Use of bacterial membrane dCACHE-type chemotaxis receptor protein family as target in preparation or screening of anti-multiple drug resistant bacteria drugs

Abstract

This invention discloses the use of a bacterial membrane dCACHE-type chemokine receptor protein family as a target in the preparation or screening of drugs against multidrug-resistant bacteria, belonging to the field of biomedical technology. This invention reveals and verifies for the first time the core regulatory mechanism of the dCACHE-type chemokine receptor protein family in the bacterial infection process: these proteins are key membrane surface receptors mediating bacterial chemotaxis towards macrophages; their loss of function can directly block bacterial chemotactic movement and significantly reduce pathogenicity. This invention, by targeting the conserved ligand binding interface, successfully inhibited the infectivity of drug-resistant pathogens such as *Yersinia pseudotuberculosis*, carbapenem-resistant *Acinetobacter baumannii*, and carbapenem-resistant *Escherichia coli*. The novel targeting strategy provided by this invention not only effectively overcomes the clinical bottleneck of "no available drugs" for carbapenem-resistant multidrug-resistant bacteria but also constructs a broad-spectrum protective barrier covering the entire homologous protein family, possessing extremely high clinical translational value and commercial development potential.

Description

Technical Field

[0001] This invention relates to a novel use of a family of bacterial membrane chemokine receptor proteins, and more particularly to the application of this dCACHE-type chemokine receptor protein family with specific functional characteristics as drug targets against multidrug-resistant bacteria. This invention belongs to the field of biomedical technology. Background Technology

[0002] Multidrug-resistant bacterial infections pose a major and serious challenge to global public health. Among them, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, and carbapenem-resistant Pseudomonas aeruginosa are prone to causing severe infections due to their strong drug resistance and wide transmission capabilities, leading to a significant increase in patient mortality and posing a great challenge to clinical treatment.

[0003] Currently used antimicrobial drugs in clinical practice mostly target classic targets such as bacterial cell wall synthesis, nucleic acid replication, and protein synthesis. Long-term and widespread use of these drugs easily induces bacterial resistance, and the development of novel antimicrobial drugs lags far behind the evolutionary rate of drug-resistant bacteria. Therefore, discovering entirely new and specific families of antimicrobial drug targets and developing novel treatment strategies against multidrug-resistant bacteria has become a key to solving the antimicrobial resistance crisis.

[0004] Bacterial chemokine receptor proteins are key transmembrane receptors on the bacterial cell membrane surface that sense environmental signals and mediate chemotaxis, participating in multiple physiological processes such as bacterial motility, colonization, biofilm formation, and infection. dCACHE-type chemokine receptors, short for Calcium Channels and Chemotaxis Receptors, are crucial sensing modules in the prokaryotic chemotactic signaling system and belong to a special branch of the chemokine family. Their core function is as "environmental sensors" for bacteria, sensing extracellular / intracellular signaling molecules through their N-terminal cache domain, triggering chemotactic behavior, and helping microorganisms move towards nutrient sources and avoid harmful stimuli. They are widely distributed in prokaryotes such as bacteria and archaea. However, the mechanisms by which the highly homologous and functionally conserved bacterial membrane dCACHE-type chemokine receptor protein family mediates bacterial chemotaxis towards macrophages and influences pathogenicity, as well as its potential as drug targets against multidrug-resistant bacteria, have not yet been fully explored and confirmed. Summary of the Invention

[0005] The purpose of this invention is to provide the use of bacterial membrane dCACHE-type chemokine receptor protein family as drug targets in the preparation or screening of drugs against multidrug-resistant bacteria. This invention solves the technical problems of existing antibacterial drugs having a lack of targets and being prone to inducing drug resistance, and provides a novel and specific target strategy and drug development direction for the treatment of multidrug-resistant bacterial infections.

