Small molecule polypeptide gp04 from phage and its application in targeting quorum sensing lasr protein

Abstract

The application relates to a bacteriophage-derived small molecule polypeptide Gp04 and application of the small molecule polypeptide Gp04 in targeting a quorum sensing LasR protein, and belongs to the technical field of biological medicines. The amino acid sequence of the bacteriophage-derived small molecule polypeptide Gp04 is shown as SEQ ID NO. 1. The small molecule polypeptide Gp04 provided in the application can inhibit QS-related phenotypes of Pseudomonas aeruginosa through overexpression or exogenous addition, and through bacterial double hybridization and electrophoretic mobility experiments, the binding and inhibiting effect of the small molecule polypeptide Gp04 on LasR are confirmed. The small molecule polypeptide can be used for preparing a preparation for inhibiting virulence and biofilm formation of Pseudomonas aeruginosa, and provides a new thought and method for developing a novel antibacterial strategy targeting QS and a bacteriophage combined prevention and treatment of pathogenic bacterial infection.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of a phage-derived small molecule polypeptide Gp04 and its targeting of the quorum sensing LasR protein. Background Technology

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa Pseudomonas aeruginosa is a Gram-negative bacterium widely found in the environment and clinical settings. It is also one of the ESKAPE pathogens listed as "critical" by the World Health Organization, posing a serious threat to human health. Its pathogenicity relies on multiple virulence factors and highly adaptive mechanisms. In the acute infection phase, the bacteria colonize host tissues via flagellar-mediated motility, while simultaneously secreting exotoxins, lipopolysaccharides, and various effector proteins to enhance its invasiveness. The quorum sensing (QS) system of Pseudomonas aeruginosa is its core regulatory mechanism. The LasR protein, as a key transcriptional regulator, directly controls the transcription of downstream virulence genes and forms a complex regulatory network with regulatory proteins such as RhlR and PqsR. In the chronic infection phase, Pseudomonas aeruginosa establishes a physical barrier through biofilms, masking surface antigens, hindering drug penetration, and providing protection for long-term colonization, making biofilms a key factor in drug resistance and chronic infection. Because Pseudomonas aeruginosa often exhibits multidrug resistance in clinical practice, traditional antibiotics face a severe challenge; therefore, there is an urgent need to develop novel control strategies targeting the QS system.

[0003] Bacteriophages, as viruses that specifically target bacteria, can not only lyse the host but also encode proteins or peptides that regulate host behavior. Previous studies have shown that some phage proteins can reduce the virulence of bacterial strains by interfering with the host's QS system. However, research on phage-derived small peptides inhibiting the LasR-mediated QS system of *Pseudomonas aeruginosa* is still limited, and their mechanisms of action and application value remain unclear. Therefore, developing novel phage-derived small peptides capable of inhibiting the *Pseudomonas aeruginosa* QS system has significant research value and application prospects. Summary of the Invention

[0004] In view of this, this application provides a phage-derived small molecule polypeptide Gp04 and its application in targeting the quorum sensing LasR protein to inhibit the QS system, virulence factors, and biofilm formation of *Pseudomonas aeruginosa*. Bacterial two-hybrid and electrophoretic mobility assays confirmed the binding and inhibitory effect of Gp04 on LasR. This small molecule polypeptide Gp04 can be used to prepare formulations that inhibit the virulence and biofilm formation of *Pseudomonas aeruginosa*, providing new ideas and methods for developing novel antibacterial strategies targeting QS and for phage-based co-prevention of pathogenic bacterial infections, effectively overcoming the shortcomings of the existing technologies.

[0005] The first aspect of this application provides the application of a phage-derived small molecule polypeptide Gp04 in the inhibition of Pseudomonas aeruginosa QS system. The phage-derived small molecule polypeptide Gp04 acts on Pseudomonas aeruginosa by exogenous addition and inhibits the QS-related phenotype of Pseudomonas aeruginosa. The amino acid sequence of the phage-derived small molecule polypeptide Gp04 is shown in SEQ ID NO. 1.

