Application of PpL in inhibiting Gram-negative bacterial biofilm formation, preparation method of inhibitor and hexameric modified body

Abstract

The present invention relates to the field of biology, and specifically to the application of PpL in inhibiting the formation of Gram-negative bacterial biofilms, inhibitors, and methods for preparing hexameric modifications. The PpL of the present invention has higher thermal stability and exhibits a biofilm inhibitory effect on Gram-negative bacteria, especially Acinetobacter baumannii. PpL solves the problem of relatively poor catalytic efficiency and thermal stability of existing acylhomoserine lactonase, and promotes its wider application in various fields, playing a vital role in effective quorum quenching strategies. The method for preparing a hexameric modification of acylhomoserine lactonase of the present invention replaces the amino acid sequence involved in inter-subunit interaction in other non-hexameric AHLases with the amino acid sequence involved in inter-subunit interaction in PpL, hexamerizes the non-hexameric AHLase, and obtains a hexameric modification of acylhomoserine lactonase, which also has strong thermal stability.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to an application of PpL in inhibiting the formation of Gram-negative bacterial biofilm, an inhibitor and a preparation method of a hexameric modified body. Background Art

[0002] Currently, bacterial antibiotic resistance is one of the most serious public health threats to humanity. It is estimated that if no effective solution is found, the annual death toll from multidrug-resistant (MDR) bacterial infections could exceed 10 million by 2050, exceeding the total number of deaths caused by heart disease and cancer. The emergence of multidrug-resistant (MDR) microorganisms is the combined result of the widespread spread of resistance genes, mainly due to the overuse of antibiotics and the exchange of resistance genes between various bacterial species. The problem becomes even more serious when microorganisms form biofilms, as biofilm formation can amplify bacterial resistance by up to 1,000 times, thereby increasing the incidence of MDR infections. Therefore, inhibiting bacterial biofilms is key to addressing bacterial antimicrobial resistance.

[0003] Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species are known to be among the most drug-resistant Gram-negative bacteria. These microorganisms employ a quorum sensing (QS) system—a cell-to-cell communication mechanism—to regulate population density and modulate the expression of genes associated with drug resistance and pathogenic behavior. One such behavior is biofilm formation, a protective mechanism that protects bacteria from adverse conditions. Acyl-homoserine lactones (AHLs) are the primary QS signaling molecules secreted by these bacteria. Numerous studies have demonstrated that QS plays a key role in regulating biofilm formation, and the introduction of exogenous AHLs can significantly influence biofilm properties, including biofilm thickness and duration. For example, Pseudomonas aeruginosa and Acinetobacter baumannii, classified as critical priority pathogens on the World Health Organization (WHO) Global Priority Pathogens (GPP) list, both rely on AHL-based mechanisms for biofilm formation and virulence factor production. In Acinetobacter baumannii, the main AHLs produced and utilized are N-dodecanoyl-L-homoserine lactone (C12-HSL) and 3-hydroxy-C12-HSL, while C4-HSL and 3-oxo-C12-HSL are the main AHLs produced in Pseudomonas aeruginosa. These AHLs play a crucial role in coordinating biofilm formation and the expression of virulence factors in bacterial species.

[0004] N-acyl-L-homoserine lactonase (AHLase) is a quorum-quenching (QQ) lactonase that disrupts bacterial behaviors that rely on AHLs for signaling. These enzymes promote the hydrolysis of the lactone ring to generate N-acyl-L-homoserine and a proton (H + This class of enzymes has been shown to inhibit biofilm formation and virulence factor secretion in Gram-negative bacteria, making them promising candidates for antimicrobial strategies. N-acyl-L-homoserine lactonases have been shown to reduce biofilm formation and increase susceptibility to multiple antibiotics in multidrug-resistant (MDR) Pseudomonas aeruginosa and Acinetobacter baumannii. Therefore, these enzymes have the potential to convert drug-resistant strains from a resistant state to an intermediate or sensitive state.

[0005] Based on the quorum sensing mechanism, quorum quenching has been highly valued as an effective biological control strategy. ] and AaL [ , which can effectively disrupt AHLs-based signaling. However, these enzymes often suffer from limitations such as low catalytic activity or insufficient thermal stability, which greatly hinder their widespread application. Therefore, discovering AHLases with both high catalytic activity and excellent thermal stability, or modifying existing AHLases to improve their thermal stability, is crucial for effective quorum quenching strategies. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide the use of PpL in inhibiting Gram-negative bacterial biofilm formation, as well as methods for preparing inhibitors and hexameric modified forms. The goal is to provide stable and highly effective PpL and inhibitors for inhibiting Gram-negative bacterial biofilm formation, while also modifying other non-hexameric acylhomoserine lactonases.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] First, the application of PpL in inhibiting the formation of Gram-negative bacterial biofilm.