[0006] To achieve the above objectives, the present invention employs the following technical means:

[0007] This invention provides the use of the bacterial membrane dCACHE-type chemokine receptor protein family as drug targets in the preparation or screening of drugs against multidrug-resistant bacteria; the bacterial membrane dCACHE-type chemokine receptor protein family refers to the dCACHE-type chemokine receptor protein family that mediates chemotaxis on the surface of bacterial cell membranes, and family members meet at least one of the following conditions:

[0008] (a) An amino acid sequence containing more than 70% sequence identity with the conserved motif shown in SEQ ID NO:1;

[0009] (b) Contains a conserved ligand-binding interface, wherein the conserved ligand-binding interface comprises at least a positively charged amino acid residue at the entrance of the binding pocket, an aromatic amino acid residue at the hydrophobic core, and a negatively charged amino acid residue at the bottom of the binding interface; wherein the positively charged amino acid residue is structurally and functionally equivalent to arginine at position 120 (R120) of Yersinia pseudotuberculosis YcrA protein (UniProt ID: A0A0H3B5X1, NCBI Protein ID: ACA69109), wherein the aromatic amino acid residue is structurally and functionally equivalent to tryptophan at position 122 (W122) and tyrosine at position 138 (Y138), and wherein the negatively charged amino acid residue is structurally and functionally equivalent to aspartic acid at position 140 (D140) and aspartic acid at position 167 (D167); wherein “functional equivalence” means that the residue can form the same type of interaction with the host ligand, and that mutating it to the specific residue does not significantly reduce the ligand-binding affinity by more than 10-fold.

[0010] Preferably, the multidrug-resistant bacteria include, but are not limited to, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and Yersinia pseudotuberculosis.

[0011] Preferably, the drug blocks the chemotactic movement of bacteria to macrophages by binding to any member of the dCACHE-type chemokine receptor protein family, thereby reducing bacterial pathogenicity and infectivity.

[0012] Preferably, the drug inhibits bacterial chemotaxis towards macrophages, biofilm formation, and infection processes by suppressing the function of the dCACHE-type chemokine receptor protein family.

[0013] Preferably, the drug is a small molecule compound, antibody, peptide, or nucleic acid drug that targets the dCACHE-type chemokine receptor protein family.

[0014] Furthermore, the present invention also proposes an antibacterial polypeptide that targets the bacterial membrane dCACHE-type chemokine receptor protein family, the amino acid sequence of which is shown in SEQ ID NO:2.

[0015] Preferably, the polypeptide binds specifically to the Yersinia pseudotuberculosis dCACHE-type chemokine receptor YcrA (UniProtID: A0A0H3B5X1, NCBI Protein ID: ACA69109) with high affinity, and its binding constant K d The value is 0.015 μM, which is much higher than the binding constant of YcrA with the host ligand.

[0016] Furthermore, the present invention also proposes the application of the aforementioned antibacterial polypeptide in the preparation of antibacterial drugs.

[0017] Preferably, the bacteria are multidrug-resistant bacteria, including but not limited to carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and Yersinia pseudotuberculosis.

[0018] Furthermore, the present invention also proposes a method for screening drugs against multidrug-resistant bacteria, which targets the bacterial membrane dCACHE-type chemokine receptor protein family and screens for active substances that can bind to or inhibit the function of any member of the family. Preferably, the multidrug-resistant bacteria include, but are not limited to, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and Yersinia pseudotuberculosis.

[0019] Finally, the present invention also proposes a method for inhibiting multidrug-resistant bacteria for non-therapeutic purposes. This method involves inhibiting the function of any member of the bacterial membrane dCACHE-type chemotactic receptor protein family, thereby blocking bacterial chemotaxis towards macrophages and inhibiting multidrug-resistant bacterial infection. Preferably, the multidrug-resistant bacteria include, but are not limited to, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and Yersinia pseudotuberculosis.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) A novel pathogenic mechanism was revealed for the first time, with outstanding innovative target.

[0022] This invention is the first to discover and demonstrate that the bacterial membrane dCACHE-type chemokine receptor protein family can mediate bacterial chemotaxis towards macrophages and induce macrophage apoptosis, serving as a key factor in regulating bacterial pathogenicity. This mechanism differs from traditional antimicrobial drug pathways, providing a novel target and intervention strategy for combating multidrug-resistant bacterial infections.

[0023] (2) The target family is highly conserved, with a broad antibacterial spectrum and wide applicability.

