Screening and application of staphylococcus aureus prophage endolysin

Abstract

The invention discloses three endolysin resisting staphylococcus aureus and application of the three endolysin. According to the invention, endolysin L1, L2 and L3 derived from the staphylococcus aureus prophage are determined. The three endolysin genes are seamlessly cloned into a pSumo-mut vector respectively, the endolysin L1, the endolysin L2 and the endolysin L3 are subjected to prokaryotic expression by using escherichia coli BL21 (DE3), and antibacterial activity evaluation is carried out. The endolysin L1, the endolysin L2 and the endolysin L3 obtained by the invention have obvious bactericidal effects on staphylococcus aureus isolates from different sources when being independently used or used in a mixed manner, so that the endolysin L1, the endolysin L2 and the endolysin L3 have good application prospects in preparation of products for resisting staphylococcus aureus and products for treating staphylococcus aureus infection.

Description

Technical Field

[0001] The present invention belongs to the field of research and development of new antibacterial drugs, and specifically relates to three endolysins against Staphylococcus aureus and their application Background Art

[0002] Staphylococcus aureus ( 金黄色葡萄球菌 ), Klebsiella pneumoniae ( 肺炎克雷伯菌 ), Enterococcus faecium ( 粪肠球菌 ), Pseudomonas aeruginosa ( 铜绿假单胞菌 ), Acinetobacter baumannii ( 鲍曼不动杆菌 ) and Enterobacter ( 肠杆菌属 ) constitute the "ESKAPE" pathogen group and are the main pathogens of healthcare-associated infections, among which Staphylococcus aureus ranks first. Staphylococcus aureus can cause infections through the skin and mucous membranes, leading to pneumonia, peritonitis, meningitis, endocarditis, urethritis and osteomyelitis. In severe cases, it can cause sepsis and even toxic shock syndrome (TSS). The morbidity and mortality of Staphylococcus aureus infections are extremely high, with more than 1 million deaths caused by Staphylococcus aureus infections each year, second only to Mycobacterium tuberculosis. In addition, Staphylococcus aureus is the main pathogen that causes mastitis in dairy cows, posing a serious public health threat to the dairy and meat industries.

[0003] Phage endolysins are enzyme products synthesized by phages in the late stage of proliferation in host bacteria. They are usually composed of an enzyme active domain (EAD) and a cell wall binding domain (CBD), which can hydrolyze the peptidoglycan layer of the host cell wall, thereby inducing bacterial lysis and releasing progeny phage particles. Endolysins are highly specific, can quickly kill target bacteria, are not easy to induce drug resistance, and do not have a destructive effect on beneficial microbial communities. Compared with antibodies, endolysins have stronger binding to bacteria, and antibodies in the body do not weaken their bactericidal activity. Therefore, endolysins are the most promising alternatives in eliminating drug-resistant bacteria. At present, five endolysins have entered the clinical trial stage. The first phase III clinical trial endolysin for the treatment of Staphylococcus aureus bacteremia is Exebacase (also known as CF-301 or PlySs2), but it failed to pass the final trial. With the rapid development of bioinformatics and artificial intelligence technologies, people have a strong interest in using new technologies to mine antimicrobial active substances. The application of high-throughput sequencing technology has led to a rapid increase in the genetic data of bacteria and viruses. How to effectively mine useful information from these data has become a key technical challenge. Therefore, a more scientific and reasonable screening method is urgently needed to quickly discover bacteriophage endolysins with stronger antibacterial ability and accelerate their clinical application. Summary of the invention

[0004] The present invention provides a method for constructing a unified pipeline EndolysinFinder for predicting endolysins. The pipeline can efficiently predict potential endolysin sequences from a large amount of bacterial genome data.

[0005] The present invention discloses three endolysins against Staphylococcus aureus and their applications. The present invention identifies endolysins L1, L2 and L3 derived from prophage of Staphylococcus aureus. The three endolysin genes are seamlessly cloned into pSumo-mut vectors respectively, and endolysins L1, L2 and L3 are expressed by prokaryotes of Escherichia coli BL21 (DE3) and their antibacterial activities are evaluated. The endolysins L1, L2 and L3 obtained by the present invention have obvious bactericidal effects on Staphylococcus aureus isolates (especially drug-resistant strains) from different sources (clinical patients, animals, environment, etc.) when used alone or in combination, showing their good application prospects in the preparation of products against Staphylococcus aureus and products for treating Staphylococcus aureus infections.

[0006] The present invention provides three Staphylococcus aureus endolysins with antibacterial activity, wherein the amino acid sequence of endolysin L1 is shown in SEQ ID No.1; the amino acid sequence of endolysin L2 is shown in SEQ ID No.2; and the amino acid sequence of endolysin L3 is shown in SEQ ID No.3.

