CN109536459B - Phage depolymerase for degrading Klebsiella pneumoniae capsular polysaccharide and biofilm - Google Patents
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Abstract
The invention discloses a phage depolymerase for degrading Klebsiella pneumoniae capsular polysaccharide and biofilm, which obtains a brand-new depo32 with high-efficiency activity by performing prokaryotic expression and purification on the 32 nd open reading frame in the genome of Klebsiella pneumoniae GH-K3. The depolymerizing enzyme has high-efficiency degradation effect on capsular polysaccharide and biofilm formed by the Klebsiella pneumoniae.
Description
Technical Field
The invention discloses a phage depolymerase for degrading Klebsiella pneumoniae capsular polysaccharide and a biofilm, relates to a method for degrading a biofilm formed by Klebsiella pneumoniae by using the phage depolymerase, and belongs to the technical field of biology.
Background
Klebsiella pneumoniae (K.) (Klebsiella pneumoniae) Is a common zoonosis pathogenic bacterium. In recent years, multidrug-resistant klebsiella pneumoniae has become one of the most major pathogens of iatrogenic infections. The bacteria not only pose a great threat to the public health industry of human beings, but also influence the healthy development of the animal breeding industry to a certain extent.
Most of the Klebsiella pneumoniae have the ability to synthesize and secrete capsular polysaccharides. Capsular polysaccharide can maintain bacterial virulence, adhesion and block penetration of some antibiotics by serving as a natural barrier of bacteria. As an important virulence factor of Klebsiella pneumoniae, capsular polysaccharide poses a great threat to public health. In addition, some klebsiella pneumoniae can form a biofilm, namely a membranous structure formed by bacterial cells wrapped by extracellular polysaccharide matrixes, lipoproteins, fibrin and the like generated by bacteria per se, so that the antibiotic resistance of the bacteria can be obviously improved, the capability of escaping from the recognition of a host immune system is avoided, and the colonization of the bacteria in a focus is accelerated. Thus, biofilms are a major causative factor in nosocomial bacterial infections. In addition, the biofilm can be stably attached to the surface of the medical appliance and is difficult to remove by the conventional physical and chemical means, which brings a serious test for preventing and controlling the secondary infection of medical treatment.
The phage depolymerase (depolymerase) degrades bacterial surface polysaccharides, thereby guiding the adsorption of phage to host outer membrane proteins. The enzyme realizes targeted degradation of capsular polysaccharide by randomly attacking glycosidic bonds to release repeating units of polymers. A plurality of domestic and foreign researches show that the phage depolymerase can effectively remove and inhibit the formation of biofilm, and has certain application potential in the public health fields of controlling pathogenic infection and the like.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for degrading bacterial capsular polysaccharide and removing and inhibiting Klebsiella pneumoniae biofilm by using phage depolymerase.
The invention also provides a Depo32 of the phage Depo, which is a brand-new Depo32 with high-efficiency activity obtained by prokaryotic expression and purification of the 32 nd open reading frame in the genome of the Klebsiella pneumoniae GH-K3. The depolymerizing enzyme has high-efficiency degradation effect on capsular polysaccharide and biofilm formed by the Klebsiella pneumoniae.
The invention discloses a Klebsiella pneumoniae phage GH-K3, wherein the bacterial strain is preserved in China center for type culture Collection in 7-6.2018 under the name ofKlebsiella pneumonia phage GH-K3, deposit number: CCTCC NO: M2018455.
The invention relates to a phage Depo32, which is characterized in that: (ii) the 32 nd open reading frame of Klebsiella pneumoniae GH-K3depo32) The nucleotide sequence of the depolymerase obtained by prokaryotic expression and purification is shown as SEQ No. 1; the amino acid sequence is shown as SEQ No. 2.