[0024] The bacterial membrane dCACHE-type chemokine receptor protein family is functionally conserved in multidrug-resistant bacteria such as carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and Yersinia pseudotuberculosis. Based on sequence and binding site conservation analysis, it is also highly conserved in pathogens with homologous conserved interfaces, such as carbapenem-resistant Pseudomonas aeruginosa. Targeting this family can achieve broad-spectrum intervention against a variety of high-risk drug-resistant bacteria in clinical practice, breaking through the limitations of single strains and demonstrating significant clinical application value.

[0025] (3) It has a unique mechanism of action and is not easy to induce bacterial resistance.

[0026] This invention reduces pathogenicity by blocking bacterial chemotaxis and host cell apoptosis pathways, without directly affecting the metabolic processes essential for bacterial survival. It is less likely to induce bacterial drug resistance mutations, can effectively alleviate the clinical drug resistance crisis, and has the potential for long-term therapeutic application.

[0027] (4) The target has strong druggability, supporting the development of multiple types of drugs.

[0028] The dCACHE family of chemokine receptor proteins is located on the surface of bacterial cell membranes and is easily targeted and bound by drugs. It can be adapted to various drug forms such as small molecule compounds, peptides, antibodies, and nucleic acids, providing a universal target platform for the screening, design, and development of drugs against various types of drug-resistant bacteria. It has extremely high drug development potential and commercialization value. Attached Figure Description

[0029] Figure 1 The bacterial membrane dCACHE-type chemotactic receptor protein mediates the chemotaxis of Yersinia pseudotuberculosis to macrophages;

[0030] Figure 2 The adhesion of Yersinia pseudotuberculosis to macrophages is mediated by the bacterial membrane dCACHE-type chemotactic receptor protein.

[0031] Figure 3 The effect of bacterial membrane dCACHE-type chemokine receptor protein on mouse survival;

[0032] Figure 4 This is a schematic diagram of the structure of the conserved ligand binding interface described in this invention, showing the spatial arrangement of the conserved amino acid motifs (R120, W122, Y138, D140, D167) in the binding pocket: R120 is located at the entrance (positive charge), W122 and Y138 are located at the hydrophobic core (aromatic), and D140 and D167 are located at the bottom (negative charge).

[0033] Figure 5A and Figure 5BThis is a diagram showing the distribution and conservation of the conserved ligand binding interface described in this invention in various pathogens; wherein, Figure 5A The results of phylogenetic analysis of YcrA homologous proteins in different bacterial species. Figure 5B The results of multiple sequence alignment of key conserved amino acid residues (R120, W122, Y138, D140, D167) in representative pathogenic bacteria;

[0034] Figure 6 The figure shows the results of detecting the binding ability of the Yersinia pseudotuberculosis dCACHE-type chemotactic receptor YcrA to the host ligand and the antimicrobial peptide of the present invention by isothermal titration calorimetry (ITC).

[0035] Figure 7 The figure shows the results of the Transwell assay used to detect the effect of the antimicrobial peptide of the present invention on the chemotactic migration ability of Yersinia pseudotuberculosis to host cells.

[0036] Figure 8 The figure shows the results of the apoptosis assay to detect the cytotoxic effects of the antimicrobial peptides of this invention on macrophages infected with Yersinia pseudotuberculosis, carbapenem-resistant multidrug-resistant Escherichia coli, and carbapenem-resistant Acinetobacter baumannii.

[0037] Figure 9 Survival curves of mice infected with Yersinia pseudotuberculosis, carbapenem-resistant multidrug-resistant Escherichia coli, or carbapenem-resistant Acinetobacter baumannii after treatment with the antimicrobial peptides of this invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] Example 1: Functional Verification of the Bacterial Membrane dCACHE-type Chemokinetic Receptor Protein Family

[0040] 1. Bacterial membrane dCACHE-type chemotactic receptor protein mediates chemotaxis of Yersinia pseudotuberculosis to macrophages.