[0009] The beneficial effects of the present invention are: the PpL of the present invention has higher thermal stability and exhibits biofilm inhibition against Gram-negative bacteria, especially Acinetobacter baumannii; therefore, PpL solves the problems of relatively poor catalytic efficiency and thermal stability of existing AHLases, promotes their wider application in various fields, and plays a vital role in effective quorum quenching strategies.

[0010] PpL (WP_082630345) is a lactonase composed of 306 amino acids with a molecular weight of 34.7 kDa; it was originally isolated from Pseudomonas thaliana. Notably, the amino acid sequence of PpL has a unique motif " 107 HXHXDH 112 -H 196 -D 217 -H 263 -", this motif is often associated with metallo-β-lactamases (MLLs).

[0011] The above-mentioned further beneficial effects of the present invention are: PpL is an N-acyl-L-homoserine lactonase family protein from Pseudomonas herba. The present invention comprehensively characterizes PpL, and studies have shown that PpL has excellent catalytic activity and thermal stability; PpL has been confirmed to be a homohexameric acyl homoserine lactone (AHL) enzyme, and its barrel-shaped structure is crucial to its high thermal stability and strong enzymatic activity.

[0012] Furthermore, the PpL inhibits Gram-negative bacterial biofilm formation by degrading acyl homoserine lactone.

[0013] Furthermore, the Gram-negative bacteria include any one or a combination of at least two of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter.

[0014] Furthermore, the PpL structure is a barrel-shaped hexamer.

[0015] In a second aspect, an inhibitor for inhibiting the formation of Gram-negative bacterial biofilm comprises PpL.

[0016] Furthermore, the inhibitor further includes pharmaceutically acceptable carriers and / or excipients.

[0017] The pharmaceutically acceptable carriers and / or excipients include pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, which depend on the administration method and the designed dosage form.

[0018] Furthermore, the dosage form of the inhibitor is any pharmaceutically acceptable dosage form, and the pharmaceutical dosage form is an oral dosage form (such as tablets, capsules, granules, suspensions, solutions, etc.) or an injectable dosage form.

[0019] In addition, the actual dosage of the active ingredient (hexameric acylhomoserine lactonase of the present invention) in the inhibitor should be determined based on a variety of relevant factors, including the severity of the disease to be treated, the route of administration, and the patient's age, sex, and weight. Therefore, the above dosage should not limit the scope of protection of the present invention in any way. The appropriate dosage of the inhibitor can be prescribed in a variety of ways based on factors such as the formulation method, administration method, the patient's age, weight, sex, disease state, diet, administration time, administration route, excretion rate, and reaction sensitivity. A skilled physician can generally easily determine a prescription and a dosage that is effective for the desired treatment.

[0020] In a third aspect, the present invention also provides a method for preparing a hexameric modified form of acylhomoserine lactonase, comprising the following steps: replacing the amino acid sequence involved in the inter-subunit interaction in acylhomoserine lactonase with the amino acid sequence involved in the inter-subunit interaction in PpL.

[0021] The amino acid sequence involved in inter-subunit interaction in other non-hexameric AHLases is replaced with the amino acid sequence involved in inter-subunit interaction in PpL, and the non-hexameric AHLase is hexamerized to obtain a hexameric acylhomoserine lactonase modified body, which also has strong thermal stability.

[0022] Furthermore, the method for preparing the hexameric modified form of the acylhomoserine lactonase comprises the following steps: replacing the amino acid sequence participating in the inter-subunit interaction in the acylhomoserine lactonase with the amino acid sequence participating in the inter-subunit interaction in PpL, wherein the amino acid sequence participating in the inter-subunit interaction in PpL includes the sequence located at positions 26-41 of the PpL amino acid sequence as shown in SEQ ID NO.1, the sequence located at positions 140-153 of the PpL amino acid sequence as shown in SEQ ID NO.2, and the sequence located at positions 226-239 of the PpL amino acid sequence as shown in SEQ ID NO.3.

[0023] In a fourth aspect, the present invention further provides a hexameric modified acylhomoserine lactonase, which is prepared by the above-mentioned method.

[0024] Furthermore, the hexameric modified form of the acylhomoserine lactonase includes that shown in SEQ ID NO.4 or SEQ ID NO.5.

