Escherichia coli resistant endolysin and application thereof

Abstract

The invention provides anti-escherichia coli endolysin E1, E2 and E3 and application thereof. Specifically, the invention discloses preparation and application of three anti-escherichia coli endolysin E1, E2 and E3. Through a gene engineering technology, E1, E2 and E3 are seamlessly cloned to a pSumo-mut vector respectively, and are subjected to prokaryotic expression in an escherichia coli BL21 (DE3) strain. Through antibacterial activity evaluation, when the endolysin E1, the endolysin E2 and the endolysin E3 are independently used or cooperatively used, the endolysin E1, the endolysin E2 and the endolysin E3 show an obvious bactericidal effect on escherichia coli isolates, particularly on multi-drug-resistant clinical isolates. The invention provides a new thought for developing a novel anti-escherichia coli drug, and has a good potential value in the aspect of developing an anti-escherichia coli infection drug.

Description

Technical Field

[0001] The present invention belongs to the field of research and development of novel antibacterial drugs, and in particular relates to anti-Escherichia coli endolysin and its application. Background Art

[0002] The overuse and misuse of antibiotics has led to a growing number of antibiotic-resistant strains, including multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pan-drug-resistant (PDR) strains. The World Health Organization has designated Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecium, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter sp. as "ESKAPE" pathogens. These bacteria are highly pathogenic and antibiotic-resistant, with some even developing resistance to all antibiotics, posing a significant threat to public health.

[0003] In recent years, pathogenic Escherichia coli (Escherichia coli) has become a major cause of foodborne illness worldwide, particularly in developing countries. Pathogenic Escherichia coli is classified into: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC). These toxicotypes are categorized based on their pathogenic characteristics, virulence factors, and their mechanisms of interaction with host cells. Different toxicotypes of E. coli are associated with specific diseases and toxin production, leading to a variety of serious illnesses, including gastroenteritis, dysentery, and hemolytic-uremic syndrome, which has become a major concern.

[0004] Bacteriophage endolysins are peptidoglycan hydrolases encoded by phage genes. They typically consist of an enzyme-active domain (EAD) and a cell wall-binding domain (CBD). During phage lysis of bacterial cells, endolysins participate in the destruction of peptidoglycan, promoting the release of phage progeny and inhibiting bacterial colonization. Therefore, they are promising antimicrobial agents against multidrug-resistant Gram-negative bacteria. Furthermore, compared to traditional broad-spectrum antibiotics, endolysins offer a significant advantage in their high specificity, enabling them to kill bacteria without harming beneficial microbiota. Over the past decade, recombinant endolysins have been used in a variety of fields to combat MDR bacteria. They can be administered parenterally, topically, or formulated as oral formulations, finding widespread applications in targeting pathogens and protecting commensal microbiota. With the rapid development of bioinformatics and artificial intelligence, the use of emerging technologies to identify antimicrobial compounds has garnered increasing attention. The widespread adoption of high-throughput sequencing technology has led to an explosive growth in the amount of bacterial and viral genetic data. Efficiently extracting valuable information from this data has become a key technical challenge. Therefore, there is an urgent need for more scientific and reasonable screening methods to quickly discover bacteriophage endolysins with stronger antibacterial abilities and accelerate their clinical application. Summary of the Invention

[0005] The present invention provides three Escherichia coli endolysins with antibacterial activity, which have the following characteristics: Endolysin E1, the amino acid sequence is shown in SEQ ID No.1.

[0006] Endolysin E2, the amino acid sequence is shown in SEQ ID No. 2.

[0007] Endolysin E3, the amino acid sequence is shown in SEQ ID No.3.

[0008] The present invention provides three recombinant Escherichia coli. The three recombinant Escherichia coli provided by the present invention can efficiently express endolysins E1, E2 and E3 that can inhibit Escherichia coli.

[0009] The preparation method of the recombinant Escherichia coli of the present invention comprises the following steps: designing a corresponding nucleotide sequence according to the amino acid sequence of endolysin, seamlessly cloning the target gene into the pSumo-mut vector, and then transforming the resultant gene into Escherichia coli BL21 (DE3).