[0006] The second aspect of this application also provides the application of the aforementioned phage-derived small molecule polypeptide Gp04 in inhibiting the LasR protein-mediated QS system of Pseudomonas aeruginosa. The phage-derived small molecule polypeptide Gp04 targets the core regulatory node of the QS system mediated by the LasR protein to achieve multi-level regulation of virulence gene expression, biofilm formation, and motility, thereby reducing the pathogenicity of the bacteria. The amino acid sequence of the phage-derived small molecule polypeptide Gp04 is shown in SEQ ID NO.1.

[0007] A third aspect of this application also provides the use of the aforementioned phage-derived small molecule polypeptide Gp04 in the preparation of an inhibitory agent for Pseudomonas aeruginosa infection, wherein the agent is used to inhibit the QS system, virulence factor expression, biofilm formation, and motility of Pseudomonas aeruginosa; the amino acid sequence of the phage-derived small molecule polypeptide Gp04 is shown in SEQ ID NO.1.

[0008] The fourth aspect of this application also provides the use of the aforementioned phage-derived small molecule polypeptide Gp04 in the preparation of a medicament for phage-based combined therapy for Pseudomonas aeruginosa infection, wherein the combined therapy is achieved by enhancing the host's protection against infection or limiting the growth of the pathogen; the amino acid sequence of the phage-derived small molecule polypeptide Gp04 is shown in SEQ ID NO.1. The small molecule polypeptide Gp04 can be used in combination with phage to enhance the inhibitory effect against Pseudomonas aeruginosa infection.

[0009] Specifically, the small molecule polypeptide Gp04 was obtained through phage genome screening and can suppress the QS system-related phenotype of Pseudomonas aeruginosa. The nucleic acid molecule encoding the phage-derived small molecule polypeptide Gp04 includes the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO.1, as shown in SEQ ID NO.2.

[0010] MTNTDLKPLFDNLRNATEFFNSVDGDLASISNGTKQIAAEWLETAALALGDALIAQRKAVGGDHE* (SEQ ID NO.1);

[0011] atgacaaacaccgacctcaaaccgctgttcgacaacctgcggaacgccaccgagttcttcaactcggtcgatggcgacctcgcctcgatctccaacgggac caaacagattgcagccgaatggctggaaactgccgcgctcgccctcggcgacgccctgatcgcccagcgcaaagctgtcgggggcgaccatgagtga (seq ID NO.2).

[0012] Compared with the prior art, this application has the following advantages:

[0013] (i) This application uses bacteriophage genome as the source of functional molecules to screen and obtain small molecule polypeptide Gp04 that can regulate the QS system of Pseudomonas aeruginosa, providing a new technical means for the development of novel anti-Pseudomonas aeruginosa drugs.

[0014] (ii) The small molecule polypeptide Gp04 in this application targets the LasR-mediated QS core regulatory node to achieve multi-level regulation of virulence gene expression, biofilm formation and motility, thereby reducing the pathogenicity of bacteria.

[0015] (iii) The small molecule polypeptide Gp04 derived from bacteriophage in this application is easy to construct and express in prokaryotic expression systems, which facilitates in vitro functional verification and mechanism research, and is beneficial to subsequent application development and translational research. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in this application or the prior art, the drawings used in the description of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 The protein sequence is Gp04;

[0018] Figure 2 For the PCR verification of the Gp04 overexpressing strain in the examples, lane 1 is the marker (DNA molecular weight standard), lane 2 is the PCR amplification result using the pBBR1-mcs5 empty vector as a template, and lane 3 is the PCR amplification result using the pBBR1-mcs5-gp04 recombinant vector as a template.

[0019] Figure 3To illustrate the effects of GpO4 overexpression on the phenotype of *Pseudomonas aeruginosa* in this example, Figure A shows the effect of GpO4 overexpression on the biofilm-forming ability of *P. aeruginosa*, Figure B shows the effect of GpO4 overexpression on the migratory ability of *P. aeruginosa*, and Figure C shows the effect of GpO4 overexpression on the aggregation and motility ability of *P. aeruginosa*. This indicates that the PAO1 levels in the experimental group were compared to those in the control group. p <0.001;

[0020] Figure 4 To illustrate the effects of Gp04 overexpression on the transcriptional levels of QS and virulence factor-related genes in Pseudomonas aeruginosa, Figure A shows the changes in the transcriptional levels of QS-related genes in Pseudomonas aeruginosa after Gp04 overexpression, and Figure B shows the changes in the transcriptional levels of virulence factor-related genes in Pseudomonas aeruginosa after Gp04 overexpression.