[0041] Homologous recombination technology was used to construct a knockout strain (ΔycrA(Vector)) of the dCACHE-type chemokine receptor protein family gene (named ycrA, UniProt ID: A0A0H3B5X1, NCBI Protein ID: ACA69109) of *Yersinia pseudotuberculosis* and its complement strain (ΔycrA(ycrA)). Transwell experiments were performed, with wild-type *Yersinia pseudotuberculosis* transfected with an empty vector (Vector) (WT(vector)) as a control. RAW 264.7 macrophages were seeded in 12-well plates at 2.0 × 10⁶ cells per well. 5 Cells were co-cultured for 48 hours to form a confluent monolayer. Overnight-cultured *Yersinia pseudotuberculosis* strains were diluted 1:100 in YLB agar and added to the upper chamber of a Transwell apparatus containing a 5.0 μm pore size polyethylene terephthalate (PET) membrane; the lower chamber contained only sterile cell culture medium (cell-free control) to assess baseline bacterial migration. After incubation at 37°C and 5% CO2 for 2 hours, the contents of the lower chamber were collected and plated onto YLB agar. After incubation at 30°C for 24 hours, colony counts were performed. The final colony count was corrected by subtracting the number of bacteria that migrated to the cell-free control.

[0042] Experimental results are as follows Figure 1 As shown, mutants lacking the ycrA gene completely lose their chemotactic ability, and the phenotype can be restored in the complementation experiment, indicating that YcrA (UniProt ID: A0A0H3B5X1, NCBI Protein ID: ACA69109) is a key chemotactic receptor for bacteria to move toward host cells.

[0043] 2. Adhesion of Yersinia pseudotuberculosis to macrophages mediated by bacterial membrane dCACHE-type chemotactic receptor protein.

[0044] To conduct quantitative adhesion experiments, mammalian cells were spaced at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 cells / well in 12-well plates. Monolayers were washed with PBS and maintained in antibiotic-free DMEM medium. Overnight cultured *Yersinia pseudotuberculosis* membrane dCACHE-type chemokine receptor protein family gene knockout strain ycrA (ΔycrA) and wild-type *Yersinia pseudotuberculosis* (WT) were resuspended in PBS and added to the cells at a 100:1 MOI. After incubation at 37°C for 1 h, non-adhesive bacteria were removed by washing with PBS. Cells were lysed with 1 ml of 0.1% Triton X-100 at room temperature for 5–10 min. The lysate was spread on YLB agar plates and incubated at 30°C for 24 h before colony counting.

[0045] For adhesion experiments in microscopic imaging, RAW 264.7 macrophages were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 g / cm³ in confocal culture dishes (Biosharp). Yersinia pseudotuberculosis membrane dCACHE-type chemokine receptor protein family gene ycrA knockout strain (ΔycrA) and wild-type Yersinia pseudotuberculosis (WT) were infected with the cells at an MOI of 100. After 30 min at 37°C, host cells were labeled with CellTracker™ Deep Red (Thermo Fisher Scientific) according to the manufacturer's instructions and incubated for another 30 min. The monolayer membrane was thoroughly washed with PBS and immediately imaged on a rotating disk confocal microscope (Andor Revolution XD).

[0046] Experimental results are as follows Figure 2 As shown, compared to the wild-type strain, the number of *Yersinia pseudotuberculosis* strains lacking the YcrA chemotactic protein that adhered to macrophages was significantly reduced, indicating that YcrA affects the adhesion of *Yersinia pseudotuberculosis* to macrophages.

[0047] 3. Bacterial membrane dCACHE-type chemokine receptor protein affects mouse survival rate.

[0048] (1) All mice were maintained and treated in accordance with the animal welfare guarantee policy promulgated by Northwest A&F University. The mice were 6-week-old SPF-grade female BALB / c mice. After purchasing the mice, they were raised for 2-3 days before infection. The ycrA knockout strain of the dCACHE type chemokine receptor protein family gene of Yersinia pseudotuberculosis (ΔycrA) and wild-type Yersinia pseudotuberculosis (WT) were cultured in YLB medium at 26°C until the initial stable stage, washed in sterile PBS and diluted to 1×10. 7 A bacterial suspension of *Yersinia pseudotuberculosis* was prepared at a concentration of *C.* / mL. Different groups were established, with 10 mice in each group. (i) Mice were intraperitoneally injected with wild-type *Yersinia pseudotuberculosis* strain (WT); (ii) Mice were intraperitoneally injected with a knockout strain of the *Yersinia pseudotuberculosis* membrane chemotactic receptor protein family gene ycrA (ΔycrA); (iii) Mice were injected with PBS as a blank control. Infected mice were observed, and the survival status of each infection group was statistically compared. The observation period was 6 days, and survival curves were plotted.

