Application of acinetobacter baumannii bacteriophage depolymerases in preparation of bacteriostatic agents

Abstract

The invention discloses application of acinetobacter baumannii bacteriophage depolymerases in preparation of bacteriostatic agents, and relates to the technical field of biology. The depolymerases disclosed by the invention can inhibit the growth of a plurality of acinetobacter baumannii, have a bacteriostatic effect on the acinetobacter baumannii, and lay a foundation for developing effective infection control strategies and treatment methods to resist infection of the multi-drug-resistant acinetobacter baumannii.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of Acinetobacter baumannii phage depolymerase in the preparation of bacteriostatic agents. Background Art

[0002] Acinetobacter baumannii is a common pathogenic bacterium causing hospital-acquired infections, which can lead to pneumonia, urinary tract inflammation and severe bloodstream infections. A major virulence determinant of Acinetobacter baumannii is the capsule that wraps around the surface of the bacterium. The capsule consists of tightly packed repeating polysaccharide units, forming a protective barrier around the bacterial cell wall, protecting against environmental stresses (such as drying and disinfectants) and host immune responses (such as serum complement). The capsule polysaccharide structures vary significantly, resulting in more than 100 different capsule types of Acinetobacter baumannii. Since most of the multi-drug resistant Acinetobacter baumannii isolated clinically have capsules, the available effective antibiotics are decreasing, and the treatment options for Acinetobacter baumannii infections are becoming increasingly limited. Therefore, to combat multi-drug resistant Acinetobacter baumannii infections, the urgency of developing effective infection control strategies and treatment methods is obvious.

[0003] Phages, also known as bacterial viruses, are isolated from sewage, patient sputum, clinical samples or marine and pond samples, and show high specificity for the lysis of bacteria, so they do not destroy the normal flora. In addition, the depolymerase encoded by the phage genome is a structural component of the adsorption device, which helps in the binding and digestion of the capsule, and can specifically bind and hydrolyze the polysaccharides on the surface of bacteria. After specifically binding to the capsular polysaccharide (CPS), exopolysaccharide (EPS) or lipopolysaccharide (LPS) on the surface of the host bacteria, the polysaccharide repeating units are specifically cleaved, and then the phage reaches the last barrier, that is, the cell wall, and then injects its DNA and infects the bacteria to produce progeny phages. Finally, the cell membrane of the host bacteria is lysed, releasing the progeny phages. Since the bactericidal effect of phages is not affected by drug resistance, phages and phage-derived enzymes are considered as alternatives to antibiotics for treating drug-resistant bacteria. In particular, the depolymerase that can hydrolyze the capsule can inhibit the growth of pathogenic bacteria by removing the capsule without killing the bacteria, so it does not exert a selective pressure on the bacteria to cause the bacteria to develop resistance to the depolymerase.

[0004] Depolymerase is a structural component of the adsorption device, which helps in the binding and digestion of the capsule. Biochemically, depolymerases are divided into two categories, lyases and hydrolases. Different from hydrolases, lyases cleave their substrates in a non-hydrolytic manner, which means that water molecules are not released after substrate cleavage.

[0005] LPS-targeting enzymes are commonly referred to as tail spike proteins (TSPs), while capsule-targeting enzymes are commonly referred to as depolymerases (Dpos).

[0006] The term depolymerase can refer to any general protein capable of degrading polymers. The therapeutic effect of depolymerases lies in their ability to hydrolyze or strip the capsule and expose the underlying bacteria to immune attack, such as complement-mediated killing. Therefore, the capsular depolymerases of bacteriophages are a promising antibacterial therapeutic tool. SUMMARY OF THE INVENTION

[0007] In view of this, the object of the present invention is to provide the application of Acinetobacter baumannii phage depolymerase in the preparation of bacteriostatic agents. This depolymerase can specifically lyse multidrug-resistant Acinetobacter baumannii, thereby achieving a bacteriostatic effect.

