the ATP state) was also consistent with expectations. Figure 2A (Table 2). In contrast, under ATP conditions, the short peptide VP5 still maintains a high affinity for DnaK-ATP ( Figure 2B Table 2 shows that the binding of short peptide VP5 to DnaK-ATP is unique under ATP conditions. Based on the unusually high affinity of short peptide VP5 for DnaK-ATP, we believe that non-fluorescent VP5 under ATP conditions strongly antagonizes the binding of FITC-NR to DnaK. To verify this hypothesis, we further investigated the antagonistic effect between the two short peptides under ATP conditions using a competitive fluorescence polarization experiment. The results showed that 200 μM non-fluorescent VP5 could completely antagonize the binding of FITC-NR to PP protein, while even 400 μM non-fluorescent NR could only reduce the binding of FITC-VP5 to PP protein to a certain extent, and the antagonistic effect was far less than that of VP5 ( Figure 2A , 2B The above experimental results once again fully demonstrate that, unlike the model short peptide NR, the short peptide VP5 can bind to DnaK with high affinity under ATP conditions.

[0069] Table 2

[0070]

[0071] Next, we further analyzed the binding characteristics of the short peptide VP5 to DnaK-ATP and PP mutations from a kinetic perspective. Figure 2CAs shown, through fluorescence polarization kinetics, we found that under ATP conditions, the model peptide NR binds relatively quickly to DnaK-ATP, and the instrument cannot capture the dynamic changes in signal value. Conversely, the binding kinetics of the short peptide VP5 with the above two proteins is relatively slow, and the dynamic change trend of signal value is relatively obvious. Furthermore, the endpoint fluorescence polarization value of the short peptide's kinetic detection results also differs from... Figure 2A , Figure 2B The results were consistent. These experiments further demonstrate, from the perspective of binding kinetics, that the binding of VP5 to DnaK-ATP has unique characteristics.

[0072] Example 4

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

[0074] The effect of short peptide VP5 on DnaK activity was tested by in vitro denaturing luciferase refolding assay.

[0075] Experimental materials for the denatured luciferase renaturation assay: 1) Buffer A: containing 25 mM Hepes-KOH (pH 7.5), 100 mM KCl, 10 mM Mg(OAC)2 and 2 mM DTT; 2) Refolding buffer: Buffer A with 3 mM ATP added.

[0076] Experimental protocol for detecting the renaturation of denatured luciferase:

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

[0078] b) Prepare the luciferase for the experiment:

[0079] i. Preparation of denatured luciferase (for negative control and experimental groups): Heat in refolding buffer at 42°C (recommended for approximately 10-20 minutes) to denature the luciferase protein. After denaturation, allow to stand at room temperature.

[0080] ii. Preparation of undenatured luciferase (for positive control): Dilute the luciferase stock solution 10-fold with refolding buffer and incubate at room temperature;

[0081] c) Prepare the reaction system

[0082] i. Positive control: Add 3 μL of the above undenatured luciferase to 30 μL of refolding buffer and perform detection immediately;

[0083] ii. Negative control: Add 3 μL of the above denatured luciferase to 30 μL of refolding buffer and perform detection immediately;

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

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

[0086] d) Testing system

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

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

[0089] Further investigation was conducted to determine whether VP5 also possesses the ability to penetrate the cell membrane of E. coli.

[0090] A single colony of wild-type Escherichia coli (E. coli ATCC 25922) was inoculated into LB broth and incubated overnight at 37°C and 200 rpm. The next day, fresh bacterial culture was inoculated at a ratio of 100× and incubated again until OD reached [the desired growth rate]. 600= Approximately 0.55. Aliquot 1 mL of bacterial culture into a 1.5 mL centrifuge tube, centrifuge at 1500 × g, 4 °C, for 5 min, and discard the supernatant. Wash the cells with 1 × PBS, centrifuge at 1500 g, 4 °C, for 5 min, and discard the supernatant. Resuspend the cells in Solution A (Takara, main component CaCl2), place on ice for 10–20 min, centrifuge at 1500 × g, 4 °C, for 5 min, discard the supernatant, resuspend the bacteria in LB filtrate, and dilute for later use. Add FITC-short peptide to the above bacterial culture to a working concentration of 30 μM, and incubate in the dark for 30 min. After the reaction is complete, centrifuge at 8000 rpm at room temperature for 3 min, discard the supernatant, then wash the cells thoroughly with PBS, and finally dissolve them in an appropriate volume of PBS solution for fluorescence microscopy. Use the blue channel (470 / 490) and a magnification of 600x for fluorescence microscopy.