The preparation method of the phage Depo32 comprises the following steps:
1) PCR, double enzyme digestion and connection molecular cloning means are adopted to obtain the 32 th open reading frame from the Klebsiella pneumoniae GH-K3depo32Gene ligation to pET28a plasmid: cleavage siteNdeI andXho I；
2) pET28a-depo32The plasmid was transformed into E.coli BL21(DE3) and screened to obtain BL21-depo32E.coli;
3) BL21-depo32In 1L of LB liquid medium containing 50. mu.g/ml kanamycin, shaking culture was carried out at 37 ℃ to logarithmic growth phase (OD)600 0.6~1.0）；
4) Adding isopropyl-beta-D-thiogalactoside into the culture solution until the final concentration is 1 mM, and performing shaking culture at 16 ℃ at 180 rpm for 16 hours;
5) BL 21-that will induce expressiondepo32Carrying out centrifugal bacteria collection and centrifugation on the bacterial liquid, and then carrying out ultrasonic crushing;
6) and (3) gently adding the supernatant sample into the balanced Ni-NTA affinity chromatography column, washing the Ni column to remove the foreign proteins, and finally collecting the eluent and performing ultrafiltration to obtain the purified depolymerizing enzyme Depo 32.
The invention discloses application of a phage Depo32 in degrading Klebsiella pneumoniae capsular polysaccharide and biofilm.
The invention relates to a medical application of a phage Depo32 in preparing antibiotic substitute preparations and medical instrument disinfectants.
The invention has the positive effects that:
the invention discloses a Klebsiella pneumoniae GH-K3, which is obtained by carrying out open reading frame(s) at the 32 th position of Klebsiella pneumoniae GH-K3depo32) Prokaryotic expression and purification are carried out to obtain phage depo32, and the depo not only can remove capsular polysaccharide and biofilm formed by Klebsiella pneumoniae, but also has high-efficiency inhibiting effect on the formation of biofilm; can be widely applied to the preparation of antibiotic substitute preparations and medical instrument disinfectants, and has remarkable clinical effect.
Drawings
FIG. 1 is a SDS-PAGE of depo32 of the phage depolymerase of the present invention;
FIG. 2 shows the results of capsular stauroscopy of Klebsiella pneumoniae K7 before and after the depo32 treatment in example;
FIG. 3 shows the results of detection of depo32 in removing Klebsiella pneumoniae biofilm in examples;
FIG. 4 shows the results of the detection of Klebsiella pneumoniae biofilm inhibition by depo32 in example.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
1) PCR, double enzyme digestion and connection molecular cloning means are adopted to obtain the 32 th open reading frame from the Klebsiella pneumoniae GH-K3depo32Gene ligation to pET28a plasmid: cleavage siteNdeI andXho I；
2) pET28a-depo32The plasmid was transformed into E.coli BL21(DE3) and screened to obtain BL21-depo32E.coli;
3) BL21-depo32In 1L of LB liquid medium containing 50. mu.g/ml kanamycin, shaking culture was carried out at 37 ℃ to logarithmic growth phase (OD)600 0.6~1.0）；
4) Adding isopropyl-beta-D-thiogalactoside into the culture solution until the final concentration is 1 mM, and performing shaking culture at 16 ℃ at 180 rpm for 16 hours;
5) BL 21-that will induce expressiondepo32Carrying out centrifugal bacteria collection and centrifugation on the bacterial liquid, and then carrying out ultrasonic crushing;
6) gently adding the supernatant sample into the balanced Ni-NTA affinity chromatography column, washing the Ni column to remove impurity proteins, collecting the eluate, and ultrafiltering to obtain purified Depo32 with nucleotide sequence shown in SEQ No. 1; the amino acid sequence is shown as SEQ No. 2.