[0008] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0009] Use of an Acinetobacter baumannii phage depolymerase in the preparation of a bacteriostatic agent, wherein the amino acid sequence of the Acinetobacter baumannii phage depolymerase is as shown in SEQ ID NO.1.

[0010] The nucleotide sequence of the coding gene of the Acinetobacter baumannii phage depolymerase is as shown in SEQ ID NO.2.

[0011] Preferably, the bacteriostatic spectrum of the bacteriostatic agent is Acinetobacter baumannii. In the embodiments of the present invention, the depolymerase Dpo164 can inhibit the growth of Acinetobacter baumannii XH1363, XH1365 and ATCC 17978.

[0012] The preparation method of the Acinetobacter baumannii phage depolymerase comprises the following steps: expressing the Acinetobacter baumannii phage depolymerase gene with the nucleotide sequence as shown in SEQ ID NO.2.

[0013] Specifically, it comprises the following steps:

[0014] (1) Construct a recombinant expression vector for expressing Acinetobacter baumannii phage lyase;

[0015] (2) Transform the recombinant expression vector into Escherichia coli competent cells, and screen to obtain an engineering bacterium expressing Acinetobacter baumannii phage lyase;

[0016] (3) Induce expression with isopropyl-β-D-thiogalactoside (IPTG) to obtain a recombinant gene expression product;

[0017] (4) The recombinant gene expression product was purified and separated by nickel column affinity chromatography to obtain recombinant Acinetobacter baumannii phage lyase.

[0018] The expression vector backbone of the recombinant expression vector is pQE60. The engineering bacterium is Escherichia coli M15. The bacteriostatic agent is a liquid agent. The concentration of the bacteriostatic agent is not less than 1 μM.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The depolymerase of the present invention can inhibit the growth of various Acinetobacter baumannii. The depolymerase has a bacteriostatic effect on Acinetobacter baumannii, laying a foundation for the development of effective infection control strategies and treatment methods to combat multi-drug resistant Acinetobacter baumannii infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the IPTG-induced expression map of recombinant depolymerase Dpo164; among them, the left figure is the rapid Coomassie brilliant blue staining, and the right figure is Western Blot (protein blot hybridization); Protein Marker: protein molecular weight standard; 1-4h: induced by IPTG for 1-4 hours respectively; Before induction: not induced by IPTG.

[0022] Figure 2 It is the elution and purification map of recombinant depolymerase Dpo164; among them, the left figure is the rapid Coomassie brilliant blue staining, and the right figure is Western Blot (protein blot hybridization); Protein Marker: protein molecular weight standard; Homogenization: the collected product after homogenization and fragmentation; Flow through: the effluent from the first pass through the nickel column; Wash3, Wash 4: the effluents after rinsing with 30 mM and 50 mM imidazole respectively; Elution: the protein collection solution treated with 250 mM imidazole.

[0023] Figure 3 It is the bacteriostatic effect diagram of recombinant depolymerase Dpo164 on multiple strains of Acinetobacter baumannii. DETAILED DESCRIPTION OF THE INVENTION

[0024] The phage Phab24 of the present invention was preserved in the China Center for Type Culture Collection (address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on February 23, 2023, and the preservation number is CCTCC NO: M 2023182.

[0025] Example 1: Extraction of phage Phab24 gene

[0026] (1) Take 300 μL of high-titer phage Phab24 lysate (PFU reaches 10 9 / mL or higher) and add it to a clean (RNase-free) 1.5 mL centrifuge tube. Then add 1 μL of DNAse I (2000 U / mL) and 2 μL of RNAse A (25 mg / mL). After mixing, incubate at 37 °C for 2 h. The purpose is to digest the residual bacterial DNA and RNA in the phage lysate;

[0027] (2) Transfer to 75 °C for 10 min to inactivate the nuclease;

[0028] (3) Add 20 μL of proteinase K (20 mg / mL) and 300 μL of Carrier RNA working solution, and immediately vortex to mix well;

