Recombinant antibiotic enzyme PlyTC37 as well as coding gene and application thereof
By modifying the C-terminal domain of the antibiotic enzyme PlyF307, a recombinant antibiotic enzyme PlyTC37 was constructed, which solved the problems of low activity and poor stability of existing antibiotic enzymes in Gram-negative bacterial infections, and achieved a highly efficient bactericidal effect against a variety of Gram bacteria, especially a significant inhibition of drug-resistant bacteria.
Patent Information
- Application Number
- CN202511626947.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-07
- Publication Date
- 2026-01-23
AI Technical Summary
Existing antibiotic enzymes have problems such as low activity, narrow spectrum and poor stability in the treatment of Gram-negative bacterial infections. In particular, the prevalence of drug-resistant Gram-negative bacteria such as extended-spectrum β-lactamase-producing Escherichia coli and carbapenem-resistant Acinetobacter baumannii is becoming increasingly serious, making it difficult to meet clinical needs.
By optimizing the C-terminus structure of the natural antibiotic enzyme PlyF307, a recombinant antibiotic enzyme PlyTC37 was constructed, which enhanced its ability to penetrate the outer membrane of Gram-negative bacteria and broadened its bactericidal spectrum while maintaining highly efficient bactericidal activity.
The recombinant antibiotic enzyme PlyTC37 exhibits significant bactericidal effects against a variety of Gram-negative bacteria and some Gram-positive bacteria under physiological conditions, with a MIC value below 25 μg/mL and a bacterial load reduction of 4-5 log units, significantly improving the therapeutic effect against drug-resistant bacteria.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of antibacterial enzymes, and particularly relates to a recombinant antibacterial enzyme PlyTC37, a coding gene thereof and application thereof. BACKGROUND
[0002] The discovery and application of antibiotics is a major milestone in the history of human medicine, which has greatly reduced the incidence and mortality of bacterial infectious diseases. However, with the widespread use and even abuse of antibiotics in medicine, agriculture and animal husbandry, the problem of bacterial drug resistance is becoming increasingly serious. The World Health Organization (WHO) has listed antibiotic resistance as one of the most urgent threats in the field of global public health. The evolution speed of drug-resistant strains far exceeds the development speed of new antibiotics, leading to the "no drug available" dilemma in clinical treatment. At present, multiple drug-resistant bacteria (MDR), extensively drug-resistant bacteria (XDR) and even pan-drug-resistant bacteria (PDR) have appeared in the clinic, such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Enterobacteriaceae (CRE) and multidrug-resistant Mycobacterium tuberculosis, etc. These "superbugs" show extensive drug resistance to existing antibiotics, significantly increasing the difficulty of treatment, medical costs and patient mortality.
[0003] In the face of this serious challenge, it is urgent to develop new antibacterial drugs. Compared with traditional antibiotics, antibacterial enzymes (such as lysozyme, lysostaphin and phage lytic enzyme) show great potential due to their unique bactericidal mechanism and biological characteristics. Antibacterial enzymes can specifically hydrolyze the peptidoglycan structure of bacterial cell wall, leading to rapid lysis and death of bacteria. Compared with antibiotics, antibacterial enzymes have the following significant advantages: (1) high specificity, only targeting specific bacterial species without affecting normal human flora; (2) fast bactericidal speed, which can take effect within minutes; (3) not easy to induce drug resistance, as the target is the conserved structure of bacterial cell wall; (4) can synergize with antibiotics to enhance bactericidal effect. Based on these characteristics, antibacterial enzymes have been successfully applied to the treatment of Gram-positive bacterial infections, such as CF-301 against Staphylococcus aureus and Cpl-1 against Streptococcus pneumoniae, which have entered the clinical trial stage.