[0021] Figure 5 To illustrate the effect of exogenous GpO4 addition on the phenotype of *Pseudomonas aeruginosa* in this example, Figure A shows the purification results of GpO4 protein, with lane 1 being the marker (DNA molecular weight standard) and lane 2 showing the purified recombinant GpO4 protein. Figure B shows the change in biofilm formation ability of *Pseudomonas aeruginosa* after exogenous GpO4 addition. Figure C shows the change in migration ability of *Pseudomonas aeruginosa* after exogenous GpO4 addition. This indicates that the PAO1 levels in the experimental group were compared to those in the control group. p <0.001;

[0022] Figure 6 To illustrate the protective effect of exogenous GpO4 on Pseudomonas aeruginosa infection of the large wax moth in the examples, Figure A shows the change in the survival rate of large wax moth larvae, and Figure B shows the phenotypic observation of large wax moth larvae after infection.

[0023] Figure 7 To illustrate the interaction between GpO4 and LasR in the examples, Figure A shows the results of the bacterial two-hybrid experiment, and Figure B shows the results of the electrophoretic mobility experiment.

[0024] Figure 8 This is a diagram of the Pseudomonas aeruginosa virulence regulation network that Gp04 may be involved in, as shown in the example. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0026] Unless otherwise specified, the experimental methods used in the embodiments of this application are all conventional methods.

[0027] In the following examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0028] I. Obtaining candidate Gp04 peptides and constructing overexpression strains

[0029] The bacteriophage HKPH_J3 used in this application is a previously obtained *Pseudomonas aeruginosa* bacteriophage that has undergone genome sequencing and annotation analysis. The genome sequence of HKPH_J3 has been submitted to the NCBI database, accession number PQ449026. Analysis was performed based on the genome annotation results of HKPH_J3. The results showed that the protein product Gp04 encoded by gene 04 in the HKPH_J3 genome has high sequence similarity to the known protein Aqs1, with BLAST homology alignment analysis showing an amino acid sequence identity of 68.12%. Since Aqs1 is associated with the regulation of QS in *Pseudomonas aeruginosa*, it suggests that Gp04 may be involved in the regulation of QS or related virulence processes in *Pseudomonas aeruginosa*. Therefore, the HKPH_J3 04 gene (gp04) was identified as a candidate functional gene, and its encoded product Gp04 was selected as the subject of subsequent research. gp04 The base sequence and amino acid sequence are as follows Figure 1 As shown.

[0030] Extract pBBR1-mcs5 plasmid, and then... Eco RI and Bam Linearized vectors were obtained by double HI digestion. The digestion system consisted of 20 μL of plasmid DNA and 10 μL of HI. Eco RI 1 μL, Bam HI 1 μL, 10×Buffer Y 4 μL, ddH2O 4 μL. Reaction conditions: 37℃ for 3 h. Using the HKPH_J3 phage genome as a template, amplification... gp04 Gene. Primers used:

[0031] gp04 -mcs5-F (forward primer): GCTTGATATC GAATTC ATGACAAACACCGACCTCAAACC (underscores) Eco RI restriction site (SEQ ID NO.3);

[0032] gp04 -mcs5-R (reverse primer): CGCTCTAGAACTAGT GGATCC TCACTCATGGTCGCCCCC (underscores) Bam HI restriction site, SEQ ID NO.4).

[0033] The PCR reaction mixture consisted of 20 μL: 1 μL template DNA, 1 μL forward primer, 1 μL reverse primer, 10 μL 2×Taq HiFi PCR mix, and 7 μL ddH2O. The PCR program was: 95℃ pre-denaturation (5 min), 95℃ denaturation (20 s), 60℃ annealing (30 s), 72℃ extension (30 s), for 35 cycles, followed by a final extension at 72℃ for 10 min. gp04 The gene was cloned into the expression vector pBBR1-mcs5 to construct a recombinant plasmid. The homologous recombination reaction system was 20 μL: 2.5 μL of digested vector, 2 μL of ligated DNA, 10 μL of homologous recombinase, and 4.5 μL of ddH2O. The reaction was carried out at 37℃ for 30 min. After verification, the plasmid was electroporated into competent *Pseudomonas aeruginosa* cells, revived, cultured, and plated on selective LB agar plates containing gentamicin. Positive clones were screened, and a *Pseudomonas aeruginosa* overexpression strain stably expressing Gp04 was finally obtained and named PAO1. gp04 Store at -80℃ for later use.