[0029] (4) Incubate at 56 °C for 30 min, and invert and mix several times during this period to fully digest the protein capsid of the phage;

[0030] (5) After cooling to room temperature, add 250 μL of absolute ethanol, immediately vortex to mix well, and let it stand at room temperature for 5 min;

[0031] (6) Transfer the above mixture to an adsorption column RA, place the adsorption column in a collection tube and centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0032] (7) Add 500 μL of protein removal solution RE, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid;

[0033] (8) Add 500 μL of washing solution (anhydrous ethanol has been added), centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid;

[0034] (9) Repeat step (8);

[0035] (10) Place the adsorption column RA back into the collection tube, centrifuge at 12000 rpm for 2 min to thoroughly remove the residual washing solution, and then place the adsorption column on a clean paper towel to dry for several minutes;

[0036] (11) Take out the adsorption column RA and place it in a clean RNase-free centrifuge tube. Drop 50 μL of preheated (65 °C) ultrapure water in the center of the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min;

[0037] (12) Use NanoDrop to measure the concentration of the extracted nucleic acid; store the extracted phage nucleic acid at -20 °C for later use.

[0038] Example 2: Cloning of depolymerase Dpo164 and construction of recombinant prokaryotic expression vector

[0039] (1) Select the expression plasmid pQE60, which contains the T5 promoter and can be induced to express by IPTG; it contains the resistance gene for ampicillin, enabling the host bacterium M15 (genotype: lac, ara, gal, mtl, recA+, uvr+ [pREP4, lacI, kanaR]) to grow on the LB plate containing ampicillin and kanamycin;

[0040] (2) Based on the cloning method of Gibson assembly, according to the coding gene ORF164 nucleotide sequence (SEQ ID NO.2) of the depolymerase Dpo164 (amino acid sequence shown in SEQ ID NO.1) and the plasmid, primers were designed respectively to amplify the target fragment and linearize the vector, and the primer information is shown in Table 1;

[0041] Table 1 Primer Sequences

[0042]

[0043]

[0044] Use the phage Phab24 genome in Example 1 as the template, and use primers 1 and 2 to amplify the target fragment to be inserted; use the plasmid pQE60 as the template, and primers 3 and 4 are used to amplify and linearize the vector;

[0045] (3) Adopt the NEB Q5 high-fidelity DNA polymerase system in Table 2;

[0046] Table 2

[0047] Component Volume (μL) <![CDATA[ddH2O]]> 16.5 5×Reaction buffer 5 100μM dNTPs 0.5 10μM Forward primer 1.25 10μM Reverse primer 1.25 Template 0.25 Q5 High-Fidelity DNA Polymerase 0.25 Total volume 25

[0048] PCR running parameters: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing at 65°C for 15 s, extension at 72°C for several minutes, a total of 34 cycles; extension at 72°C for 5 min, and then store at 12°C.

[0049] In each cycle, the extension time at 72°C depends on the size of the fragment to be amplified, and the extension time is determined according to the synthesis speed of 1 kb / min. The extension time in this example is 2 minutes;

[0050] (4) Prepare a 1% agarose gel with TBE buffer:

[0051] Weigh 0.3 g of agarose powder into a conical flask, add 30 mL of TBE buffer, heat and boil it in a microwave oven, add 3 μL of Gelgreen dye, and finally pour it into the mold to cool and form;

[0052] (5) After mixing the PCR-linearized plasmid and the target fragment with the loading buffer respectively, perform electrophoresis at 100 V for 60 min;

[0053] (6) After the electrophoresis is completed, take out the gel, place it on a clean blue light plate to observe the size of the target band, and cut the gel area where the target band is located into a clean centrifuge tube with a clean blade;

[0054] (7) Purify the PCR product using the QIAGEN Gel Extraction Kit:

[0055] ① Add 300 μL of orange Buffer G to the tube, place it on a 50 °C metal bath and heat until the gel is completely dissolved. During this period, the sample can be shaken several times to help dissolve the gel;