Test example 1
Removal of bacterial capsular polysaccharides by depolymerase
1. Expression purification of phage depo32
The plasmid adopted by the prokaryotic expression is pET-28a (+), and the molecular weight of the expressed depolymerizing enzyme is about 98 kDa;
1) recombinant DNAEnterobacter BL21-depo32Induced expression of (1). Through molecular cloning means such as PCR, double enzyme digestion, connection and the likedepo32Gene ligation into pET28a plasmid (restriction site)NdeI andXhoI) (ii) a Then pET28a-depo32The plasmid was transformed into E.coli BL21(DE3) and screened on solid LB plate containing 50. mu.g/ml Kanamycin (Kanamycin, Kan) to obtain BL21-depo32Escherichia coli. 10 ml of overnight-cultured bacterial liquid was inoculated into 1L of LB liquid medium containing 50. mu.g/ml kanamycin and shake-cultured at 37 ℃ for 2 to 3 hours) to logarithmic phase (OD)600 0.6 to 1.0); then adding isopropyl-beta-D-thiogalactoside into the culture solution until the final concentration is 1 mM, and carrying out shaking culture at 16 ℃ at 180 rpm for 16 hours;
2) purification of depo 32. BL 21-that will induce expressiondepo32The cells were collected by centrifugation (6000 rpm, 10 minutes), washed 2-3 times with precooled PBS, and then suspended in an appropriate amount of protein purification buffer. Suspended BL21-depo32After the bacterial liquid is subjected to ice bath, ultrasonic crushing is carried out (working for 5 seconds, intermittent operation for 5 seconds, and ice bath is carried out in the whole process), centrifugation is carried out, and supernatant is sucked. And (3) lightly adding the supernatant sample into the balanced Ni-NTA affinity chromatography column, and collecting effluent liquid for subsequent analysis after the supernatant is fully combined with the Ni column. The Ni column was washed to remove the contaminating proteins using 4 column volumes of wash buffer containing 20 mM imidazole and 50 mM imidazole in sequence. The protein of interest is then eluted with 4 column volumes of 500 mM imidazole in the eluent. Collecting eluate, and ultrafiltering to obtain purified depo 32. Using a BCA protein quantitative kit to carry out concentration determination on the purified depo32, subpackaging and storing in a refrigerator at-80 ℃;
3) SDS-PAGE electrophoretic analysis: 12% of separation glue and 5% of concentrated glue are prepared. The purified sample is boiled for 10 minutes and then loaded, the gel voltage is concentrated for 90V, the gel voltage is separated for 120V, electrophoresis is carried out for 2 hours, and Coomassie brilliant blue staining is carried out for 45 minutes. A band was evident at 98 kDa upon destaining. The electrophoretogram of the purified protein is shown in FIG. 1 (Marker in lane 1, supernatant in lane 2, 20 mM imidazole rinse effluent in lane 3, purified protein in lane 4);
the SDS-PAGE gel formulation was as follows:
SDS-PAGE separating gel (12%)
| Composition of solution | Volume (mL) |
| ddH2O | 3.3 |
| 30% acrylamide | 4.0 |
| 1.5 mol/L Tris·HCl（pH8.8） | 2.5 |
| 10％SDS | 0.1 |
| 10% Ammonium Persulfate (AP) | 0.1 |
| TEMED | 0.004 |
| Total | 10 |
SDS-PAGE gel concentrate (5% acrylamide)
| Composition of solution | Volume (mL) |
| ddH2O | 6.8 |
| 30% acrylamide | 1.7 |
| 1.0 mol/L Tris·HCl（pH6.8） | 1.25 |
| 10％SDS | 0.1 |
| 10% Ammonium Persulfate (AP) | 0.1 |
| TEMED | 0.01 |
| Total | 10 |
2. The overnight cultured Klebsiella pneumoniae K7 was treated with depo32 at 37 ℃ for 3 hours to a final concentration of 200. mu.g/ml, and K7 without enzyme treatment was used as a control;
3. 100 mul of bacterial liquid is taken and fully mixed with 1 percent Congo red aqueous solution with the same volume for 1 minute. 5 mul of the mixed solution was uniformly spread on a glass slide and air-dried to form a thin film. Then gently spread onto the film area using 5 μ l Maneval solution and air dried thoroughly;
4. observing and taking a picture by using an optical microscope imaging system;
as shown in fig. 2, the capsular structure around K7 was clearly visible (left panel), while the bacterial capsular structure treated with depo32 disappeared (right panel).