[0049] Experimental results are as follows Figure 3 As shown, mice infected with the ycrA knockout strain (ΔycrA) of the dCACHE chemokine family gene of Yersinia pseudotuberculosis showed a significant survival advantage compared to mice infected with the wild-type strain (WT), indicating that YcrA is a key protein that enhances bacterial pathogenicity.

[0050] In summary, the bacterial membrane dCACHE-type chemokine receptor protein family is a key protein family that mediates bacterial chemotaxis, adhesion to macrophages, and enhances bacterial pathogenicity.

[0051] Example 2: Analysis of the widespread and conserved nature of conserved ligand binding interfaces of the dCACHE-type chemokine receptor protein family in bacterial membranes

[0052] This invention reveals and verifies for the first time that the conserved ligand-binding interface in bacterial dCACHE-type chemokine receptor proteins is a key functional site for signal recognition in the chemotactic systems of various pathogenic bacteria. Represented by the Yersinia pseudotuberculosis dCACHE-type chemokine receptor protein (named YcrA, UniProt ID: A0A0H3B5X1, NCBI Protein ID: ACA69109), this receptor mediates chemotactic directional movement of bacteria by sensing host ligands, and is a key molecule for bacteria to lock onto and move toward host cells.

[0053] The conserved ligand-binding interface comprises a conserved amino acid motif as shown in SEQ ID NO:1 (TGRPWYLQAVNAGKPVVTPPYIDAGTQQLVVTFAWSIVQDGVLKGVIAADVT). Specifically, this sequence contains five key amino acid residues corresponding to arginine (R120), tryptophan (W122), tyrosine (Y138), aspartic acid (D140), and aspartic acid (D167) at position 120 of the complete YcrA amino acid sequence. This motif is highly conserved in dCACHE-type chemokine receptors of various pathogens, constituting the core functional interface for chemokine-host ligand binding. Figure 4 ).

[0054] To verify the widespread distribution and high conservation of this conserved binding interface in various pathogens, this invention used BLASTP software to search for homologous proteins of YcrA in the NCBI non-redundant protein database, and randomly selected 200 homologous proteins from different species for phylogenetic analysis. Multiple sequence alignment was performed using ClustalW in MEGA software, and a phylogenetic tree was constructed based on the Jones-Taylor-Thornton model using the maximum likelihood method. The results were visualized using the ChiPlot web server. The results are as follows: Figure 5A As shown, homologous proteins of YcrA are widely distributed in different species of bacteria, including a variety of pathogens. Among them, pathogens of the Enterobacterial and Pseudomonas orders (such as Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa) contain highly homologous dCACHE-type chemotactic receptors.

[0055] Furthermore, this invention randomly selects pathogenic bacteria from different species in the phylogenetic tree, uses multiple sequence alignment analysis to determine the conservation of key amino acid residues (R120, W122, Y138, D140, D167), and calculates the sequence identity of each species with SEQ ID NO:1. The results are as follows: Figure 5B As shown, the five key amino acid residues mentioned above are highly conserved in various pathogenic bacteria, including *Yersinia pestis*, *Acinetobacter baumannii*, *Escherichia coli*, and *Pseudomonas aeruginosa*. In the homologous proteins of these bacteria, arginine at position 120 (R120), tryptophan at position 122 (W122), tyrosine at position 138 (Y138), aspartic acid at position 140 (D140), and aspartic acid at position 167 (D167) are strictly conserved at their corresponding positions, consistent with the functional positions of the *Yersinia pseudotuberculosis* YcrA protein. Sequence identity analysis results showed that *Yersinia pestis* had 100% identity with SEQ ID NO:1, *Escherichia coli* 72%, *Pseudomonas aeruginosa* 65%, and *Acinetobacter baumannii* 61%. Although Acinetobacter baumannii has low overall identity, its five key residues are strictly conserved. Furthermore, the functional effectiveness of this bacterium has been verified in Example 4 of this invention, indicating that the function of the conserved ligand binding interface does not depend on the high overall sequence identity, but rather on the chemical properties and spatial arrangement of these five key residues.