[0025] SEQ ID NO.4 (AaL variant):

[0026] MTNIAKAQPKLYVMDNGRMRMAKDFFGGAGIFSNSGTIEFPIYTVLIDHPEGKILFDTS

[0027] CNPDSMGAQGRWGEATQSMFPWTASEECYLHNRLEQLKVRPEDIKFVIASHLHLDHA

[0028] GCLEMFTNATIIVHEDEFSGALQTYARNHETEEEKEWVGAYIWGDIDAWIKNNLNWRT

[0029] IKRDEDNIVLAEGIKILNFGSGHAWGMLGLHVQLPEKGGIILASDAVYSAESYGMWPPGYHQGDSLGFVRSVEKIKRIAKETNSEVWFGHDSEQFKRFRKSTEGYYE;

[0030] SEQ ID NO.5 (AiiB modified body) MGNKLFVLDLGEIRVAKDFFGGAGIFSNSGTIDIPVSAYLIQCTDATVLYDTGCHPECMGTNGRWPAQSQLNAPYIGASECNLPERLRQLGLSPDDISTVVLSHLHNDHAGCVEYFGKSRLIAHEDEFATA VRYFAHETEEEKEWVSPYIVKDIEAWLATPRNWDLVGRDERERELAPGVNLLNFGTGHASGMLGLAVRLEKQPGFLLVSDACYTATNYGPPMWPPGYHQGDTIGYDRTVSHIRQYAESRSLTVLFGHDREQFASLIKSTDGFYE. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The AHLs enzyme-catalyzed reaction and substrate of the present invention; (A) AHLs enzyme-catalyzed reaction: In this reaction, the AHLs enzyme promotes the hydrolysis of N-acyl-L-homoserine lactone in the presence of water (H2O); this enzymatic process leads to the cleavage of the lactone ring to form N-acyl-L-homoserine and a proton (H + ) ions; (B) substrates tested in the present invention: a variety of substrates were tested in the study to evaluate AHLase activity and substrate preference;

[0032] Figure 2Sequence and structure comparison of representative enzymes of the metallo-β-lactamase subfamily with PpL; (A) Sequence analysis: Amino acid sequences of different metallo-β-lactamase-like (MLL) enzymes, including GcL from Geobacterium thermoxylosuccinate, AaL from Cyanobacterium acidolimitans, AiiA from Bacillus thuringiensis, AiiB from Agrobacterium tumefaciens, AidC from Flavobacterium sp. strain StRB126, and PpL; The active site of PpL consists of five histidine residues and two aspartic acid residues involved in metal coordination. , indicated by asterisks, and the amino acids responsible for subunit interactions are underlined. The figure shows the key secondary structures of PpL, including α-helices and β-sheets, and the percentage of identity between PpL and other MLLs sequences is marked; (B) PpL monomer structure: The protein structure of the PpL monomer is shown in cartoon form, which contains αβ / βα folds and two metal cations (indicated by red spheres); (C) Structural alignment: Comparison of the structure of PpL with the typical MLLs representative AiiA;

[0033] Figure 3 Structural analysis of PpL according to the present invention; (A) The polymerization of three dimers ultimately forms a barreled hexamer; (B) The amino acids forming a salt bridge between the D and C subunits of PpL, showing the specific atoms involved in the salt bridge formation and the distances between them; (C) The amino acids forming a hydrogen bond between the D and C subunits of PpL, showing the donor and acceptor atoms involved in the hydrogen bond formation and the distances between them; (D) The amino acids forming a hydrogen bond between the B and C subunits of PpL, showing the donor and acceptor atoms involved in the hydrogen bond formation and the distances between them;

[0034] Figure 4 Analysis of molecular weight and thermal stability of PpL; (A) Molecular weight analysis of wild-type PpL (WT); blue indicates PpL, and red indicates protein markers; (B) DSF analysis of the thermal stability of wild-type PpL;

[0035] Figure 5 Physicochemical parameters affecting PpL activity; (A) Effect of buffer pH on enzyme activity; 100% relative activity was defined as the enzyme activity in a pH 7.0 buffer at 30°C; (B) Effect of temperature on PpL activity during enzyme catalysis; 100% relative activity was defined as the enzyme activity in a pH 8.0 buffer at 30°C; (C) Effect of metal ions on PpL activity; the control group was subjected to the same reaction conditions but without the metal ions in the reaction buffer; data are expressed as mean ± standard deviation (SD).