[0007] SEQ ID No. 1 is as follows: MKTYSEARARLRWYQGRYIDFDGWYGYQCADLAVDYIYWLLEIRMWGNAKDAINNDFKNMATVYENTPSFVPQIGDVAVFTKGIYKQYGHIGLVFNGGNTNQFLILEQNYDGNANTPAKLRWDNYY GCTHFIRPKYKSEGLMNKITNKVKPPAQKAVGKSASKITVGSKAPYNLKWSKGAYFNAKIDGLGATSATRYGDNRTNYRFDVGQAVYAPGTLIYVFEIIDGWCRIYWNNHNEWIWHERLIVKEVF.

[0008] SEQ ID No. 2 is as follows: MLITKNQAEKWFDNSLGKQFNPDLFYGFQCYDYANMFFMLATGERLQGLYAYNIPFDNKAKIEKYGQIIKNYDSFLPQKLDIVVFPSKYGGGAGHVEIVESANLNTFTSFGQNWNGKGWTNGVAQPGWGPETVTRHVHYYDNPMYFIRLNFPNNLSVGNKAKGIIKQATTKKEAVIKPKKIMLVAGHGYNDPGAVGNGTNERDFIRKYITPNIAKYLRHAGHEVALYGGSSQSQDMYQDTAYGVNVGNKKDYGLYWVKSQGYDIVLEIHLDAAGESASGGHVIISSQFNADTIDKSIQDVIKNNLGQIRGVTPRNDLLNVNVSAEININYRLSELGFITNKNDMDWIKKNYDLYSKLIAGAIHGKPIGGVVASEVKAPVKNEKNPPVPAGYTLDKNNVPYKKEQGNYTVANVKGNNVRDGYSTNSRITGVLPNNTTITYDGAYCINGYRWITYIANSGQRRYIATGEVDIAGNRISSFGKFSAV。

[0009] SEQ ID No. 3 is as follows: MQAKLTKKEFIEWLKTSEGKQYNADGWYGFQCFDYANAGWQVLFGYNLKGVGAKDIPSANDFNGLATVYQNTPDFLAQPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWT DGVQQPGSGWEKVTRRQHAYDFPMWFIRPNFKSETAPRSVQSPTQASKKETAKPQPKAVELKIIKDVVKGYDLPKRGSNPKFIVIHNDAGSKGATAEAYRNGLVNAPLSRLEAGIAHSYVS GNTVWQALDESQVGWHTANQIGNKYGYGIEVCQSMGADNATFLKNEQATFQECARLLKKWGLPANRNTIRLHNEFTSTSCPHRSSVLHTGFDPVTRGLLPEDKRLQLKDYFIKQIRAYMD GKIPVATVSNESSASSNTVKPVASAWKRNKYGTYYMEESARFTNGNQPITVRKVGPFLSCPVGYQFQPGGYCDYTEVMLQDGHVWVGYTWEGQRYYLPIRTWNGSAPPNQILGDLWGEIS.

[0010] The invention provides three recombinant Escherichia coli, and the three recombinant Escherichia coli provided by the invention can efficiently express endolysins L1, L2 and L3 capable of inhibiting Staphylococcus aureus.

[0011] The preparation method of the recombinant Escherichia coli of the present invention is as follows: the nucleotide sequence of the endolysin is designed according to the amino acid sequence of the endolysin, the target gene is seamlessly cloned into the pSumo-mut vector, and then transferred into the Escherichia coli BL21 (DE3).

[0012] The preparation method of the endolysin with antibacterial activity of the present invention is to express it with recombinant Escherichia coli and then separate and purify it from cell lysate.

[0013] The endolysins L1, L2 and L3 of the present invention have obvious bactericidal effects on Staphylococcus aureus.

[0014] The endolysins L1, L2 and L3 of the present invention show good therapeutic effects in a mouse skin wound infection model. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the expression and purification of endolysin protein.

[0016] Figure 2is the in vitro bactericidal activity of endolysin proteins; where: A Staphylococcus aureus strains were treated with L1, L2 and L3 respectively and incubated at 37°C for 1 hour. 10-fold serial dilutions of each sample after different treatments were spotted on BHI agar and incubated at 37°C for 18 hours. The control group was treated with PBS alone; B 100ug / mL of L1 (ATCC 43300), L2 (SAU52) and L3 (SAU14) against Staphylococcus aureus Figure 3 It is the hemolytic activity of endolysin protein.