[0056] Example 3: Design and synthesis of specific antimicrobial peptides targeting the bacterial membrane dCACHE-type chemokine receptor protein family

[0057] Based on the conserved ligand-binding interface determined in Example 2 (containing five key residues: R120, W122, Y138, D140, and D167, as well as the conserved motif shown in SEQ ID NO:1), this invention utilizes computer-aided drug design to obtain an antimicrobial polypeptide capable of binding to the conserved ligand-binding interface with high affinity. Its amino acid sequence is shown in SEQ ID NO:2 (GKKYYTIGKYDYEK). This antimicrobial polypeptide was synthesized by Hefei Peptide Library Biotechnology Co., Ltd.

[0058] Example 4: Validation of the inhibitory activity of specific antimicrobial peptides targeting the bacterial membrane dCACHE-type chemokine receptor protein family

[0059] 1. Isothermal titration calorimetry (ITC) for detecting the binding affinity of antimicrobial peptides to the bacterial membrane dCACHE-type chemokine receptor.

[0060] The specific operating steps are as follows: The antimicrobial peptide (SEQ ID NO:2) and the purified dCACHE-type chemokine receptor (Yersinia pseudotuberculosis YcrA) were diluted in ITC buffer (50 mM Tris, 150 mM NaCl, 10% glycerol, pH 8.0). The antimicrobial peptide or host ligand was diluted to 100 μM, and the chemokine receptor to 10 μM. The sample was degassed for 15 min before titration. After degassed, 250 μL of the peptide or host ligand was aspirated into the titration syringe, and 1 mL of the chemokine was added to the reaction chamber. Each reaction was titrated 25 times under stirring at 200 rpm at 25°C. Each sample underwent three independent experiments. Protein dialysis buffer was used as a control. The results were analyzed using Nano Analyze software, and the affinity constant K was calculated. d .

[0061] Test results as follows Figure 6 As shown, the antimicrobial peptide of the present invention can bind specifically to the Yersinia pseudotuberculosis dCACHE-type chemotactic receptor YcrA with high affinity, and its binding constant K d The concentration was 0.015 μM, which is much higher than the binding constant of YcrA with the host ligand (0.17 μM). This result indicates that the antimicrobial peptide of the present invention can efficiently and competitively bind to the conserved ligand-binding interface of YcrA, blocking its interaction with the host ligand, thereby interfering with the signal recognition of the bacterial chemotactic system.

[0062] 2. Detection of the effect of specific antimicrobial peptides targeting the bacterial membrane dCACHE-type chemokine receptor protein family on bacterial chemotaxis

[0063] RAW 264.7 macrophages were seeded in 12-well plates at a density of 2.0 × 10⁶ cells per well. 5 Cells were co-cultured for 48 hours to form a confluent monolayer. Overnight cultured *Yersinia pseudotuberculosis* was diluted 1:100 in YLB. Experimental group: A final concentration of 5 µg / ml antimicrobial peptide (SEQ ID NO:2) and diluted bacterial suspension (total volume 0.8 mL) were simultaneously added to the upper chamber of a Transwell apparatus containing a 5.0 μm pore size polyethylene terephthalate (PET) membrane. Control group: An equal volume of PBS and diluted bacterial suspension were added to the upper chamber. The lower chamber contained only sterile cell culture medium (cell-free control) to assess baseline bacterial migration. After incubation at 37°C and 5% CO2 for 2 h, the contents of the lower chamber were collected and plated onto YLB agar plates. After incubation at 30°C for 24 h, colony counts were performed. The final colony count was corrected by subtracting the number of bacteria that migrated to the cell-free control.