[0036] Figure 6Figure 3 is the mass spectrometry (MS) analysis of AHLs by PpL; (A) In the positive electrospray ionization (ESI+) mode, the mass spectrometry (MS) result of C6-HSL has a mass-to-charge ratio (m / z) of 200.16; (B) In the negative electrospray ionization (ESI-) mode, it shows the generation of an ion with m / z of 216.10, corresponding to C6-HS, the product of the hydrolysis of C6-HSL by PpL; (C) In the ESI- mode, the MS result of C4-HSL has an m / z of 170.08; (D) In ​​the ESI- mode, MS shows The deprotonated ion with m / z of 188.09 was generated, corresponding to C4-HS, the product of the hydrolysis of C4-HSL by PpL; (EF) respectively show the mass spectrometry results of 3-oxo-C8-HSL and its hydrolysis product 3-oxo-C8-HS; (GH) respectively show the mass spectrometry results of C8-HSL and its hydrolysis product C8-HS; (IJ) respectively show the mass spectrometry results of C10-HSL and its hydrolysis product C10-HS; (KL) respectively show the mass spectrometry results of C12-HSL and its hydrolysis product C12-HS;

[0037] Figure 7 Effects of PpL on biofilm formation of Acinetobacter baumannii (A. baumannii) were investigated. Biofilms were quantified using crystal violet staining, and the amount of biofilm was expressed as absorbance (550 nm). Bovine serum albumin (BSA) was used as a negative control, and the quorum sensing inhibitor (QSI) 5-fluorouracil was used as a positive control.

[0038] Figure 8 To transform the homodimer AaL reported in the literature into a hexamer; (A) Structural alignment of PpL and AaL. The amino acids with a blue background in the figure represent the amino acids involved in hexamerization in PpL, and the corresponding amino acids in AaL are in green; (B) The new sequence formed by replacing the amino acids corresponding to the green background in AaL with the amino acids with a blue background in PpL, and the hexamer predicted by AlphaFold Multimer; (C) The amino acids forming hydrogen bonds between the D and B subunits in the hexameric AaL are shown, as well as the donor and acceptor atoms involved in hydrogen bond formation and the distance between them; (D) The amino acids forming hydrogen bonds between the B and C subunits in the hexameric AaL are shown, as well as the donor and acceptor atoms involved in hydrogen bond formation and the distance between them;

[0039] Figure 9To transform the homologous dimer AiiB reported in the literature into a hexamer; (A) The structures of PpL and AiiB were aligned. The amino acids with a blue background in the figure represent the amino acids involved in hexamerization in PpL, and the corresponding amino acids in AiiB are in green; (B) The new sequence formed by replacing the amino acids corresponding to the green background in AiiB with the amino acids with a blue background in PpL, and the hexamer predicted by AlphaFold Multimer; (C): The amino acids that form hydrogen bonds between the D and C subunits in the hexamer AiiB are shown, as well as the donor and acceptor atoms involved in the hydrogen bond formation, and the distance between them; (D) The amino acids that form hydrogen bonds between the D and E subunits in the hexamer AiiB are shown, as well as the donor and acceptor atoms involved in the hydrogen bond formation, and the distance between them. DETAILED DESCRIPTION

[0040] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0041] 1. Materials and Methods

[0042] (1) Sequence alignment of PpL and representative MLLs

[0043] The present invention compared the PpL sequence with protein databases and used the BLAST search program on the NCBI website (www.ncbi.nlm.nih.gov) to identify previously identified lactonase enzymes. Representative metallo-β-lactamases (MLLs) of known structure were aligned with PpL using MUSCLE in the MEGA software suite.

[0044] (2) Expression and purification of PpL protein

[0045] The pET28a plasmid containing PpL was synthesized by Sangon Biotech (Shanghai, China) and PpL was expressed using the BL21 (DE3) strain (TransGen Biotech, China) in Luria-Bertani (LB) medium supplemented with kanamycin (50 mg / ml) and 0.2 mM ZnCl. To induce PpL expression, 0.5 mM IPTG was added when the optical density (OD600) reached 0.6-0.8. After overnight shaking culture at 16°C, cells were harvested by centrifugation at 4°C (4000 g for 10 minutes) and resuspended in lysis buffer (25 mM Tris, 500 mM NaCl, 10 mM imidazole, 0.2 mM ZnCl, pH 8.0). Subsequently, cells were lysed at 6°C using a high-pressure homogenizer (JNBIO, China). The lysate was centrifuged at 4°C (26,000 g, 40 minutes) and the supernatant was applied to Ni-NTA affinity chromatography (GE Healthcare, USA). Thrombin (Sigma-Aldrich, Burlington, MA, USA) was used to cleave the 6xHis tag overnight at 4°C. PpL was further purified by anion exchange chromatography and gel filtration chromatography using a Superdex 200 10 / 300 GL column (GE Healthcare) in a buffer (50 mM Tris, 200 mM NaCl, 5% glycerol, 0.2 mM zinc chloride, pH 8.0). Finally, the purity of the protein was confirmed by SDS-PAGE.