Test example 2
Removal of Klebsiella pneumoniae biofilm by purified depo32 depolymerase
1) 20. mu.l of overnight-cultured Klebsiella pneumoniae was inoculated into a 96-well cell culture plate (purchased from NEST) containing 200. mu.l of LB medium per well, and cultured in a 37 ℃ incubator for 72 hours;
2) culturing for 72 hours, taking out the cell culture plate, removing the culture medium by using a pipette gun, and washing twice by using a sterilized PBS buffer solution to remove free bacteria, thus obtaining a mature biofilm;
3) purified depo32 was diluted to 1 mg/ml with protein buffer and 40. mu.l of depo32 dilution and 160. mu.l of PBS buffer were added to each well of a 96-well biofilm-containing cell culture plate to a final concentration of 200. mu.g/ml. After the plate was incubated at 37 ℃ for 6 hours, the liquid was discarded, and the plate was gently washed twice with a sterilized PBS buffer to remove free biofilm. Then 200. mu.l of methanol was added to each well and fixed for 30 minutes. After the fixation solution was discarded to be naturally air-dried, the biofilm was stained with 200. mu.l of 0.1% crystal violet (available from Kyoto Biotech Co., Ltd.) at room temperature for 30 minutes. After staining was completed, the staining solution was discarded, and washed twice with PBS buffer to remove free staining solution. The cell culture plate was dried in an oven at 70 deg.C, taken out and added to 200. mu.l of 33% strength acetic acid solution per well, and placed in a shaker for 30 minutes to release crystal violet from the biofilm. Finally, the absorbance at a wavelength of 600 nm was measured using a microplate reader. According to OD600The size of the value determines the residual amount of biofilm;
as shown in FIG. 3, the clearance rates of the depolymerizing enzyme depo32 on biofilms formed by K1, K7, KPP6 and KPP7 were 53.17%, 41.53%, 58.57% and 62.00%, respectively.
Test example 3
Inhibition of Klebsiella pneumoniae biofilms by purified depo32 depo
20. mu.l of overnight-cultured Klebsiella pneumoniae was inoculated into each well containing 140. mu.l of LB mediumMedium 96 well cell culture plates (purchased from NEST). Purified depo32 was diluted to 1 mg/ml with protein buffer and 40. mu.l of depo32 dilution was added per well to a 96 well cell culture plate containing biofilm to a final concentration of 200. mu.g/ml. After 72 hours of incubation, the cell culture plates were removed, the medium was discarded using a pipette and washed twice with sterile PBS buffer to remove free bacteria and enzymes. Then 200. mu.l of methanol was added to each well and fixed for 30 minutes. After the fixation solution was discarded to be naturally air-dried, the biofilm was stained with 200. mu.l of 0.1% crystal violet (available from Kyoto Biotech Co., Ltd.) at room temperature for 30 minutes. After staining was completed, the staining solution was discarded, and washed twice with PBS buffer to remove free staining solution. The cell culture plate was dried in an oven at 70 deg.C, taken out and added to 200. mu.l of 33% strength acetic acid solution per well, and placed in a shaker for 30 minutes to release crystal violet from the biofilm. Finally, the absorbance at a wavelength of 600 nm was measured using a microplate reader. According to OD600The size of the value determines the residual amount of biofilm;
as shown in FIG. 4, the inhibition rates of the depolymerizing enzyme depo32 on biofilms formed by K1, K7, KPP6 and KPP7 were 76.45%, 77.96%, 76.65% and 83.58%, respectively.