[0064] Experimental results are as follows Figure 7 As shown, after adding the antimicrobial peptide of the present invention, the number of Yersinia pseudotuberculosis bacteria migrating to macrophages was significantly reduced, proving that the antimicrobial peptide can effectively interfere with the bacterial chemotaxis system, causing bacteria to lose their ability to directionally chemotactically attract macrophages.

[0065] 3. Detection of the reduction in cytotoxicity of pathogenic bacteria by specific antimicrobial peptides targeting the bacterial membrane dCACHE-type chemokine receptor protein family.

[0066] Macrophages were digested with Raw264.7 trypsin and then... 6 Cells were seeded in 6-well plates at a density of [number] cells per well. Experimental group: After cell adhesion, cells were infected with *Yersinia pseudotuberculosis*, carbapenem-resistant *Escherichia coli*, or carbapenem-resistant *Acinetobacter baumannii* at an MOI of 100:1. An antimicrobial peptide (SEQ ID NO:2) was added simultaneously with the bacteria at a final concentration of 5 µg / mL. Control group: Cells were infected with the above pathogens at an MOI of 100:1. An equal volume of PBS was added simultaneously with the bacteria. Blank group: Only an antimicrobial peptide at a final concentration of 5 µg / mL was added to test its cytotoxicity. Cells were collected 2 h after infection, washed with PBS, resuspended in 195 μL binding buffer, and stained with Annexin V-FITC and PI in the dark for 10 min at room temperature. Samples were immediately analyzed on a CytoFLEX flow cytometer, and data were processed using CytExpert software.

[0067] Experimental results are as follows Figure 8 As shown, regardless of the pathogenic bacteria, the apoptosis rate in the experimental group where both bacteria and antimicrobial peptides were added was significantly lower than that in the control group, indicating that the specific antimicrobial peptides targeting the bacterial membrane dCACHE-type chemokine receptor protein family can effectively reduce the toxicity of pathogenic bacteria to host cells. Meanwhile, the apoptosis rate in the blank group (containing only antimicrobial peptides) was not significantly different from that in the untreated group, demonstrating that the antimicrobial peptides have good biosafety to host cells at the working concentration.

[0068] 4. Detection of the effect of specific antimicrobial peptides targeting the bacterial membrane dCACHE-type chemokine receptor protein family on reducing the pathogenicity of pathogenic bacteria in mice.

[0069] (1) All mice were maintained and treated in accordance with the animal welfare guarantee policy promulgated by Northwest A&F University. The mice were 6-week-old SPF-grade female BALB / c mice, which were kept for 2-3 days after purchase before infection.

[0070] (2) Yersinia pseudotuberculosis was cultured in YLB medium at 26°C, while multidrug-resistant Escherichia coli and multidrug-resistant Acinetobacter baumannii were cultured in LB medium at 37°C. After reaching the initial stable stage, the strains were washed twice in sterile PBS, and Yersinia pseudotuberculosis, multidrug-resistant Escherichia coli, and multidrug-resistant Acinetobacter baumannii were diluted to 1×10⁻⁶. 7 cells / mL, 2×10 7 cells / mL, 5×10 5 A bacterial suspension of cells / mL.

[0071] (3) Different groups were set up, with 10 mice in each group. Treatment intervention: Mice were simultaneously injected intraperitoneally with bacteria and antimicrobial peptide (SEQ ID NO:2) (10 mg / kg body weight), and the peptide was injected once every 24 hours thereafter. The control group included: (i) mixed infection with bacteria and peptide using PBS instead of peptide; (ii) injection of antimicrobial peptide alone (10 mg / kg every 24 h) without bacterial infection; (iii) injection of PBS alone as a blank control. The infected mice were observed, and the survival status of each infection group was statistically compared. The observation lasted for 6 days, and survival curves were plotted.

[0072] Experimental results are as follows Figure 9 As shown, regardless of the pathogenic bacteria, mice treated with the antimicrobial peptide at the time of infection and every 24 hours thereafter showed a significant survival advantage compared to mice not treated with the antimicrobial peptide. Control mice injected with the antimicrobial peptide alone without bacterial infection did not die, further demonstrating the good biocompatibility of the peptide at the working concentration.