[0046] (3) Crystal diffraction data collection and structure determination

[0047] A commercial crystallization kit (Hampton Research CA, USA) was used for initial crystal screening. PpL protein crystals were obtained under conditions of 0.1 M BIS-TRIS propane at pH 9.0 and 30% w / v polyethylene glycol 6000, and crystals formed within 2 days at 20°C. Protein crystals were optimized using the hanging drop method, and 1 μL of protein solution at a concentration of 18 mg / ml was mixed with 1 μL of pool solution for crystallization. The present invention ultimately successfully obtained high-quality PpL protein crystals under conditions containing 0.1 M BIS-TRIS propane, pH 8.9 and 24% w / v polyethylene glycol 8000. Subsequently, to prevent frostbite, these PpL protein crystals were transferred to a mother liquor containing 20% ​​glycerol, and the samples were frozen in liquid nitrogen. Crystal diffraction data were collected by beamline BL19U1 (Shanghai) of the Shanghai Synchrotron Radiation Facility (SSRF). The data were processed using HKL3000 software. Alphafold2 was used to predict the initial structural model of PpL, and the structure of PpL was solved by molecular replacement. The model was further refined manually using COOT, guided by the electron density map. The model was refined and quality assessed using PHENIX software. Table 1 summarizes the data collection and structure refinement statistics.

[0048] Table 1 PpL crystal data collection and optimization

[0049]

[0050] In Table 1, Rmerge=∑ hkl ∑ i |I i (hkl)-<I(hkl)> | / ∑hkl∑iI i (hkl), where I i (hkl) is a single intensity measurement,<I(hkl)> is the average intensity of all i reflections.

[0051] (4) Determination of enzyme kinetic parameters

[0052] PpL enzyme activity was determined using a pH-sensitive colorimetric assay. In this experiment, a 200 μl reaction system containing 100 μM BTB, 2.5 mM MOPS (pH 7.1), and varying amounts of AHL (0–5 mM) was used. To ensure data quality, the enzyme concentration used in the kinetic experiments was adjusted based on the observed velocities. Enzyme concentrations ranging from 0.04 to 0.25 μM were used for different substrates. The MOPS / BTB system used in this experiment operates on the principle that the hydrolysis of AHL releases protons, resulting in a change in the pH of the solution. BTB is a pH-sensitive dye whose color changes with changes in solution pH. This color change was monitored by continuously tracking absorbance at 630 nm using a CLARIOstar Plus plate reader (Ottenberg, Germany) at room temperature (37°C). Km and Vmax values ​​were calculated using OriginPro software (version 9.80). A standard curve was constructed using HCl to plot the absorbance change versus proton concentration.

[0053] (5) HPLC analysis of the effects of physical and chemical factors on PpL activity

[0054] The present invention used high-performance liquid chromatography (HPLC) to analyze the effects of temperature, pH, and metal ions on PpL activity. The reaction mixture consisted of 5 mM C6-HSL, 10 μM PpL, and 25 mM Tris (pH 8.0). The mixture was incubated for 20 min at various temperatures (4°C, 16°C, 25°C, 37°C, 40°C, 45°C, 50°C, 60°C, and 70°C). After incubation, the protein was precipitated by adding SDS to a final concentration of 2%, and the precipitate was removed by centrifugation. A 20 μL sample was injected onto an equilibrated Agilent 1260 (Agilent Technologies, CA, USA) HPLC system equipped with a ZORBAX Eclipse Plus C18 column (size: 4.6 × 100 mm, diameter: 3.5 μm). The mobile phase was acetonitrile-water (volume ratio: 27:73) at an elution rate of 0.7 mL / min. Because C6-HSL has an absorbance at 210 nm, the HPLC method is used to monitor changes in absorbance at 210 nm to reflect changes in C6-HSL concentration for the evaluation of enzyme activity. Similarly, the optimal pH for PpL activity is determined by preparing reaction mixtures with different pH values. The present invention uses Na2HPO4 / citric acid to prepare a buffer with a pH value of 2.0-7.0, uses Tris-HCl to prepare a buffer with a pH value of 8.0-9.0, and uses Na2CO3 / NaHCO3 to prepare a buffer with a pH value of 10.0-11.0. In addition, to evaluate the effect of metal ions on PpL activity, as described above, the present invention adds 0.1 mM or 0.5 mM of a specific metal ion to the reaction mixture. Each experiment is repeated 3 times.