[0056] ② After adding 100 μL of isopropanol and mixing well, transfer it to the adsorption column and centrifuge at 13000 rpm for 1 min;

[0057] ③ Pour out the liquid at the bottom of the tube, add 700 μL of Buffer E to the adsorption column, centrifuge at 13000 rpm for 1 min. After pouring out the eluate again, put the adsorption column back into the tube and centrifuge at 13000 rpm for 1 min;

[0058] ④ Take out the adsorption column and place it on a clean paper towel for several minutes to help the evaporation of organic solvents such as alcohol in the eluate. Then place the adsorption column into a clean EP tube, add 40 μL of ultrapure water preheated on a 65 °C metal bath to the center of the adsorption column, let it stand at room temperature for several minutes, and then centrifuge at 13000 rpm for 1 min;

[0059] ⑤ Measure the concentration of the purified product on the NanoDrop;

[0060] (8) Calculate the required volume according to the concentrations of the target fragment and the vector, construct the Gibson seamless ligation system, and then incubate it at 50 °C for 30 min; Add the components as shown in Table 3 in sequence;

[0061] Table 3

[0062] Component Volume (μL) Target fragment to be inserted 3 Linearized vector fragment 2 2×Seamless Clone Mixture 5 Total volume 10

[0063] (9) Pipette 5 μL of the product after seamless ligation into the competent XL10-Gold (product number BC109-01, Beijing Bomed Gene Technology Co., Ltd.), mix gently, ice bath for 15 min, then incubate at 42 °C for 90 s, immediately ice bath for 2 min, and finally add 600 μL of LB, resuscitate at 37 °C and 200 rpm for 1 h;

[0064] (10) Pipette 100 μL of the resuscitated bacterial solution and spread it on an LB plate containing 100 mg / L ampicillin, and incubate overnight at 37°C;

[0065] (11) Randomly pick monoclonal colonies the next day into 3 mL of LB containing 100 mg / L ampicillin, incubate at 37°C and 200 rpm until the bacterial solution becomes turbid. Then, pipette 500 μL of the bacterial solution and send it to Shanghai Sangon Biotech Co., Ltd. for first-generation sequencing. The sequencing primers are the universal primers pQE30+ and pQE30-, and the remaining bacterial solution is stored at 4°C;

[0066] pQE30+: 5′-GTGAGCGGATAACAATTTCAC-3′,

[0067] pQE30-: 5′-CTGAACAAATCCAGATGGAG-3′;

[0068] (12) After the sequencing results are obtained, compare them with the constructed plasmid map, and select the sample with a completely matching sequence for plasmid extraction. Use the plasmid miniprep kit from Beijing Bomed to extract the plasmid and name it plasmid pQE60-carO with inner 6×His;

[0069] (13) Pipette 1 μL of the constructed plasmid pQE60-carO with inner 6×His into the host bacterium M15 melted on ice. Gently flick the bottom of the tube to help mix, then immediately place it on ice for 15 min, then place it in a 42°C metal bath for 90 s, and then place it on ice for 2 min. Add 500 μL of LB, incubate at 37°C and 200 rpm for 1 h;

[0070] (14) Pipette 100 μL of the bacterial solution and evenly spread it on a medium containing ampicillin (100 mg / L) and kanamycin (50 mg / L) using glass beads, and incubate overnight at 37°C;

[0071] (15) Pick a single colony the next day into LB containing ampicillin (100 mg / L) and kanamycin (50 mg / L), and incubate overnight at 37°C and 200 rpm. This is M15 containing the recombinant prokaryotic expression vector pQE60-ORF164.