[0010] The preparation method of the endolysin with antibacterial activity of the present invention comprises the following steps: expressing the endolysin by recombinant Escherichia coli, and then separating and purifying the endolysin from cell lysates.

[0011] The endolysins E1, E2 and E3 of the present invention have obvious bactericidal effects on Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Expression and purification of endolysin protein Figure 2 Endolysin inhibits E. coli at the minimum inhibitory concentration (MIC); Figure 3 In vitro antibacterial activity of endolysin proteins; all were cultured in an inverted manner at 37°C for 10 h, among which, A shows the co-treatment of E. coli ETEC K88 with endolysin E1 and EDTA. The left part of the figure shows the EDTA treatment alone, and the inhibition zone on the right is the E1 + EDTA treatment. B shows E. coli ETEC K88 treated with endolysin E2 and EDTA. The left part of the figure shows the EDTA treatment alone, and the inhibition zone on the right is the E2 + EDTA treatment. C shows E. coli ETEC K88 treated with endolysin E3 and EDTA. The left part of the figure shows the EDTA treatment alone, and the inhibition zone on the right is the E3+EDTA treatment. DETAILED DESCRIPTION

[0013] The above content of the present invention is further described in detail below through examples. Obviously, the described examples are only some of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0014] Example 1: Mining the most frequently occurring prophage-derived endolysins in Escherichia coli 32779 Escherichia coli genome data were obtained from the NCBI database, and endolysin prediction analysis of prophage origin was performed on them. A total of 6636 endolysins were identified, with an average of 1 endolysin sequence in every 5 Escherichia coli genomes. In order to improve the credibility of the predicted sequences as endolysins, the present invention only retains those protein sequences accompanied by holin genes in the adjacent regions (upstream or downstream) of the endolysin genes. Subsequently, the ClustalW tool was used to cluster protein sequences with a similarity of ≥ 90%. The screened protein sequences were matched with the corresponding bacterial genome IDs, and the distribution frequencies of various endolysin sequences in the Escherichia coli genome were statistically analyzed, and the top three endolysin sequences with the highest frequencies were screened out, as follows: SEQ ID No. 1: MPPSLRKAVAAAIGGGAIAIASVLITGPSGNDGLEGVSYIPYKDIVGVWTVCHGHTGKDIMLGKTYTKAECKALLNKDLATVARQINPYIKVDIPETMRGALYSFVYNVGAGNFRTSTLLRKINQGDIKGACDQLRRWTYAGGKQWKGLMTRREIEREICLWGQQ; SEQ ID No. 2: MPVINTHQNIAAFLDMLAVSEGTANHPLTKNRGYDVIVTGLDGKPEIFTDYSDHPFAHGRPAKVFNRRGEKSTASGRYQQLYLFWPHYRKQLALPDFSPLSQDRLAIQLIRERGALDDIRAGRIERAISRCRNIWASLPGAGYGQREHSLEKLVTVWRTAGGVPA; SEQ ID No.3: MNPSIVKRCLVGAVLAIAATLPGFQQLHTSVEGLKLIADYEGCRLQPYQCSAGVWTDGIGNTSGVIPGKTITERQAAEGLISNVLRVERALERCVKQQPPQKVYDAAVSFAFNVGTGNACSSTLVKLLNQRRWADACRQLPRWVYVKGVFNQGLDNRRAREMAWCLQGAN.