[0034] pBBR1-mcs5- gp04 The PCR verification results are shown in Figure 2 Among them, the swimlane marker is the DNA molecular weight standard.

[0035] II. Effects of Gp04 overexpression on the phenotype of Pseudomonas aeruginosa

[0036] (1) Detection of biofilm formation ability

[0037] Pseudomonas aeruginosa PAO1 and overexpression strain PAO1 ( gp04 ) Cultured separately to the logarithmic growth phase, and OD 600 Dilute to 0.01 and inoculate into 96-well plates, incubating overnight at 37°C. Discard the supernatant, stain with 0.5% crystal violet, discard the staining solution, wash and dry, dissolve in anhydrous ethanol, and measure OD. 570 The absorbance value at that location.

[0038] (2) Motion performance testing

[0039] Prepare separate culture media for migratory motility (2 g tryptone, 1 g sodium chloride, 0.6 g agar powder dissolved in ddH2O and brought to a final volume of 200 mL, then autoclaved) and for aggregation motility (10 g tryptone, 5 g glucose, 5 g sodium chloride, 5 g agar powder dissolved in ddH2O and brought to a final volume of 1 L, then autoclaved). Pour these media into petri dishes and allow them to cool to room temperature and solidify. Then, take 2 μL of each of the following strains: Pseudomonas aeruginosa PAO1 and the overexpressing strain PAO1. gp04 The sample was placed at the center of the culture medium and incubated overnight at 37°C. The diameter of bacterial diffusion was then measured.

[0040] Crystal violet staining results showed that the overexpressing strain PAO1 ( gp04 The biofilm formation in *Pseudomonas aeruginosa* was significantly reduced compared to the control group P. aeruginosa PAO1, indicating that GpO4 overexpression can inhibit biofilm formation in *P. aeruginosa*. Figure 3 A in the middle). Meanwhile, swimming mobility and cluster migration experiments showed that PAO1 ( gp04 The diffusion diameter of the GpO4 was also significantly smaller than that of the control group Pseudomonas aeruginosa PAO1, indicating that overexpression of the small molecule polypeptide GpO4 can inhibit the colonization and migration ability of Pseudomonas aeruginosa. Figure 3 B in Figure 3 (C in the middle). Figure 3 The control group PAO1 was obtained from laboratory preservation, and the experimental procedures were the same as those for the experimental group, i.e., PAO1 ( gp04 (Same as above.) (See diagram) This indicates that the PAO1 levels in the experimental group were compared to those in the control group. p <0.001. In summary, overexpression of the small molecule polypeptide Gp04 can reduce biofilm formation and motility in Pseudomonas aeruginosa, thereby weakening its pathogenic phenotype.

[0041] III. Effects of small molecule polypeptide Gp04 overexpression on transcription of QS and virulence factor-related genes in Pseudomonas aeruginosa

[0042] To investigate the effects of Gp04 overexpression on the QS system and transcriptional levels of virulence-related genes in Pseudomonas aeruginosa, samples were extracted from the overexpressing strain PAO1. gp04 Total RNA was synthesized into cDNA, and then detected using real-time quantitative PCR (RT-qPCR). The designed specific primers used in the experiment are shown in Tables 1 and 2. All samples were prepared with reaction systems at low temperatures before amplification and fluorescence acquisition. The relative expression levels of the target genes were calculated using a 2^(1-2) ratio, with the internal reference gene as the standard. -ΔΔCT The method employs standardized calculations to assess the transcriptional levels of different genes.