Sequence listing
<110> Jilin university
<120> phage depolymerase for degrading Klebsiella pneumoniae capsular polysaccharide and biofilm
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 930
<212> DNA
<213> phage depolymerase
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ctatctacaa taaaactcat gaataacaca tataattatg agattggtgg gttcactcca 60
gacgaggcat taaaatataa cgtatgggat gctaatggat tggcaacaaa cagaatatct 120
ggagtaatac acccaaggct tgttaactcg cggcttggga taaacagtgt cgcatttgat 180
aacatgtcaa ataaacttga tgtttcatcc cttattcaca atgaaacatc acaaattata 240
gggctaacac caagcactgg ttcaaatgtc cctcacacaa gaataatgtg gagcaatgga 300
gcaatgtata gttcaactga cttgaacaac ggtttcaggc ttaattatct aagcaaccat 360
aacgaaccgc ttacacctat gcatctatac aatgagtttt ctgtttctga gtttggagga 420
tcagtaacag aatcaaacgc cttggatgaa attaaataca tattcattca aacgacttat 480
gcaaactcag gtgatgggag gtttataatt caggcgcttg atgccagtgg gtctgttttg 540
tcatcaaact ggtattcgcc tcaaagcttt aactcaacgt tcccgataag tggattcgtt 600
aggtttgatg ttccgacagg tgcgaagaaa ataagatatg gatttgttaa cagtgccaat 660
tacactggct cacttagatc gcacttcatg tctggttttg catataataa aaggttcttc 720
cttaaaatat atgctgtata caatgactta ggtagatatg ggcaattcga accgccatac 780
tcggtagcta ttgataggtt tagagttggt gataatacaa cgcaaatgcc atcaatacct 840
gcgagttcag ctacagatgt agctggagtg aacgaggtta taaactcact acttgcatct 900
ttgaaagcaa atggatttat gagtagctaa 930
<210> 2
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<212> PRT
<213> phage depolymerase
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Asn Ile Asp Thr Ile Glu Ser Asn Tyr Leu Lys Asp Ile Ser Gly Ala
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Ala Ile Val Val Ser Ser Cys Leu Gly Phe Lys Ile Asp Ser Pro His
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Claims (4)
1. A phage Depo32, comprising: the 32 th open reading frame of Klebsiella pneumoniae GH-K3depo32And the depolymerizing enzyme obtained by prokaryotic expression and purification has the amino acid sequence as follows: shown in SEQ ID No. 2.
2. The method for preparing the Depo32 phage Depo32 according to claim 1, comprising the following steps:
1) PCR, double enzyme digestion and connection molecular cloning means are adopted to obtain the 32 th open reading frame from the Klebsiella pneumoniae GH-K3depo32The gene passes through the enzyme cutting siteNdeI andXhoi was ligated into pET28a plasmid;
2) pET28a-depo32The plasmid was transformed into E.coli BL21(DE3) and screened to obtain BL21-depo32E.coli;
3) BL21-depo32In 1L containing 50 u g/ml kanamycin LB liquid medium, 37 degrees C shaking culture to OD600 0.6~1.0；
4) Adding isopropyl-beta-D-thiogalactoside into the culture solution until the final concentration is 1 mM, and performing shaking culture at 16 ℃ at 180 rpm for 16 hours;
5) BL 21-that will induce expressiondepo32Carrying out centrifugal bacteria collection and centrifugation on the bacterial liquid, and then carrying out ultrasonic crushing;
6) and (3) gently adding the supernatant sample into the balanced Ni-NTA affinity chromatography column, washing the Ni column to remove the foreign proteins, and finally collecting the eluent and performing ultrafiltration to obtain the purified depolymerizing enzyme Depo 32.
3. Use of a phage Depo32 according to claim 1 in the preparation of a preparation for degrading Klebsiella pneumoniae capsular polysaccharide and biofilm-associated agents.
4. The medical use of a phage Depo32 according to claim 1 in the preparation of antibiotic replacement preparations and disinfectants for medical devices.
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