[0004] However, existing antibiotics face significant technical bottlenecks in treating gram-negative bacterial infections. Gram-negative bacteria have a unique cell wall structure, with their outer membrane composed of lipopolysaccharide (LPS) and porin proteins, forming a natural barrier that hinders the contact of antibiotics with the inner layer of peptidoglycan. In addition, the peptidoglycan layer of gram-negative bacteria is thin and has low cross-linking, further limiting the bactericidal efficiency of antibiotics. The few antibiotics reported against gram-negative bacteria (such as PlyF307 against Acinetobacter baumannii, LysKp against Klebsiella pneumoniae, and LysPA26 against Pseudomonas aeruginosa) generally have low activity, narrow spectrum, poor stability, and other problems, making it difficult to meet clinical needs. In particular, the prevalence of clinically important drug-resistant gram-negative bacteria (such as extended-spectrum beta-lactamase-producing Escherichia coli and carbapenem-resistant Acinetobacter baumannii) is increasingly serious, and new types of antibacterial preparations need to be developed urgently.
[0005] To address the above problems, researchers have attempted to modify antibacterial enzymes through protein engineering techniques in recent years, mainly including: (1) adding active peptides that penetrate the outer membrane (such as antibacterial peptides or phage tail fiber proteins); (2) constructing fusion proteins to enhance targeting; (3) optimizing enzymatic properties to improve stability.
[0006] Although PlyF307 is reported to have activity against Acinetobacter baumannii, its activity is severely dependent on low ionic strength environments. In the presence of physiological concentrations of salt ions (such as 150 mM NaCl), its activity is significantly inhibited, with a bactericidal effect (about 2 logs reduction) far from meeting clinical treatment needs (usually requiring ≥ 3-4 logs reduction). More importantly, PlyF307 is essentially ineffective against other clinically important gram-negative bacteria (such as Pseudomonas aeruginosa and Klebsiella pneumoniae) and gram-positive bacteria, and its narrow bactericidal spectrum and harsh activity conditions severely limit its clinical application. It is generally believed by those skilled in the art that it is difficult to simultaneously solve the dual problems of penetrating the outer membrane of gram-negative bacteria and maintaining high activity under physiological salt conditions through simple modifications.
[0007] Therefore, the development of a new type of recombinant antibacterial enzyme that can efficiently penetrate the outer membrane of gram-negative bacteria, has broad-spectrum bactericidal activity, and has good stability is of great significance for solving the clinical problem of drug-resistant gram-negative bacterial infections. SUMMARY
[0008] To overcome the above technical problems, the present application constructs an artificially modified recombinant antibacterial enzyme through an innovative molecular design strategy. This enzyme not only retains the catalytic activity of natural antibacterial enzymes, but also significantly improves the penetration ability of the outer membrane of gram-negative bacteria and the bactericidal spectrum through structural optimization, providing a new solution for the clinical treatment of drug-resistant bacterial infections.
[0009] In a first aspect, the present application provides a recombinant lysin, wherein the amino acid sequence of the recombinant lysin is any one of the following, (1) the amino acid sequence shown in SEQ ID NO. 1; (2) an amino acid sequence derived from (1) by substitution and / or deletion and / or addition of one or more amino acid residues, having 95% sequence identity and having the same function.
[0010] In the present application, the lysin PlyF307 (GenBank: AJG41873.1, aa1-146) from Acinetobacter phage RL-2015 has muramidase activity, but in the presence of physiological concentration of NaCl, PlyF307 reduces the load of Acinetobacter baumannii by only about 2 logarithmic units, and has limited bactericidal ability, and has no significant activity on Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. In the present application, the C-terminal of PlyF307 sequence is modified, and the recombinant lysin obtained after modification is named PlyTC37, and the amino acid sequence is shown in SEQ ID NO. 1.
[0011] Further, the recombinant lysin is obtained by modifying the C-terminal of the natural lysin PlyF307 derived from phage, and the amino acid sequence of the natural lysin PlyF307 is shown in SEQ ID NO. 3.
[0012] Further, the recombinant lysin can reduce the bacterial load of Acinetobacter baumannii by at least 3 logarithmic units in the presence of physiological concentration of NaCl.