[0043] Table 1. Primers used in real-time quantitative PCR reactions

[0044]

[0045] Table 2. Primers used in real-time quantitative PCR reactions

[0046]

[0047] To investigate the inhibitory effect of the small molecule polypeptide Gp04 on the QS system and related pathogenic factors of Pseudomonas aeruginosa, genes closely related to the QS system were selected. rhlI , rhlR , lasI , lasB , lasR , pqsR , pqsH Virulence genes associated with biofilm formation pelE , pelF , pelG , pslA , pslB , pslC , algD Transcriptional analysis was performed on various pathogenic and movement-related genes. toxA It encodes exotoxin A, an important virulence factor of Pseudomonas aeruginosa, which can mediate cell damage by inhibiting host cell protein synthesis; lecA Responsible for encoding lectins and promoting bacterial aggregation; pilB , pilD , pilT This is closely related to the motility of bacteria; phzM It participates in the synthesis of pyocyanin. RT-qPCR results show that ( Figure 4 ), overexpressing strain PAO1 ( gp04 Most of the genes mentioned above were downregulated to varying degrees, indicating that the small molecule polypeptide Gp04 can effectively inhibit the expression of the QS system and related pathogenic factors in Pseudomonas aeruginosa. Figure 4 The A in the figure indicates that Gp04 can downregulate the expression of genes related to the QS system in Pseudomonas aeruginosa; Figure 4 The B in the data indicates that Gp04 downregulates the expression of most genes related to biofilm formation, motility, and virulence in *Pseudomonas aeruginosa*. This is consistent with the phenotype of reduced biofilm formation and decreased motility observed in *Pseudomonas aeruginosa* after overexpression of the small molecule peptide Gp04, suggesting that the small molecule peptide Gp04 may weaken the pathogenicity of *Pseudomonas aeruginosa* by interfering with the QS regulatory network and thus affecting multiple pathogenicity-related phenotypes.

[0048] IV. Effects of exogenous addition of small molecule polypeptide Gp04 on the phenotype of Pseudomonas aeruginosa

[0049] To obtain the recombinant small molecule polypeptide Gp04 protein, the recombinant plasmid pET32a- was constructed. gp04 The pET32a plasmid was extracted and processed... Bam HI and Hin Linearized vectors were obtained by double digestion with dIII. The digestion system consisted of 20 μL of plasmid DNA and 10 μL of dIII. Bam HI 1 μL, HindIII 1 μL, 10×Buffer Y 2 μL, ddH2O 6 μL. The reaction conditions were 37℃ for 3 h. The HKPH_J3 phage genome was used as a template for amplification. gp04 The primers used for the gene are:

[0050] gp04 -pET32a-F (forward primer): GCCATGGCTGATATC GGATCC ATGACAAACACCGACCTCAAACC (underscores) Bam HI restriction site (SEQ ID NO.47).

[0051] gp04 -pET32a-R (reverse primer): CTCGAGTGCGGCCGC AAGCTT TCACTCATGGTCGCCCCC (underscores) Hin dIII restriction site (SEQ ID NO.48).

[0052] PCR reaction system and procedure and construction of pBBR1-mcs5- gp04 Same. gp04 The gene was cloned into the expression vector pET32a to construct the recombinant plasmid. The homologous recombination reaction system and conditions were the same as those used in constructing pBBR1-mcs5- gp04 The same procedure was followed. After verification to ensure the sequence was free of mutations, the recombinant plasmid was transformed into BL21(DE3) competent cells, and expression was induced by IPTG (final concentration 1 mM). The bacterial cells were harvested by centrifugation, and the supernatant was purified by Ni-NTA affinity chromatography. Subsequently, the purified small molecule polypeptide Gp04 protein was added exogenously to the *Pseudomonas aeruginosa* culture system at concentrations of 3 μg / mL and 6 μg / mL, respectively, with PBS-treated *Pseudomonas aeruginosa* as the control group. The effects on biofilm formation and motility-related phenotypes of *Pseudomonas aeruginosa* were examined, with all other experimental conditions remaining the same as described above.