[0091] The results show ( Figure 3B Unlike the model short peptide NR, VP5 can penetrate the cell membrane and enter the interior of E. coli: E. coli incubated with FITC-NR, after washing with PBS, could not be observed under a fluorescence microscope with fluorescently labeled E. coli cells; conversely, a large number of fluorescently labeled E. coli cells remained in the FITC-VP5 group. Based on this experiment, we preliminarily conclude that VP5 has the ability to penetrate the cell membrane of E. coli.

[0092] Observation using fluorescence microscopy initially determined that the short peptide VP5 has good ability to penetrate the cell membrane of E. coli. In order to fundamentally reveal the reason for the cell membrane penetration of the short peptide VP5, 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 the cell membrane penetration of VP5.

[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 Biotech Co., Ltd., with a purity greater than 95%. The initial sample preparation process was consistent with the sample preparation process described above for the assay of ability to penetrate the *E. coli* cell membrane. The difference was that after dissolving the samples in an appropriate volume of PBS solution, the membrane penetration efficiency of the short peptides was quantitatively analyzed using flow cytometry. During flow cytometry analysis, the FITC channel was selected, and the voltage was set to 407V.

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

[0097] Further investigation was conducted to determine whether VP5 could penetrate the cell membranes of Gram-positive bacteria with higher peptidoglycan content. Similar to the methods described above, Staphylococcus aureus (BMZ137680) was used as the research subject. Fluorescence microscopy observations showed that ( Figure 5A When Staphylococcus aureus cells were co-incubated with FITC-VP5 for a period of time, most cells emitted fluorescence, indicating that VP5's membrane-penetrating ability is not limited to Gram-negative bacteria. Furthermore, quantitative analysis using flow cytometry showed that the short peptide VP5 penetrated approximately 75% of Staphylococcus aureus cells (e.g., ...). Figure 5B (As shown). In summary, our experiments have once again demonstrated that VP5 is a short peptide with superior membrane-penetrating ability. Unlike small molecules such as antibiotics that are passively transported into the bacterial interior, VP5 once again shows its application potential of crossing the membrane to enter the bacterial interior and thus exert its function.

[0098] Example 5

[0099] This embodiment further analyzes whether VP5, after penetrating the membrane and entering the interior of bacteria, will damage the integrity of the bacterial cell membrane.

[0100] The assay was validated using propidium iodide fluorescent dye (Beyotime, catalog number: ST511). Preliminary bacterial cell treatment followed the same protocol as the membrane penetration assay described above. 1) Propidium iodide staining positive control group treatment: The bacterial culture was treated with 70% isopropanol solution and incubated at 39°C for 30 min. The treated sample was then centrifuged, washed with 1×PBS, and resuspended. Afterward, it was incubated in the dark with 10 μL propidium iodide (working concentration 50 μg / mL) for 15 min, washed with 1×PBS, and resuspended. 5 μL was transferred to a glass slide and observed using a Texas Red (595 / 613) fluorescence microscope. 2) VP5 group treatment: 30 μM and 120 μM short peptides were added, and the mixture was incubated at 37°C for 60 min. Then, the same propidium iodide (working concentration 50 μg / mL) labeling, washing, and observation procedures as the positive group were performed.

[0101] Propidine iodide can penetrate damaged cell membranes and bind to intracellular nucleic acid substances (DNA and RNA), emitting red fluorescence under a fluorescence microscope. Figure 6Microscopic observation revealed that when different concentrations (30 μM, 120 μM) of FITC-VP5 were incubated with *E. coli* cells at 37°C for a certain period, fluorescently labeled *E. coli* cells could be detected via the green fluorescence channel, while the red fluorescence channel failed to capture clearly labeled *E. coli* cells. These experimental results indicate that VP5 can penetrate the cell membrane without disrupting its integrity, a mechanism of action different from most antimicrobial peptides, demonstrating its unique properties.

[0102] Example 6

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

[0104] A single colony of wild-type Escherichia coli (E. coli ATCC 25922) was inoculated into LB broth and incubated overnight at 37°C and 200 rpm. The next day, fresh bacterial culture was inoculated at a ratio of 100× and incubated again until OD reached [the desired growth rate]. 600 = Approximately 0.55. Dilute the bacterial culture with LB to OD. 600 =0.2% was used as the stock solution and diluted 10-fold to the working concentration. Then, different working concentrations of VP5 (40μM, 80μM, 160μM, 320μM) were added to 96-well plates containing bacterial culture, with the VP5-free group serving as a positive control. The bacterial cultures in the 96-well plates were then statically incubated at 37℃ and 42℃ for 16–20 hours, respectively. The absorbance (OD) of the bacterial culture in the 96-well plates was then measured using a SynergyHTX multi-mode microplate reader. 600 ).