[0072] Example 3: Induced Expression and Purification of Recombinant Depolymerase

[0073] (1) Inoculate a single colony of M15 containing the recombinant prokaryotic expression vector pQE60-ORF164 into 20 ml of LB broth containing ampicillin and kanamycin, incubate at 37°C and 200 rpm overnight;

[0074] (2) Inoculate 20 ml of the overnight culture into 800 ml of LB broth containing ampicillin at 37 °C and 220 rpm until the OD600nm reaches 0.6 - 0.8;

[0075] (3) Add 200 μL of 1 M IPTG and induce the culture at 30 °C and 220 rpm for 2 to 4 h. The incubation time depends on the experimental results of small-scale induction of recombinant protein expression;

[0076] (4) Centrifuge at 5000 g for 25 min at 4 °C to collect the bacterial cells and discard the supernatant;

[0077] (5) Resuspend the bacterial cell pellet in 40 ml of pre-cooled PBS, transfer it to a clean 50 ml centrifuge tube, centrifuge at 4200 rpm for 25 min at 4 °C, and discard the supernatant;

[0078] (6) Quickly freeze the bacterial cell pellet in liquid nitrogen and finally store it in a -80 °C ultra-low temperature freezer for later use;

[0079] (7) Take out the frozen bacterial cells and let them thaw at room temperature. Add pre-cooled Binding buffer to a total volume of 40 mL, and then add 400 μL of 0.1 M phenylmethylsulfonyl fluoride (PMSF) to resuspend the bacterial cells;

[0080] (8) Pour the resuspended bacterial cells into the sample collection tank of the homogenizer and homogenize until the final bacterial liquid becomes transparent. Collect the lysed bacterial cells into a clean Beckman centrifuge tube and centrifuge at 18000 rpm for 45 min at 4 °C;

[0081] (9) Take 80 μL of the supernatant for subsequent SDS-PAGE electrophoresis. Pour the remaining supernatant into 1 mL of Ni-NTA resin that has been pre-washed with PBS and Binding buffer, and incubate at 4 °C and 10 rpm for 3 h to allow the tagged protein in the supernatant to bind fully to the resin;

[0082] (10) Install the column and rinse the column with 10 ml of Binding buffer, then pour in the mixture of supernatant and resin, and collect 80 μL of the liquid flowing out of the column;

[0083] (11) Wash the resin successively with Washing buffer containing different concentrations of imidazole and collect 80 μL of the liquid flowing out of the column;

[0084] (12) Elute the resin with 5 mL of Elution buffer for 20 min and collect the final protein to obtain the expression product Dpo164. Take 80 μL of the purified depolymerase Dpo164 product for subsequent SDS-PAGE electrophoresis, and store the eluted protein at 4 °C;

[0085] (13) Add 20 μL of 5×PBS to 80 μL of the sample collected at different time intervals and boil for 10 min;

[0086] (14) Prepare two precast SDS-PAGE gels, 10 μL per well, perform vertical electrophoresis at 150 V for 45 min;

[0087] (15) Place one gel in Coomassie Brilliant Blue Fast Staining Solution and let it stand at room temperature for 30 min until the protein bands are clearly visible. Then pour out the staining solution, put the gel in deionized water for preservation and take pictures;

[0088] (16) Transfer the other parallel gel, at 180 V for 90 min, block it in 5% skim milk for 30 min, incubate with the primary antibody for 1 h, and rinse with PBS-T for 30 min; then incubate with the secondary antibody for 1 h, rinse with PBS-T for 30 min, and finally place it in PBS;

[0089] (17) Use clean forceps to take out the NC membrane and place it on the tray of the exposure instrument. Drop a freshly premixed hypersensitive ECL luminescent solution A and B with a volume ratio of 1:1 on the surface of the NC membrane and expose for an appropriate time.