[0053] SDS-PAGE test results show that ( Figure 5 In Figure A), the small molecule peptide Gp04 protein is 6.90 kDa. After adding a lysis-promoting tag, a target protein band of the expected size appeared at approximately 25 kDa, indicating successful expression and purification of the Gp04 fusion protein. Using *Pseudomonas aeruginosa* as a model strain, the effect of exogenous addition of the small molecule peptide Gp04 on its related phenotypes was further evaluated. Figure 5 The B-molecule, the small polypeptide GpO4, significantly inhibited biofilm formation in *Pseudomonas aeruginosa*, with the most pronounced inhibitory effect observed at 6 μg / mL. (Movement experiment...) Figure 5C) further shows that, compared with the control group, the small molecule peptide Gp04 treatment group showed a smaller movement radius and decreased migration ability, indicating that the exogenous addition of small molecule peptide Gp04 has an inhibitory effect on bacterial motility. Figure 5 In the figure, PAO1 refers to *Pseudomonas aeruginosa* treated with PBS as a control group. All other experimental conditions were the same as the experimental group (Gp04 protein levels were 3 μg / mL and 6 μg / mL, corresponding to PAO1+3 μg / mL Gp04 and PAO1+6 μg / mL Gp04 in the figure). This indicates that the PAO1 levels in the experimental group were compared to those in the control group. p <0.001. In summary, the exogenous addition of the small molecule polypeptide Gp04 can simultaneously inhibit biofilm formation and motility of Pseudomonas aeruginosa, suggesting that it may reduce bacterial virulence by interfering with QS-related regulatory pathways.

[0054] V. Protective effect of exogenous addition of small molecule polypeptide Gp04 on Pseudomonas aeruginosa infection of the large wax moth.

[0055] To evaluate the therapeutic effect of the small molecule peptide Gp04 on Pseudomonas aeruginosa infection, a preliminary experiment was conducted to determine the absolute lethal dose (LD50) of Pseudomonas aeruginosa against the larvae of the large wax moth within 24 hours. 100 ) is 1×10 3 CFU / mL. The experiment was set up with 5 groups, each containing 10 larvae. Three of these groups were infection groups, treated by injecting 5 μL of *Pseudomonas aeruginosa* (1×10⁻⁶ CFU / mL) into the left forelimb. 3 An infection model was established using CFU / mL. One hour later, 5 μL of PBS (infection model control group, PAO1 group) or 5 μL of 150 μg / mL or 300 μg / mL small molecule peptide Gp04 (low-dose treatment groups, PAO1+150 μg / mL Gp04 group and PAO1+300 μg / mL Gp04 group) was injected into the right forelimb. Two uninfected control groups were also established. The left forelimb was injected with 5 μL of sterile PBS, and one hour later, the right forelimb was injected with either 5 μL of PBS (blank control group, PBS group) or 300 μg / mL Gp04 (drug toxicity control group, Gp04 group) to eliminate the potential influence of solvent, injection technique, and the peptide itself on larval survival. All groups were cultured at 37℃ in the dark, and the survival of the large wax moth was recorded every 6 hours.

[0056] Experimental results are as follows Figure 6As shown in the figure, the survival rate of *Heliotropium indicum* larvae injected with PBS or the small molecule peptide Gp04 alone was 100%, indicating that the small molecule peptide Gp04 had no significant toxicity to *Heliotropium indicum*. Compared with the control group injected with *Pseudomonas aeruginosa* alone, the survival rate of *Heliotropium indicum* larvae treated with the small molecule peptide Gp04 was significantly improved. Specifically, when the concentration of the small molecule peptide Gp04 protein was 300 μg / mL, the survival rate of *Heliotropium indicum* increased to 30%. This indicates that the small molecule peptide Gp04 has a certain protective effect against *Pseudomonas aeruginosa* infection. These results suggest that the small molecule peptide Gp04 may reduce the virulence of *Pseudomonas aeruginosa*, thereby alleviating host infection damage and improving host survival. Figure 6 The A in the figure indicates that Gp04 itself has no obvious toxicity to the giant wax moth and can improve the survival rate of the giant wax moth after PAO1 infection; Figure 6 The B in the figure indicates that the overall condition of PAO1-infected giant wax moth improved after Gp04 treatment.