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

[0106] Example 7

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

[0108] Based on the model *E. coli*, several antibiotic resistance mechanisms were simulated, and the synergistic antibacterial ability of VP5 was tested. For initial bacterial culture and preparation, refer to Example 6. The prepared bacterial cells were inoculated at 200 μL per well in a 96-well plate and cultured overnight at 37°C. For the antibiotic group and the VP5 synergistic antibiotic group, the corresponding working concentration of short peptide and antibiotic solution was added and mixed before further culture. After mixing, the 96-well plate was transferred to a 37°C biochemical incubator and cultured for approximately 15 hours. After culture, the absorbance (OD) in each well was measured using a Synergy HTX multi-mode microplate reader. 600 ).

[0109] The tested antibiotics included aminoglycosides, macrolides, and tetracyclines. Among these, aminoglycoside antibiotics often fail to penetrate the bacterial interior due to mutations in bacterial membrane porins (e.g., kanamycin). + Gentamicin GM + Macrolide antibiotics (azithromycin AMZ) + ) and tetracycline antibiotics (tetracycline TC) + Antibiotic resistance often results from the overexpression of efflux pump proteins on bacterial cell membranes, causing large amounts of antibiotics to be pumped out of the bacteria, preventing their accumulation within the bacteria. The result is... Figure 8 As shown, we first screened different concentrations of antibiotics that could cause approximately half of E. coli to die (simulating a simple antibiotic resistance scenario), and found that in Kana... + 15 μg / mL Figure 8 (Figure A in the middle), GM + 2.5 μg / mL Figure 8 (C diagram), AMZ + 10 μg / mL Figure 8 (China E-map), TC + 1 μg / mL Figure 8 The G-plot (or similar diagram) can meet our needs. Secondly, after determining the low-efficiency working concentrations of different antibiotics, we added VP5 short peptides at different concentration gradients during bacterial growth to test the synergistic antibacterial activity of VP5 with low-concentration antibiotics, such as... Figure 8 As shown in Figures B, D, F, and H, adding different concentrations (down to 40 μM) of VP5 can achieve complete inhibition of E. coli growth, while simultaneously... Figure 7The results showed that the minimum antibacterial activity of VP5 alone (40-120 μM) at 37℃ was only about 45%, indicating that the antibacterial effect of VP5 alone was limited. However, when it was added to an environment with low concentrations of antibiotics (bacterial viability above 40%), the overall antibacterial effect was significantly improved, and the bacterial viability dropped to below 10%. This indicates that VP5 not only has a certain antibacterial function, but it can also break the drug resistance mechanism (antibiotics cannot enter bacteria efficiently or cannot accumulate at high concentrations inside bacteria), and restore the antibacterial effect of antibiotics. This proves the potential of this short peptide as a novel synergistic antibiotic adjuvant for Escherichia coli. The experimental results also further demonstrate the reality of the short peptide VP5 exerting a synergistic antibacterial function by targeting the important antibacterial target DnaK.

[0110] Example 8

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

[0112] Using a multidrug-resistant strain of *Escherichia coli* (Shanghai Center for Microbiology, D24652, resistant to tetracycline, chloramphenicol, streptomycin, and spectinomycin) as the test subject, the inhibitory effect of short peptide VP5 synergistic heat shock on its growth was investigated. The detection method is the same as in Example 7, except that the strain cultured this time was a multidrug-resistant *Escherichia coli*.

[0113] turn out( Figure 9A At 37°C, 320 μM VP5 could inhibit the growth of multidrug-resistant bacteria by half; under heat shock conditions, 160 μM VP5 could almost completely inhibit the growth of drug-resistant bacteria. Furthermore, we investigated the synergistic effect of VP5 with tetracycline (TC). + ), spectinomycin (STM) + Streptomycin (SM) + ), chloramphenicol (CAP) + The inhibitory effect of VP5 on the growth of multidrug-resistant Escherichia coli was observed when 20 μM VP5 was used in combination with 50 μg / mL of various antibiotics. Figure 9B 20 μM VP5 can synergistically inhibit the growth of multidrug-resistant Escherichia coli with 50 μg / mL tetracycline and spectinomycin, but due to the complexity of antibiotic resistance mechanisms, the antibacterial effect of VP5 synergistically with streptomycin and chloramphenicol is not significant. Furthermore, we also found ( Figure 9C40 μM VP5, in combination with 10 μg / mL tetracycline (the normal working concentration for non-drug-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 bacterium, VP5 alone is not ideal, requiring higher concentrations of short peptides to achieve antibacterial effects. However, when combined with heat shock conditions (simulating bacterial growth under stress) or with low concentrations of antibiotics (especially tetracycline and spectinomycin), the inhibitory effect of VP5 on multidrug-resistant Escherichia coli is significantly enhanced. These experimental results further demonstrate the high compatibility and rationality of VP5 as a synergistic antibiotic adjuvant for Escherichia coli, rather than its ability to exert highly effective antibacterial functions alone as a traditional antimicrobial peptide.