[0073] In summary, the specific antimicrobial peptides screened in this invention can target and bind to highly conserved ligand-binding interfaces in the bacterial membrane dCACHE-type chemokine receptor protein family, effectively blocking the pathogenic process of bacterial chemotaxis and apoptosis induction, and exhibiting excellent anti-multidrug-resistant bacterial infection activity both in vivo and in vitro.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. The use of the bacterial membrane dCACHE chemokine receptor protein family as drug targets in the preparation or screening of drugs against multidrug-resistant bacteria; wherein the bacterial membrane dCACHE chemokine receptor protein family refers to the dCACHE chemokine receptor protein family that mediates chemotaxis on the surface of bacterial cell membranes, and family members meet at least one of the following conditions: (a) An amino acid sequence containing more than 70% sequence identity with the conserved motif shown in SEQ ID NO:1; (b) Contains a conserved ligand-binding interface, wherein the conserved ligand-binding interface comprises at least a positively charged amino acid residue at the entrance of the binding pocket, an aromatic amino acid residue at the hydrophobic core, and a negatively charged amino acid residue at the bottom of the binding interface; wherein the positively charged amino acid residue is structurally and functionally equivalent to arginine at position 120 (R120) of the dCACHE-type chemokine receptor protein YcrA of *Yersinia pseudotuberculosis* bacterial membrane, wherein the aromatic amino acid residue is structurally and functionally equivalent to tryptophan at position 122 (W122) and tyrosine at position 138 (Y138), and wherein the negatively charged amino acid residue is structurally and functionally equivalent to aspartic acid at position 140 (D140) and aspartic acid at position 167 (D167); wherein "functional equivalence" means that the residue can form the same type of interaction with the host ligand, and that mutating it to the specific residue does not significantly reduce the ligand-binding affinity by more than 10-fold.

2. The use according to claim 1, characterized in that, The multidrug-resistant bacteria mentioned include, but are not limited to, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and Yersinia pseudotuberculosis.

3. The use according to claim 1, characterized in that, The drug blocks the chemotactic movement of bacteria to macrophages by binding to any member of the dCACHE-type chemokine receptor protein family, thereby reducing bacterial pathogenicity and infectivity.

4. The use according to claim 1, characterized in that, The drug inhibits bacterial chemotaxis towards macrophages, biofilm formation, and infection processes by suppressing the function of the dCACHE-type chemokine receptor protein family.

5. The use according to claim 1, characterized in that, The drug is a small molecule compound, antibody, peptide, or nucleic acid drug that targets the dCACHE-type chemokine receptor protein family.

6. An antibacterial polypeptide targeting the bacterial membrane dCACHE-type chemokine receptor protein family, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

2.

7. The antibacterial polypeptide according to claim 6, characterized in that, The polypeptide binds specifically to the Yersinia pseudotuberculosis dCACHE-type chemotactic receptor YcrA with high affinity, and its binding constant K d The value is 0.015 μM, which is much higher than the binding constant of YcrA with the host ligand.

8. The use of the antibacterial polypeptide according to claim 6 or 7 in the preparation of antibacterial drugs, preferably, the bacteria are multidrug-resistant bacteria, including but not limited to carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and Yersinia pseudotuberculosis.

9. A method for screening drugs against multidrug-resistant bacteria, characterized in that, Using the bacterial membrane dCACHE-type chemokine receptor protein family as described in claim 1 as a target, active substances that can bind to or inhibit the function of any member of this family are screened. Preferably, the multidrug-resistant bacteria include, but are not limited to, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and Yersinia pseudotuberculosis.

10. A method for inhibiting multidrug-resistant bacteria for non-therapeutic purposes, characterized in that, By inhibiting the function of any member of the bacterial membrane dCACHE-type chemotactic receptor protein family as described in claim 1, the chemotaxis of bacteria to macrophages is blocked, thereby inhibiting multidrug-resistant bacterial infections. Preferably, the multidrug-resistant bacteria include, but are not limited to, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa, and Yersinia pseudotuberculosis.

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