[0057] VI. Interaction analysis between small molecule peptide GpO4 and LasR

[0058] (1) Bacterial two-hybrid experiment

[0059] To analyze the interaction between the small molecule peptide Gp04 and the LasR protein, a bacterial two-hybrid assay was used. gp04 and lasR The genes were cloned into the vectors pKNT25 and pUT18C, respectively, to construct the corresponding fusion expression plasmid pKNT25. gp04 ) and pUT18C ( lasR pKNT25 plasmid was extracted and processed... Hin dIII and Bam Linearized vectors were obtained by double HI digestion. The digestion system consisted of 20 μL of plasmid DNA and 10 μL of HI. Hin dIII 1 μL, Bam HI 1 μL, 10×Buffer Y 2 μL, ddH2O 6 μL. The reaction conditions were 37℃ for 3 h. The pUT18C plasmid was extracted and linearized by HindIII digestion. The digestion system was 20 μL: plasmid DNA 10 μL, Hin dIII 1 μL, 10×Buffer R 2 μL, ddH2O 7 μL. The reaction conditions were 37℃ for 3 h. pKNT25 ( gp04 Using the HKPH_J3 phage genome as a template, amplification gp04 Gene. pUT18C ( lasR Using the PAO1 genome as a template, amplification lasR Gene. The primers used in the construction process are as follows:

[0060] gp04 -pKNT25-F (forward primer): GATTACGCC AAGCTT ATGACAAACACCGACCTCAAACC (underscores) Hin dIII restriction site (SEQ ID NO.49)

[0061] gp04 -pKNT25-R (reverse primer): AGCTCGGTACCCGG GGATCC TCACTCATGGTCGCCCCC (underscores) Bam HI restriction site (SEQ ID NO.50)

[0062] lasR -pUT18C-F (forward primer): CCATGATTACGCC AAGCTT ATGGCCTTGGTTGACGGTTT (underscore is...) Hin dIII restriction site (SEQ ID NO.51).

[0063] lasR -pUT18C-R (reverse primer): CGGCT AAGCTT TCAGAGAGTAATAAGACCCAAATTAACG (underscore is...) Hin dIII restriction site (SEQ ID NO.52).

[0064] PCR reaction system and procedure and construction of pBBR1-mcs5- gp04 The same. The amplified samples were obtained separately. gp04 The gene was cloned into the linearized pKNT25 vector to construct the recombinant plasmid pKNT25. gp04 ). The amplified lasR The gene was cloned into the linearized pUT18C vector to construct the recombinant plasmid pUT18C( lasR Homologous recombination reaction system and conditions and construction of pBBR1-mcs5- gp04 The same. After verifying that the recombinant plasmid sequence was correct, the recombinant plasmid pKNT25( gp04 ) and pUT18C ( lasR The bacteria were transformed individually and co-transformed in BTH101 competent E. coli cells, and positive clones were obtained by antibiotic selection. The bacteria containing... gp04 and lasR The BTH101 strain was cultured on a selective medium containing X-gal, IPTG and corresponding antibiotics, and colony color changes were observed to determine whether there were interactions between proteins.

[0065] The results show that ( Figure 7 In the A section, only strain BTH101, which co-expresses the small molecule peptides Gp04 and LasR (i.e., simultaneously contains pKNT25), is included. gp04 pUT18C lasR Blue colonies appeared in both plasmids, while those expressing pKNT25 alone... gp04 ) or pUT18C ( lasR Neither the BTH101 strain nor the negative control produced blue, indicating that the small molecule polypeptide Gp04 interacts with the LasR protein.

[0066] Figure 7 In section A, both the positive and negative controls were derived from a bacterial adenylate cyclase two-hybrid system kit. The strain expressing the enzyme alone was labeled pKNT25. gp04 ) and pUT18C ( lasR The co-expressed strain is identified as pKNT25. gp04 )+pUT18C( lasR ).

[0067] (2) Electrophoretic mobility experiment

[0068] To further verify the effect of the small molecule peptide GpO4 on LasR-DNA binding activity, electrophoretic mobility assays were used for analysis. The LasR protein is composed of pET-21a- lasR The expression vector was induced and purified in BL21(DE3) competent cells, and the pET-21a plasmid was extracted and processed... Bam HI and Hin Linearized vectors were obtained by double digestion with dIII. The digestion system consisted of 20 μL of plasmid DNA and 10 μL of dIII. Bam HI 1 μL, Hin dIII 1 μL, 10×Buffer Y 2 μL, ddH2O 6 μL. The reaction conditions were 37℃ for 3 h. The primers used in the vector construction process are as follows:

[0069] lasR -pET21a-F (forward primer): GCAAATGGGTCGC GGATCC ATGGCCTTGGTTGACGGTTT (underscore is...) Bam HI restriction site (SEQ ID NO.53).