[0090] The results of SDS-PAGE analysis are as Figure 1 shown. After the M15 bacteria containing the recombinant prokaryotic expression vector pQE60-ORF164 were induced by IPTG, there was an induced protein band in the supernatant at around 80 KD, which was consistent with the expected size. This indicates that the M15 engineering bacteria containing the recombinant prokaryotic expression vector pQE60-ORF164 can be induced to express the target protein, and the expressed depolymerase protein product Dpo164 is a soluble protein. Figure 2 shown. There were target protein bands in the effluent after homogenization and fragmentation, the first nickel column passing, and elution with 30 mM imidazole, and the size was close to 80 KD; the collected solution after treatment with 250 mM imidazole finally contained a high concentration of the target protein; indicating that the constructed engineering bacteria M15 can be homogenized and fragmented after induced expression, and high-concentration target protein can be collected after elution and treatment with different concentrations of imidazole.

[0091] Example 6: Bacteriostatic effect of depolymerase Dp0164 on Acinetobacter baumannii

[0092] Acinetobacter baumannii XH194, XH1363, and XH1365 were isolated from Sir Run Run Shaw Hospital, Zhejiang University.

[0093] Inoculate an appropriate amount of overnight cultured bacterial solution into fresh LB to make the initial OD600nm 0.8, then add the purified depolymerase Dpo164 product to a final concentration of 1 μM, place it in a microplate reader set at 37 °C, shake at 180 rpm, and read OD600nm at different time points. XH194 is Acinetobacter baumannii that is insensitive to phage Phab24, and XH1363, XH1365, and ATCC 17978 are all Acinetobacter baumannii sensitive to phage Phab24.

[0094] Figure 3 It is the bacteriostatic effect diagram of depolymerase Dpo164 on multiple strains of Acinetobacter baumannii. The above 4 strains of Acinetobacter baumannii (XH194 is insensitive to phage Phab24, and XH1363, XH1365, and ATCC 17978 are all sensitive to phage Phab24) were respectively inoculated into fresh LB, shake-cultured (37 °C, 200 rpm) until OD600 reached 0.5, then add the purified depolymerase Dpo164 to a final concentration of 1 μM, continue shake-culturing, and detect OD600 at different time nodes. The results showed that for XH194, which is insensitive to phage Phab24, its OD600 increased to nearly 1 after adding depolymerase Dpo164 and grew well; while the growth curves of the other three strains were flat after adding depolymerase Dpo164, indicating that depolymerase Dpo164 can inhibit the growth of XH1363, XH1365, and ATCC 17978.

Claims

1. Use of Acinetobacter baumannii phage depolymerase in the preparation of an antibacterial agent, wherein the amino acid sequence of the Acinetobacter baumannii phage depolymerase is as shown in SEQ ID NO.

1.

2. The application according to claim 1, wherein The nucleotide sequence of the coding gene of the Acinetobacter baumannii phage depolymerase is as shown in SEQ ID NO.

2.

3. The application according to claim 1 or 2, characterized in that, The antibacterial spectrum of the antibacterial agent is Acinetobacter baumannii.

4. The application according to claim 1 or 2, characterized in that, The preparation method of the Acinetobacter baumannii phage depolymerase comprises the following steps: expressing the Acinetobacter baumannii phage depolymerase gene with the nucleotide sequence as shown in SEQ ID NO.

2.

5. The application according to claim 4, wherein The preparation method of the Acinetobacter baumannii phage depolymerase specifically comprises the following steps: (1) Construct a recombinant expression vector expressing Acinetobacter baumannii phage lyase; (2) Transform the recombinant expression vector into Escherichia coli competent cells, and screen to obtain an engineered bacterium expressing Acinetobacter baumannii phage lyase; (3) Induce expression with isopropyl-β-D-thiogalactoside to obtain a recombinant gene expression product; (4) Purify and isolate the recombinant gene expression product by nickel column affinity chromatography to obtain recombinant Acinetobacter baumannii phage lyase.

6. The application according to claim 5, characterized in that, The expression vector backbone of the recombinant expression vector is pQE60.

7. The application according to claim 5, characterized in that The engineered bacterium is Escherichia coli M15.

8. The application according to claim 1, wherein The antibacterial agent is a liquid agent.

9. The application according to claim 1, characterized in that, The concentration of the antibacterial agent is not less than 1 μM.

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