[0017] Figure 4 It is the bactericidal activity of endolysin L1 / L2 / L3 cocktail against 226 strains of Staphylococcus aureus. DETAILED DESCRIPTION

[0018] The above contents of the present invention are further described in detail below through embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1: Mining the most frequently occurring prophage-derived endolysins in Staphylococcus aureus The 15,405 Staphylococcus aureus genomes from the NCBI database were used to predict prophages and prophage-derived endolysins, and 27,552 prophages and 34,192 endolysin sequences were found, with an average of 2.2 endolysin sequences per bacterial genome. In order to improve the reliability of predicting sequences as endolysins, only protein sequences containing holin genes upstream or downstream of endolysin genes were retained. The ClustalW tool was used to perform cluster analysis on protein sequences with a similarity greater than or equal to 90%. Finally, the obtained protein sequences were matched with the bacterial genome ID to obtain the frequency of occurrence of different types of endolysin sequences in the Staphylococcus aureus genome. The top three endolysin sequences with high frequencies were screened out as follows: SEQ ID No.1: MKTYSEARARLRWYQGRYIDFDGWYGYQCADLAVDYIYWLLEIRMWGNAKDAINNDFKNMATVYENTPSFVPQIGDVAVFTKGIYKQYGHIGLVFNGGNTNQFLILEQNYDGNANTPAKLRWDNYYGCTHFIRPKYKSEGLMNKITNKVKPPAQKAVGKSASKITVGSKAPYNLKWSKGAYFNAKIDGLGATSATRYGDNRTNYRFDVGQAVYAPGTLIYVFEIIDGWCRIYWNNHNEWIWHERLIVKEVF; SEQ ID No.2: MLITKNQAEKWFDNSLGKQFNPDLFYGFQCYDYANMFFMLATGERLQGLYAYNIPFDNKAKIEKYGQIIKNYDSFLPQKLDIVVFPSKYGGGAGHVEIVESANLNTFTSFGQNWNGKGWTNGVAQPGWGPETVTRHVHYYDNPMYFIRLNFPNNLSVGNKAKGIIKQATTKKEAVIKPKKIMLVAGHGYNDPGAVGNGTNERDFIRKYITPNIAKYLRHAGHEVALYGGSSQSQDMYQDTAYGVNVGNKKDYGLYWVKSQGYDIVLEIHLDAAGESASGGHVIISSQFNADTIDKSIQDVIKNNLGQIRGVTPRNDLLNVNVSAEININYRLSELGFITNKNDMDWIKKNYDLYSKLIAGAIHGKPIGGVVASEVKAPVKNEKNPPVPAGYTLDKNNVPYKKEQGNYTVANVKGNNVRDGYSTNSRITGVLPNNTTITYDGAYCINGYRWITYIANSGQRRYIATGEVDIAGNRISSFGKFSAV; SEQ ID No.3: MQAKLTKKEFIEWLKTSEGKQYNADGWYGFQCFDYANAGWQVLFGYNLKGVGAKDIPSANDFNGLATVYQNTPDFLAQPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWT DGVQQPGSGWEKVTRRQHAYDFPMWFIRPNFKSETAPRSVQSPTQASKKETAKPQPKAVELKIIKDVVKGYDLPKRGSNPKFIVIHNDAGSKGATAEAYRNGLVNAPLSRLEAGIAHSYVS GNTVWQALDESQVGWHTANQIGNKYGYGIEVCQSMGADNATFLKNEQATFQECARLLKKWGLPANRNTIRLHNEFTSTSCPHRSSVLHTGFDPVTRGLLPEDKRLQLKDYFIKQIRAYMD GKIPVATVSNESSASSNTVKPVASAWKRNKYGTYYMEESARFTNGNQPITVRKVGPFLSCPVGYQFQPGGYCDYTEVMLQDGHVWVGYTWEGQRYYLPIRTWNGSAPPNQILGDLWGEIS.

[0020] Example 2: Expression and purification of Staphylococcus aureus prophage endolysin The predicted endolysin gene was seamlessly cloned into the pSumo-mut vector. The plasmid was transformed into Escherichia coli BL21 (DE3) using CaCl2. The expression of the fusion protein was induced with 0.2 mM IPTG at 15°C for 18 hours. The bacteria were centrifuged at 8000 rpm for 20 minutes, the bacterial pellet was resuspended with PBS, and after ultrasonic disruption, the supernatant was collected by centrifugation at 4°C at 12000 rpm for 30 minutes. The supernatant solution was loaded into the pre-equilibrated 5mL HisTrapTM FF crud at a flow rate of 0.5 mL / min, washed with Washing-Buffer (20mM Tris-HCl, 30 mM imidazole, 0.15 M NaCl, pH8.0) at a flow rate of 1 mL / min, and then eluted with Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH8.0) at a flow rate of 1 mL / min, and the effluent was collected. The collected protein solution was added to a dialysis bag, dialyzed with PBS overnight, and concentrated by ultrafiltration centrifuge tube. The protein concentration was determined using a BCA Protein Assay Kit, L1: 0.5 mg / mL, L2: 0.5 mg / mL, L3: 1 mg / mL.