[0015] Example 2: Purification and expression of Escherichia coli prophage endolysin The three predicted endolysin sequences were integrated into the pSumo-mut vector using seamless cloning technology, and the constructed plasmid was introduced into the Escherichia coli BL21 (DE3) strain using CaCl2. IPTG was added at a final concentration of 0.2 mM to induce fusion protein expression for 18 h at 15 °C. The bacteria were collected by centrifugation (8000 rpm, 20 min) and the precipitate was resuspended in PBS. After ultrasonic disruption, the supernatant was separated by centrifugation at 4 °C (12000 rpm, 30 min). The supernatant was loaded into a pre-equilibrated 5 mL His Trap at a flow rate of 0.5 mL / min. TMFF crud was washed with Washing Buffer (20 mM Tris-HCl, 30 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min, followed by elution with Elution Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0). The eluate was collected. The resulting protein solution was dialyzed against PBS overnight and concentrated using an ultrafiltration centrifuge tube. Protein concentrations were determined using a BCA Protein Assay Kit. The concentrations of the three endolysins tested were 0.4 mg / mL for E1, 0.3 mg / mL for E2, and 0.1 mg / mL for E3.

[0016] Example 3: Analysis of antibacterial activity of endolysins derived from Escherichia coli prophages In vitro antibacterial activity assay The antibacterial activity of 12 E. coli isolates was tested by growing them in MH broth at 37 °C to OD 600 =0.6. Take a sterile 96-well plate, dilute the bacterial solution and add a final concentration of 5×10 5 coli with a final concentration of 1 mM EDTA. E1, E2, and E3 were serially diluted using the broth microdilution method, and an equal amount of PBS and EDTA was added to the untreated control group. After incubation at 37 °C for 10 h, the OD 600 The minimum inhibitory concentrations (MICs) of the endolysins E1, E2, and E3 were 100 µg / mL, 75-150 µg / mL, and 12.5-50 µg / mL, respectively.

[0017] E. coli isolates were grown in MH broth at 37 °C to an OD 600 =0.6. Collect the cells and resuspend them in PBS. Dilute E. coli to 5×10 5 CFU / mL was evenly spread onto a MH solid plate. The endolysin was mixed with EDTA and dripped vertically onto the plate. An equal amount of PBS and EDTA was added to the control group. After incubation at 37°C for 10 hours, the endolysin inhibition zone was observed. The results showed that the control group treated with EDTA alone had no antibacterial effect, while endolysins E1, E2, and E3 all exhibited antibacterial activity against 12 E. coli isolates.

Claims

1. Anti-E. coli endolysin, with the following characteristics: Endolysin E1, the amino acid sequence of which is specifically shown in SEQ ID No. 1; or a similar sequence obtained by substitution, insertion, or deletion of one or more amino acids as shown in SEQ ID No. 1 and still having the same or similar function; or a sequence having an amino acid sequence similarity of more than 90% to that shown in SEQ ID No. 1 and still having the same or similar function; Endolysin E2, the amino acid sequence of which is specifically shown in SEQ ID No. 2; or a similar sequence obtained by substitution, insertion, or deletion of one or more amino acids as shown in SEQ ID No. 2 and still having the same or similar function; or a sequence having an amino acid sequence similarity of more than 90% to that shown in SEQ ID No. 2 and still having the same or similar function; Endolysin E3, the amino acid sequence of which is specifically shown in SEQ ID No. 3; or a similar sequence obtained by replacing, inserting, or deleting one or more amino acids as shown in SEQ ID No. 3 and still having the same or similar function; or, a sequence that has more than 90% similarity to the amino acid sequence shown in SEQ ID No. 3 and still has the same or similar function.

2. A method for preparing recombinant Escherichia coli that efficiently expresses the endolysin according to claim 1, having the following characteristics: the nucleotide sequence corresponding to the amino acid sequence of the endolysin according to claim 1 is seamlessly cloned into the pSumo-mut vector, and transformed into Escherichia coli BL21 (DE3).

3. The method for preparing endolysin according to claim 1, characterized in that endolysin E1, endolysin E2 and endolysin E3 are separated and purified from Escherichia coli cell lysates.

4. The use of endolysin according to claim 1, characterized in that: 1) For the preparation of anti-Escherichia coli products; 2) For the preparation of products for the treatment of Escherichia coli infections.

5. The use according to claim 1, characterized in that: 1) Use one or more endolysins, alone or in combination, in the preparation of anti-E. coli products; 2) Preparation of drugs for treating Escherichia coli infections using one or more endolysins alone or in combination.

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