[0055] (6) Biofilm inhibition experiment

[0056] The present invention uses Acinetobacter baumannii (A.baumannii) ATCC 19606 TM . A plastic microplate was used for the biofilm inhibition test. Specifically, Acinetobacter baumannii cultured overnight in LB medium was diluted 1000 times with MOPS minimal medium. Subsequently, 0.1 mL of the diluted bacterial solution was distributed into a 96-well polystyrene microtiter plate. Different volumes of PpL were added to different wells and then incubated at 37°C. After incubation for 12 hours, the culture plate was rinsed with distilled water, dried at 25°C for 30 minutes, and then 200 μL of 0.1% crystal violet solution was added to each well. The crystal violet was then discarded and the wells were rinsed with distilled water. 250 μL of 30% acetone was added to dissolve the crystal violet, and 200 μL was taken from each well and transferred to a fresh flat-bottom 96-well plate. The absorbance value was measured at 550 nm.

[0057] (7) Hexameric transformation of non-hexameric MLLs

[0058] First, the non-hexameric MLLs protein structure and the PpL protein structure were structurally aligned using structural analysis software. Secondly, the amino acids involved in hexamerization in the PpL protein are mainly composed of three parts: amino acids 26-41 (AKDFFGGAGIFSNSGT, SEQ ID NO.1), amino acids 140-153 (SSHQHETEEEKEWV, SEQ ID NO.2) and amino acids 226-239 (ERMWPPGYHQGNAF, SEQ ID NO.3). The corresponding amino acids in the non-hexameric MLLs are marked. Again, the amino acid sequence involved in hexamerization in the PpL protein is used to replace the corresponding sequence in the non-hexameric MLLs. Finally, the hexameric structure of the new sequence is predicted by AlphaFold Multimer. If the pTM+ipTM score of the predicted structure is greater than 0.75 (range 0-1), it indicates that the interaction between the subunits is strong and a hexamer can be formed. In addition, the predicted hexameric structure was loaded into the PDBePISA website (https: / / www.ebi.ac.uk / msd-srv / prot_int / cgi-bin / piserver) to analyze the interaction forces between subunits to see whether multiple chemical bonds such as hydrogen bonds, salt bridges and disulfide bonds were generated.

[0059] 2. Experimental results

[0060] (1) Brief description of PpL protein

[0061] PpL (WP_082630345) is a lactonase composed of 306 amino acids with a molecular weight of 34.7 kDa. It was originally isolated from Pseudomonas thaliana. Notably, the amino acid sequence of PpL has a unique motif " 107 HXHXDH 112 -H 196 -D 217 -H 263 -", this motif is a hallmark of metallo-β-lactamase (MLL). The protein sequence of PpL was aligned with the MLL sequence of known structure. It was found that these enzymes had very low sequence identity with PpL. The highest sequence identity was only 27.3%, which was compared with AiiA (see Figure 2 A). The most significant difference between the PpL sequence and the other MLLs is the presence of an additional C-terminal extension of approximately 20 amino acids (see Figure 2 A). The monomeric structure of PpL is roughly spherical, conforming to the expected αβ / βα sandwich fold characteristic of the metallo-β-lactamase (MLL) superfamily ( Figure 2 B). The structure of PpL is very similar to that of other MLL family members (including AiiA, AiiB, AidC, GcL, and AaL), but PpL has two additional β-sheet structures (β13, β14) at its C-terminus.

[0062] (2) Crystal structure characteristics of PpL hexamer

[0063] In the crystal structure, PpL assembles as a homohexamer, and its overall structure appears to be a barrel with a central pore (see Figure 3 A) For a clearer description, the present invention divides this homologous hexamer into three dimers. When performing protein structure comparison, the present invention observed significant structural consistency between these three dimers. The formation of dimers mainly depends on interactions involving multiple hydrogen bonds and salt bridges. (See Figure 3 BD).