[0070] lasR -pET21a-R (reverse primer): GCCGC AAGCTT TCAGAGAGTAATAAGACCCAAATTAACG (underscore is...) HindIII restriction site (SEQ ID NO.54).

[0071] PCR reaction system and procedure and construction of pBBR1-mcs5- gp04 Same. The amplified result gp04 The gene was cloned into the linearized pET-21a vector to construct the recombinant plasmid pET-21a- gp04 Homologous recombination reaction system and conditions and construction of pBBR1-mcs5- gp04 The process was the same. The recombinant plasmid was transformed into BL21(DE3) competent cells, and expression was induced by IPTG (final concentration 1 mM). The small molecule polypeptide Gp04 was prepared using the purified sample from the previous experiments. Using the PAO1 genome as a template, PCR amplification was performed. lasB Promoter, PCR reaction system and procedure and construction of pBBR1-mcs5- gp04 The primers used are the same as those used in this study:

[0072] EMSA- lasB -F (forward primer): TTGTTCAGTTCTCCTGGTTTTTTCA (SEQ ID NO.55).

[0073] EMSA- lasB -R (reverse primer): ATCGCCCCATGGCCCCT (SEQ ID NO.56).

[0074] The purified LasR protein was combined with lasB Promoter DNA probes were incubated, and an experimental group containing the small molecule peptide GpO4 was also set up. After reacting at room temperature for 30 min, loading buffer was added, and non-denaturing polyacrylamide gel electrophoresis was performed. After electrophoresis, staining was performed, and changes in the bands of the binding complex were observed. Lane 1 contained only GpO4. lasB Blank control for the probe; lane 2 was infused with 20 μM LasR protein and lasB Probe; Lane 3 was filled with 20 μM Gp04 protein and lasB Probe; Lane 4 was simultaneously supplemented with 20 μM LasR protein and 20 μM Gp04 protein. lasB Probe.

[0075] Research results are as follows Figure 7 As shown in Figure B, a clear migration arrest band appears in lane 2, indicating that the LasR protein can specifically bind to [the target protein]. lasB The promoter; however, no migration band was observed when the small molecule polypeptide Gp04 was incubated alone with the DNA probe (lane 3), indicating that Gp04 does not bind directly. lasB Promoter; in lane 4, LasR- lasBThe signal of the complex band was significantly weaker than that in lane 2, indicating that GpO4 inhibited the interaction between LasR and... lasB Promoter binding. In summary, the small molecule peptide GpO4 itself does not directly bind to the promoter. lasB Instead of targeting the promoter, it interferes with the LasR-mediated QS transcriptional regulation process by affecting the binding ability of LasR to the target promoter.

[0076] Based on the above experimental results, a network diagram of the possible regulatory role of the small molecule polypeptide Gp04 in the virulence regulation of Pseudomonas aeruginosa was drawn. Figure 8 This further clarifies the pathogenic mechanism of *Pseudomonas aeruginosa*, namely that the small molecule polypeptide Gp04 can inhibit the function of LasR, the core regulatory protein of the *P. aeruginosa* quorum sensing system, thereby inhibiting the cascade reaction of the downstream Rhl and PQS systems, globally downregulating the expression of genes related to quorum sensing, biofilm formation, and virulence factors, and ultimately reducing the biofilm formation ability, motility, virulence, pathogenicity, and colonization ability of *P. aeruginosa*. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them; although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. The application of phage-derived small molecule polypeptide Gp04 in the preparation of agents to inhibit Pseudomonas aeruginosa infection, characterized in that, The formulation is used to inhibit the quorum sensing system, virulence factor expression, biofilm formation, and motility of Pseudomonas aeruginosa; the amino acid sequence of the phage-derived small molecule polypeptide Gp04 is shown in SEQ ID NO.

1.

2. The application of the phage-derived small molecule polypeptide Gp04 in the preparation of a drug for the combined treatment of Pseudomonas aeruginosa infection using bacteriophages, characterized in that, The combined treatment is achieved by enhancing the host's protection against infection or limiting the growth of pathogens; the amino acid sequence of the phage-derived small molecule polypeptide Gp04 is shown in SEQ ID NO.1.

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