[0021] Example 3: Analysis of the antibacterial activity of endolysins derived from Staphylococcus aureus prophage 1. In vitro bactericidal activity assay S. aureus strains were grown in BHI broth at 37°C to OD600 = 0.6. The cells were collected and resuspended in PBS. Endolysins were added to the bacterial suspension at a final concentration of 100 µg / mL, and untreated controls were incubated in PBS. After incubation at 37°C for 1 hour, each sample was serially diluted and plated, and 10 µL of the serially diluted samples were spotted on BHI agar. The minimum inhibitory concentrations of endolysins L1, L2, and L3 were 31.2 µg / mL, 62.5 µg / mL, and 25 µg / mL, respectively.

[0022] 2. Hemolytic activity assay Rabbit erythrocyte suspension (4% (w / v)) in PBS (pH 7.4) was incubated with various concentrations of endolysin at 37°C for 1 h. The samples were centrifuged at 500 g for 10 min, and the absorbance of the supernatant was measured at 415 nm using a microplate reader. 1% TritonX-100 (Solarbio, Beijing, China) was used as a positive control. The hemolysis rate was calculated using the following formula: hemolysis rate = (OD value of the experimental group - OD value of the negative control group) / (OD value of the positive control group - OD value of the negative control) × 100%. Endolysins L1, L2, and L3 have no hemolytic activity and are safe.

[0023] Example 4: Antibacterial activity analysis of endolysin L1 / L2 / 3 cocktail Endolysin L1 / L2 / 3 were mixed in equal amounts and tested for antimicrobial activity against 226 isolates of Staphylococcus aureus from patients, animals, and the environment. Grow in BHI broth at 37°C to OD600=0.6. Collect the cells and resuspend in PBS. Endolysin was added to the bacterial suspension at a final concentration of 100 µg / mL, and the untreated control group was incubated in PBS. After incubation at 37°C for 1 hour, each sample was serially diluted and plated, and 10 µL of the serially diluted samples were spotted on BHI agar. The results showed that the endolysin L1 / L2 / 3 cocktail method had antimicrobial activity against 100% of the 226 isolates.

Claims

1. An anti-Staphylococcus aureus endolysin, characterized in that: Endolysin L1, the amino acid sequence is as shown in SEQ ID No.1; or, as shown in a similar sequence having the same or similar function obtained by replacing, inserting or deleting one or more amino acids as SEQ ID No.1; or, a sequence having a similarity of more than 90% with the amino acid sequence shown in SEQ ID No.1 and still having the same or similar function; Endolysin L2, the amino acid sequence is as shown in SEQ ID No.2; or, as shown in a similar sequence having the same or similar function obtained by replacing, inserting or deleting one or more amino acids as SEQ ID No.2; or, a sequence having a similarity of more than 90% to the amino acid sequence shown in SEQ ID No.2 and still having the same or similar function; Endolysin L3, the amino acid sequence is as shown in SEQ ID No.3; or, as shown in a similar sequence having the same or similar function obtained by replacing, inserting or deleting one or more amino acids as SEQ ID No.3, or, a sequence having a similarity of more than 90% to the amino acid sequence shown in SEQ ID No.3 and still having the same or similar function.

2. A method for preparing a recombinant Escherichia coli expressing the endolysin of claim 1, characterized in that: The nucleotide sequence of the endolysin was designed according to the amino acid sequence of claim 1, and the target gene was seamlessly cloned into the pSumo-mut vector and transformed into Escherichia coli BL21 (DE3).

3. The method for preparing recombinant Escherichia coli according to claim 2, characterized in that: Endolysin L1, endolysin L2 and endolysin L3 were isolated and purified from Escherichia coli cell lysate.

4. Use of the endolysin according to claim 1 in the preparation of a product against Staphylococcus aureus.

5. Use of the endolysin according to claim 1 in the preparation of a product for treating Staphylococcus aureus infection.

6. The use according to claim 4, characterized in that: Application of any one or several endolysins in the preparation of products against Staphylococcus aureus.

7. The use according to claim 5, characterized in that: Use of any one or several endolysins in the preparation of products for treating Staphylococcus aureus infection.

Images