[0064] (3) State of PpL hexamer in solution

[0065] In order to evaluate the polymerization state in solution, the present invention uses gel filtration chromatography to analyze the polymerization form of PpL. The molecular weight is determined to be 214.6 kDa, which is close to the theoretical molecular weight of hexamer (34730×6=208.38 kDa). Figure 4 A), while other reported homoserine lactonase enzymes appear in the form of monomers or dimers. The thermal stability of wild-type PpL was further evaluated by differential scanning fluorimetry (DSF). The results showed that wild-type PpL has a high thermal stability (Tm = 71.9 ° C) ( Figure 4 B), which is much higher than that of other reported homoserine lactonase (Tm is around 51.8°C). Therefore, we believe that it is this hexameric assembly that leads to its significantly higher thermal stability than other homoserine lactonases.

[0066] (4) PpL enzyme activity

[0067] The present invention studied several physical and chemical parameters that may affect the activity of PpL, including temperature, pH, and metal ions. The results showed that PpL showed high activity in alkaline reaction systems, with its optimal activity occurring at pH 8.0 ( Figure 5 A). PpL showed relatively high activity in the temperature range of 30 to 60°C, reaching its peak at 37°C ( Figure 5 B). Different metal ions have different effects on PpL activity. Ni 2+ Mg 2+ and Zn 2+ enhanced the activity of PpL, while Ca 2+ 、Cu 2+ and Fe 2 + Reduced its activity. 2+ Little effect on enzyme activity ( Figure 5 C).

[0068] (5) Specificity of PpL for AHL substrates

[0069] To elucidate the mechanism of AHL degradation by PpL, the present invention tested the enzymatic activity of PpL against various AHLs ( Figure 1 A), including N-butyryl-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL), N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL), N-decanoyl-L-homoserine lactone (C10-HSL) and N-dodecanoyl-L-homoserine lactone (C12-HSL) ( Figure 1 B). First, mass spectrometry (MS) was used to confirm whether PpL could hydrolyze N-acyl-L-homoserine lactone to N-acyl-L-homoserine. The results showed that PpL could catalyze the hydrolysis of C6-HSL to C6-HS (see Figure 6 A and 6B). Similarly, PpL also effectively degraded C4-HSL, 3-oxo-C8-HSL, C8-HSL, C10-HSL and C12-HSL to generate C4-HS ( Figure 6 C and 6D), 3-oxo-C8-HS ( Figure 6 E and 6F), C8-HS( Figure 6G and 6H), C10-HS( Figure 6 I and 6J) and C12-HS ( Figure 6 K and 6L). This result strongly demonstrates the ability of PpL to act as an AHLase and to hydrolyze the ester bond in the homoserine lactone ring of AHLs.

[0070] To gain a deeper understanding of the substrate specificity of PpL, we determined the kinetic constants for the hydrolysis of AHL substrates with different N-acyl side chain lengths or substitutions at the C-3 carbon (see Table 2). With the exception of C4-HSL, the kcat values ​​for the other substrates ranged from 32.4 to 278.8 s -1 The Km value gradually decreases with the increase of N-acyl side chain length. These kinetic studies show that PpL is highly efficient in hydrolyzing medium- and long-chain AHLs, showing a 10 5 -10 6 M -1 ·s -1 This catalytic efficiency is similar to that of GcL and Aal, and significantly exceeds that of AiiA, AiiB, and MomL in the AHLase family. It is worth noting that C8-HSL is the most favorable substrate for PpL, with a kcat / Km value of 2.1×10 6 M -1 ·s -1 Regarding C4-HSL, mass spectrometry results showed that a small amount of C4-HS was produced in the control group (the reaction mixture lacked enzyme) compared to the experimental group. Some studies have suggested that C4-HSL undergoes slow spontaneous hydrolysis at 37°C. Therefore, we speculate that the small amount of C4-HS produced may be the result of self-hydrolysis. In contrast, PpL exhibited lower affinity (Km = 2.17 mM) and catalytic efficiency (1.8 x 10^3 M-1 S-1) for C4-HSL, similar to the characteristics exhibited by AiiB.

[0071] Table 2 Kinetic constants of PpL hydrolysis of AHLs

[0072]

[0073] Note: Enzyme activity reactions were performed at pH 8.0 and 37°C. Initial reaction rates were calculated by subtracting the background hydrolysis rate of AHLs in the absence of enzyme. Due to the poor solubility of C12-HSL, the hydrolysis kinetic constant was not determined. ND: not determined; SD: standard deviation; AHL: acylhomoserine lactonase.