[0114] Example 9

[0115] This embodiment tests VP5 cytotoxicity.

[0116] Using HEK293 human embryonic kidney cells, a commonly used laboratory cell line, as the test subject, the effect of different concentrations of VP5 short peptide on cell growth was investigated. HEK293 cells in the logarithmic growth phase were collected, and the accurate cell density was determined by counting using a hemocytometer. The cells were then diluted to a specific density using fresh DMEM medium (10% fetal bovine serum, FBS). This cell suspension was seeded into 96-well plates (10,000 cells / well) and cultured overnight at 37°C with 5% CO2 until complete adhesion. The next day, the medium was replaced with fresh DMEM medium (10% fetal bovine serum, FBS), and different concentrations of puromycin (Beyotime Biotechnology Co., Ltd., ST551) or VP5 short peptide were added to the medium. The cells were then cultured for another 2 days at 37°C with 5% CO2. Positive controls (normally cultured cells, without the tested antibiotics and VP5) and background control wells (containing only cell culture medium and the different test drugs, without cells) were also set up. After culturing, the cell culture medium in the 96-well plate was completely aspirated, and fresh cell culture medium (containing 10% CCK8 reaction solution, purchased from Beyotime Biotechnology Co., Ltd., C0037) was immediately added. The plate was then incubated at 37°C with 5% CO2 for 1 hour. The absorbance (OD) in each well was then measured using a Synergy HTX multi-mode microplate reader. 450 The cell growth inhibition curves caused by the drug were analyzed and plotted using PRISM 7.0 (GraphPad).

[0117] The results are as follows Figure 10As shown, compared to puromycin (PURO)'s highly effective inhibition of HEK293 cell growth, VP5 has very little effect on the growth of this eukaryotic cell. Even at 320 μM, VP5 can still maintain HEK293 cell viability above 90%. This indicates that the VP5 short peptide, when used as a synergistic antibiotic adjuvant to address antibiotic resistance, has a certain degree of safety, further demonstrating the promising application prospects of this short peptide.

[0118] In summary, this invention has conducted in-depth research and screening to obtain a short peptide that specifically binds to the bacterial Hsp70s protein. It was discovered that this peptide binds to the Hsp70s protein through a traditional substrate-binding pocket, but exhibits an unusually high affinity for the protein in the DnaK-ATP state. The short peptide can inhibit Hsp70s protein activity to a certain extent, preventing it from aiding in the renaturation of denatured luciferase. Furthermore, while maintaining the integrity of the bacterial cell membrane, this short peptide possesses superior ability to cross the bacterial cell membrane. Its application potential in the preparation of delivery vectors, the development of antibacterial drugs, and the preparation of antibiotic adjuvants is further discussed, providing new ideas for solving current problems such as bacterial resistance and the difficulty in discovering new antibiotics.

[0119] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. Use of a polypeptide for the manufacture of a transmembrane polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.

3.

2. Use of the transmembrane polypeptide according to claim 1 in the preparation of a delivery carrier.

3. Use of the transmembrane polypeptide according to claim 1 in the preparation of an antibiotic adjuvant, the antibiotic being tetracycline, spectinomycin, kanamycin, gentamicin and azithromycin.

4. A drug resistant bacteria inhibitor, characterized by, The drug-resistant bacterial inhibitor comprises an antibiotic; and the delivery carrier according to claim 2 or the antibiotic adjuvant according to claim 3; The antibiotic in the drug-resistant bacterial inhibitor is tetracycline, spectinomycin, kanamycin, gentamicin and azithromycin; and the drug-resistant bacteria are drug-resistant Escherichia coli and / or Staphylococcus aureus.

5. The drug resistant bacteriostatic agent of claim 4, wherein, The drug-resistant bacterial inhibitor is a compound preparation.

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