[0013] Further, the minimum inhibitory concentration (MIC) of the recombinant lysin on at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Salmonella and Pseudomonas aeruginosa is not higher than 25 μg / mL.
[0014] In a second aspect, the present application also provides a polynucleotide encoding the recombinant lysin of the first aspect, and the sequence of the polynucleotide is shown in SEQ ID NO. 2.
[0015] In a third aspect, the present application also provides a biological material containing the polynucleotide of the second aspect, and the biological material is any one of the following, 1) an expression cassette containing the polynucleotide of the second aspect; 2) an expression vector containing the polynucleotide of the second aspect; 3) an expression vector containing the expression cassette of 1); 4) a recombinant microorganism containing the expression vector of 2); 5) a recombinant microorganism comprising the expression vector of 3).
[0016] In a fourth aspect, the present application also provides the use of the recombinant lysin of the first aspect, or the polynucleotide of the second aspect, or the biomaterial of the third aspect in the preparation of a medicament for treating a disease associated with bacterial infection.
[0017] Further, the bacteria are one or more of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Salmonella and Pseudomonas aeruginosa.
[0018] In a fifth aspect, the present application also provides a pharmaceutical composition for treating a disease associated with bacterial infection, the pharmaceutical composition comprising the recombinant lysin of the first aspect, or the polynucleotide of the second aspect, or the biomaterial of the third aspect, and a pharmaceutically acceptable carrier.
[0019] The present application finds that by rationally designing the C-terminal domain of the natural lysin PlyF307, the outer membrane barrier can be significantly overcome. Without significantly affecting the activity of its original catalytic domain, the modification endows the recombinant lysin with the ability to efficiently localize and penetrate the outer membrane of Gram-negative bacteria, so as to contact the inner peptidoglycan substrate and exert the lysis effect. This modification strategy unexpectedly not only enhances the activity against Gram-negative bacteria, but also widens the bactericidal spectrum to part of Gram-positive bacteria. The recombinant lysin PlyTC37 shown in SEQ ID NO. 1 is a preferred embodiment obtained based on the strategy.
[0020] The present application has the following technical effects relative to the prior art.
[0021] 1) The recombinant lysin PlyTC37 of the present application expands the bactericidal spectrum compared with PlyF307, so that it has killing effect on Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Salmonella, Pseudomonas aeruginosa and other Gram-negative bacteria, as well as Staphylococcus aureus, Streptococcus agalactiae, Streptococcus haemolyticus and other Gram-positive bacteria. Among them, the MIC value of the recombinant lysin PlyTC37 against Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Salmonella and Pseudomonas aeruginosa is about 20 μg / ml.
[0022] 2) The recombinant lysin PlyTC37 of the present application has strong antibacterial effect in a complex physiological environment; in the presence of physiological concentration of NaCl, the recombinant lysin PlyTC37 reduces the load of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Salmonella and Pseudomonas aeruginosa by 4-5 logarithmic units, significantly improving its clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 SDS-PAGE of recombinant antibacterial PlyTC37 and antibacterial PlyF307; where, left: antibacterial PlyTC37, middle: antibacterial PlyF307, right: protein Marker. Specific embodiments
[0024] The application will be further described in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not used to limit the scope of the application. The experimental methods not specified in the following examples are generally according to the conventional conditions, the conditions described in the laboratory manual or the conditions suggested by the manufacturer.
[0025] Example Preparation of recombinant antibacterial PlyTC37
[0026] 1. Construction of engineering strain: the polynucleotide sequence encoding antibacterial PlyTC37 was artificially synthesized and ligated between the specific enzyme cutting sites of expression vector ppic9k to obtain a recombinant expression plasmid, which was transformed into competent cells of yeast KM71 to obtain an engineering strain expressing recombinant antibacterial PlyTC37.