[0074] (6) PpL inhibits A. baumannii biofilm formation

[0075] The present invention evaluates the potential of PpL as a lactonase to inhibit biofilm formation in Acinetobacter baumannii, a pathogen known to produce and utilize AHLs. The present invention uses 96-well microtiter plates to form biofilms of Acinetobacter baumannii and quantifies the biofilms by crystal violet staining. Compared with the untreated group, when the concentration of PpL protein was 5μg / ml, the formation of biofilm was reduced to 52%. As the concentration of PpL protein increased, biofilm formation gradually decreased, and when the protein concentration reached 160μg / ml, it was reduced to 22%, indicating that PpL, as a quorum sensing inhibitory enzyme, can effectively inhibit the formation of biofilms of Acinetobacter baumannii in a dose-dependent manner. In the present invention, experimental groups with the addition of serum albumin (BSA) and 5-fluorouracil were used as negative and positive controls.

[0076] (7) Hexameric transformation of non-hexameric MLLs

[0077] Hexamerization of non-hexameric MLLs can effectively improve the thermal stability of MLLs. The present invention provides a hexamerization modification scheme. This scheme uses AiiB and AaL as examples for modification. Figure 8 B and Figure 9 B shows the new sequences formed after the modification of AiiB and AaL according to the present invention, and the homologous hexamer predicted by AlphaFold Multimer. Their pTM+ipTM are 0.88 and 0.94 respectively, indicating that the interaction between the subunits is strong and can form a stable hexamer. In addition, the amino acids involved in the interaction between the subunits were analyzed by PDBePISA, and it was found that a single subunit in AiiB and AaL formed multiple chemical bonds with the adjacent subunits on the left and right ( Figure 8 CD and Figure 9 CD), further demonstrating the formation of hexamer.

[0078] 3. Conclusion and Discussion

[0079] The lactonase PpL from Pseudomonas phage has a specific substrate range, specifically and efficiently hydrolyzing medium- and long-chain AHLs while showing minimal catalytic activity against short-chain AHLs (C4-HSL). This substrate specificity distinguishes it from most MLL lactonases, which can hydrolyze both short- and long-chain AHLs. PpL's ability to hydrolyze AHLs can inhibit biofilm formation in Acinetobacter baumannii.

[0080] PpL is the first AHLase discovered to date that exists in the form of a hexamer. Other reported AHLases are characterized as monomers and dimers. Structural comparisons revealed that PpL and reported AHLases have low sequence similarity and high structural similarity. Experimental results show that PpL has high thermal stability, with a Tm value of 71.2°C, which is significantly higher than that of other AHLases (Tm values ​​of approximately 43-58.2°C). At 70°C, PpL maintains 80% of its catalytic activity, while the enzymatic activity of other MLLs decreases significantly starting at 60°C. It is speculated that the high thermal stability of PpL is due to its existence in the form of a hexamer. Hexamerization of non-hexameric AHLases can effectively improve their thermal stability. Therefore, the present invention provides a method for hexamerizing AHLases and successfully hexamerized AiiB and AaL.

[0081] The present invention has conducted a comprehensive study on PpL, including its substrate specificity, enzyme catalytic efficiency, its protein structural characteristics, and various physical and chemical factors affecting its enzyme activity. In addition, the results show that PpL can inhibit the formation of Acinetobacter baumannii biofilm in a dose-dependent manner. Enzyme kinetics results show that PpL has a high hydrolysis efficiency for medium- and long-chain AHLs (kcat / kM≈10 5 ~10 6 M -1 ·s -1 In conclusion, PpL represents a new class of AHLases with high thermostability and high catalytic activity.

[0082] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Application of PpL in the preparation of an inhibitor for inhibiting the formation of Gram-negative bacterial biofilm, characterized in that: PpL is a lactonase composed of 306 amino acids with accession number WP_082630345, and the Gram-negative bacteria is Acinetobacter baumannii.

2. The use of PpL according to claim 1 in the preparation of an inhibitor for inhibiting gram-negative bacterial biofilm formation, characterized in that: The amino acid sequences in the PpL involved in the inter-subunit interaction include the sequence located at positions 26-41 of the PpL amino acid sequence as shown in SEQ ID NO.1, the sequence located at positions 140-153 of the PpL amino acid sequence as shown in SEQ ID NO.2, and the sequence located at positions 226-239 of the PpL amino acid sequence as shown in SEQ ID NO.

3.

3. The use of PpL according to claim 1 in the preparation of an inhibitor for inhibiting the formation of Gram-negative bacterial biofilm, characterized in that: The PpL inhibits A. baumannii biofilm formation by degrading acylhomoserine lactones.

4. The use of PpL according to claim 1 in the preparation of an inhibitor for inhibiting gram-negative bacterial biofilm formation, characterized in that: The PpL structure is a barrel-shaped hexamer.

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