[0027] 2. Expression: the positive yeast strain selected was inoculated into 100 mL of BMGY medium and cultured at 30°C and 250 r / min until OD600 reached 2-6. The bacterial cells were collected by centrifugation and resuspended in 500 mL of BmMY medium for induction expression. The culture was incubated at 28°C and 250 r / min for 96 h, and 0.5% (V / V) of methanol was added every 24 h during the incubation. After 96 h of induction expression, the culture supernatant was collected by centrifugation at 10000 r / min for 5 min.
[0028] 3. Purification: the fermentation supernatant was added with purified water to control the conductivity below 10 ms / cm, and then loaded onto a CMFF cation exchange column. The column was washed with 50 mM Tris buffer at pH 8.0 at 0M, 0.2M and 0.6M NaCl gradient, respectively, to obtain the sample by washing, eluting and dialysis. The sample was stored at -20°C for standby use. The SDS-PAGE of the purified sample is shown in Figure 1 .
[0029] Comparative example
[0030] The difference between the comparative example and the example is that in step 1, the polynucleotide sequence encoding antibacterial PlyF307 (SEQ ID NO. 4) was artificially synthesized according to the amino acid sequence of antibacterial PlyF307 (SEQ ID NO. 3, GenBank: AJG41873.1, aa1-146), and the remaining steps were the same as the example. The SDS-PAGE of the purified sample is shown in Figure 1 .
[0031] Example 1 The overnight cultured Acinetobacter baumannii (ATCC 19606), Klebsiella pneumoniae (ATCC 700603), Escherichia coli (ATCC 25922), Salmonella enterica (ATCC 14028), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213) and other strains were subcultured to the mid-log phase, centrifuged to collect the bacterial cells and resuspended in PBS buffer (the concentration of the bacterial solution was adjusted to 1 x 10 6 The bacterial solution, the recombinant bacteriocin PlyTC37 prepared in the example (final concentration 50 μg / mL), the bacteriocin PlyF307 prepared in the comparative example (final concentration 50 μg / mL) and the control protein (bovine serum albumin BSA, final concentration 50 μg / mL) were added to the 96-well plate, and the 96-well plate was placed in a constant temperature shaker and incubated at 37°C, 200 rpm. At 0, 15, 30, 45 and 60 minutes after the start of incubation, the absorbance (OD600) value of each well was measured at 600 nm wavelength using a microplate reader. The wells containing only the bacterial solution and PBS were used as blank controls to correct the baseline.
[0032] Table 1 Effect of different treatments on the OD600 value of bacteria (after 60 minutes of action)
[0033] As shown in Table 1, compared with the blank control and the PlyF307 treatment group, PlyTC37 showed significant bactericidal effect (OD600 value decreased by more than 90%) on all test strains after 60 minutes of treatment, while PlyF307 only had weak activity on Acinetobacter baumannii, and the BSA control group had no bactericidal effect. This indicates that PlyTC37 has rapid and broad-spectrum bactericidal ability.
[0034] Example 2 In vitro MIC experiment The bacterial solution of Acinetobacter baumannii (ATCC 19606), Klebsiella pneumoniae (ATCC 700603), Escherichia coli (ATCC 25922), Salmonella enterica (ATCC 14028), Pseudomonas aeruginosa (ATCC 27853) was diluted to about 1 x 10 5CFU / mL, the prepared recombinant antibacterial PlyTC37, antibacterial PlyF307 prepared in the comparative example and the control protein (lysozyme, Sigma) were serially diluted by two times (concentration gradient was 100, 50, 25, 12.5, 6.25, 3.125 μg / mL, etc.), and 100 μL of the diluted drug solution was added to each well. Then 100 μL of the prepared bacterial solution (the final inoculation amount was about 5×10 5 CFU / mL) was added to each well, and a positive control (bacterial solution without drug) and a negative control (culture medium without bacteria) were set. After the 96-well plate was sealed, it was incubated at 37°C for 18-20 hours.
[0035] The results showed that the MIC value of the recombinant antibacterial PlyTC37 against Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Salmonella and Pseudomonas aeruginosa was about 20 μg / ml, while the antibacterial activity of the comparative example PlyF307 and the control protein lysozyme was not detected in the measured concentration range (MIC > 100 μg / mL). The specific data are shown in Table 2.
[0036] Table 2 MIC values of different antibacterial agents against Gram-negative bacteria (μg / mL)
[0037] Example 3 Bactericidal effect under physiological salt concentration
[0038] In the PBS buffer containing 150 mM NaCl, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Salmonella and Pseudomonas aeruginosa were added (the initial bacterial amount was about 1×10 6 CFU / mL), and the prepared recombinant antibacterial PlyTC37 (final concentration 50 μg / mL) or the comparative example PlyF307 (final concentration 50 μg / mL) was added. After 2 hours of action at 37°C, serial dilution was performed and the number of surviving bacteria (CFU / mL) was counted by plating, and the logarithmic reduction value (Log10 Reduction) of bacterial load was calculated. The results are shown in Table 3.
[0039] Table 3 Bactericidal effect of recombinant antibacterial under physiological salt concentration (150 mM NaCl)
[0040] As shown in Table 3 above, in the presence of physiological concentration of NaCl, the recombinant antibacterial enzyme PlyTC37 reduced the bacterial load of A.baumannii, K.pneumoniae, E.coli, Salmonella and P.aeruginosa by 4-5 Log10 Reduction, showing strong bactericidal activity. The comparative example PlyF307 only had about 2 Log10 Reduction against A.baumannii, and had essentially no activity against other tested gram-negative bacteria (Log10 Reduction <0.3), which fully demonstrates the significant advantage of the recombinant antibacterial enzyme PlyTC37 in physiological environment.
[0041] The specific embodiments are only an explanation of the present application, which is not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.
Claims
1. A recombinant antibacterial enzyme PlyTC37, characterized in that, The amino acid sequence of the recombinant lysin is any one of the following, (1) the amino acid sequence shown in SEQ ID NO. 1; (2) an amino acid sequence derived from (1) by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid shown in SEQ ID NO. 1 and having 95% sequence identity and having the same function.
2. The recombinant bacteriocin PlyTC37 of claim 1, wherein, The recombinant lysin is obtained by modifying the C-terminal end of the natural lysin PlyF307 derived from bacteriophage, and the amino acid sequence of the natural lysin PlyF307 is shown in SEQ ID NO.
3.
3. The recombinant antibacterial enzyme PlyTC37 of claim 1 or 2, wherein, The recombinant lysin can reduce the bacterial load of Acinetobacter baumannii by at least 3 logarithmic units in the presence of physiological concentration of NaCl.
4. The recombinant bacteriocin PlyTC37 of claim 3, wherein, The minimum inhibitory concentration (MIC) of the recombinant lysin to at least one of Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Salmonella and Pseudomonas aeruginosa is not higher than 25 μg / mL.
5. A polynucleotide encoding the recombinant antibacterial PlyTC37 of any one of claims 1-4, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.
2.
6. A biological material comprising the polynucleotide of claim 5. The biological material is any one of the following, 1) an expression cassette containing the polynucleotide of claim 5; 2) an expression vector containing the polynucleotide of claim 5; 3) an expression vector containing the expression cassette of 1); 4) a recombinant microorganism containing the expression vector of 2); 5) a recombinant microorganism containing the expression vector of 3).
7. Use of the recombinant lysin PlyTC37 of any one of claims 1-4, or the polynucleotide of claim 5, or the biological material of claim 6 in the preparation of a medicament for treating diseases related to bacterial infection.
8. A pharmaceutical composition for treating a disease associated with a bacterial infection, wherein the composition comprises a compound of claim 1 or 2. The pharmaceutical composition comprises the recombinant lysin of any one of claims 1-4, or the polynucleotide of claim 5, or the biological material of claim 6, and a pharmaceutically acceptable carrier.