Anti-salmonella endolysin and application thereof

Abstract

The invention discloses four kinds of endolysin SMLYS-1, SMLYS-2, SMLYS-3 and SMLYS-4 derived from salmonella prophage, and the application values of the four kinds of endolysin SMLYS-1, SMLYS-2, SMLYS-3 and SMLYS-4 are elaborated in detail. Four endolysin genes are seamlessly cloned into a pSumo-mut vector, and efficient expression is realized by utilizing an escherichia coli BL21 (Arctic-Express) system, so that the endolysin protein with remarkable antibacterial activity is successfully obtained. Experimental results show that the endolysin SMLYS-1, the endolysin SMLYS-2, the endolysin SMLYS-3 and the endolysin SMLYS-4 can be used for effectively killing salmonella isolates from various sources, and the endophysin SMLYS-1, the endolysin SMLYS-2, the endolysin SMLYS-3 and the endolysin SMLYS-4 have great potential as an anti-salmonella preparation and a salmonella infection treatment drug. The invention provides a new technical means for preventing and treating salmonella infection, and has important scientific significance and practical application value.

Description

Technical Field

[0001] This invention belongs to the field of novel antibacterial drug development, specifically relating to anti-Salmonella endosomalin and its application. Background Technology

[0002] Salmonella spp. is a genus of Gram-negative short rod-shaped bacteria that widely colonizes the gastrointestinal tract of warm-blooded animals, particularly poultry and livestock. These bacteria are rod-shaped, non-spore-forming, and most strains exhibit motility with peritrichous flagella. As an important zoonotic pathogen, its pathogenic serotypes can cause human infections through contaminated food and water, demonstrating significant ecological adaptability. They persist in soil, water, and the food processing chain, exhibiting phenotypic evolution characterized by multidrug resistance (MDR).

[0003] In poultry farming, Salmonella infection can induce three typical avian diseases: pullorum disease caused by Salmonella Gallinarum, fowl typhoid mediated by Salmonella Heidelberg, and fowl paratyphoid caused by Salmonella Enteritidis. These pathological processes lead to stunted growth, reduced egg production efficiency, and high mortality rates in chicks, causing significant economic losses to the livestock industry. More importantly, contaminated poultry products (poultry meat and eggs) act as carriers of pathogens, enabling cross-species transmission through the food chain, significantly increasing the public health risk of zoonotic diseases.

[0004] While current food preservation systems widely employ chemically synthesized preservatives, long-term use raises safety concerns, such as teratogenicity and metabolic toxicity. Meanwhile, the irrational use of antibiotics in animal husbandry has accelerated the global spread of multidrug-resistant Salmonella strains. In light of this, phage lysin therapy, due to its low resistance-inducing properties, has become a key area of ​​interdisciplinary research.

[0005] Bacteriophages are a class of functional viruses that obligately infect prokaryotes, with a host range encompassing bacteria, actinomycetes, and archaea. At the end of the phage lysis cycle, the genome encodes endolysin, which targets and degrades the peptidoglycan layer of the host cell wall, achieving cell lysis and releasing progeny viral particles. This phage is named for its unique intracellular site of action. Due to its rapid lysis, high substrate specificity, and low resistance induction, this enzyme is defined as an "enzyme antibiotic" and has demonstrated broad-spectrum antibacterial potential in clinical medicine, agricultural product preservation, and food microbial control.

[0006] Although endosomalin exhibits superior antibacterial efficacy against Gram-positive bacteria, its lytic effect against Gram-negative bacteria is severely limited by its two-membrane structure: the outer membrane permeability barrier and the thin periplasmic peptidoglycan layer significantly weaken the transmembrane delivery efficiency of exogenous endosomalin. To overcome this barrier mechanism, current research generally employs a synergistic strategy between outer membrane deconstructors (such as EDTA and weak organic acids) and endosomalin molecules. However, the biocompatibility and safety issues of exogenous synergists necessitate the development of novel membrane permeability enhancement technologies (such as short peptide-based membrane permeability effect molecular design, liposome nanocarrier technology, or charge-modified protein engineering) to achieve highly efficient control of Gram-negative pathogens (such as Salmonella and Escherichia coli), ultimately aiming to completely replace traditional antibacterial agents. Summary of the Invention

[0007] This invention provides an anti-Salmonella endosomalin and its application.

[0008] This invention discloses four anti-Salmonella endosomalins and their applications. This invention identifies four endosomalins derived from Salmonella prophages: SMLYS-1, SMLYS-2, SMLYS-3, and SMLYS-4. The genes of these four endosomalins were seamlessly cloned into the pSumo-mut vector, and their antibacterial activity was evaluated by expressing SMLYS-1, SMLYS-2, SMLYS-3, and SMLYS-4 in the prokaryotic cell line *Escherichia coli* BL21 (Arctic-Express). The endosomalins SMLYS-1, SMLYS-2, SMLYS-3, and SMLYS-4 obtained in this invention, when used individually, exhibit significant bactericidal activity against Salmonella isolates from different sources (animals, environment, etc.), demonstrating their promising application prospects in the preparation of anti-Salmonella products and products for treating Salmonella infections.

[0009] This invention provides four Salmonella endosomalins with antibacterial activity: the amino acid sequence of endosomalin SMLYS-1 is shown in SEQ ID No. 1; the amino acid sequence of endosomalin SMLYS-2 is shown in SEQ ID No. 2; the amino acid sequence of endosomalin SMLYS-3 is shown in SEQ ID No. 3; and the amino acid sequence of endosomalin SMLYS-4 is shown in SEQ ID No. 4.

[0010] SEQ ID No. 1 is as follows: MNQQQFQQAAGISAGLSARWYPYITAAMSEFGITAPLDQAMFIAQAGHESAGFTVLKESFNYSVEALKKTFGKRLTPYQCEMLGRIDGRQVAHQPQIANLVYGGRMGNKDAGDGWKYRGRGLIQITGLENYTRCGVALKLDLVANPGQLELERHAARSAAWFFVTKGCLKYSGDLVRVTQIINGGQNGFGDRRERFEKAKSVLV.

[0011] SEQ ID No. 2 is as follows: MQISSNGITRLKREEGERLKAYPDSRGIPTIGVGHTGKVDGNPVVSGMTITSEKSSELLKEDLQWVEDAISSLVRVTLNQNQNQYDALCSLIFNIGKSAFAGSTVLRQLNLKNYQAAADAFLLWKKAGKDPDILLPRRRRRERALFLS.

[0012] SEQ ID No. 3 is as follows: MSNTFKFSSRSEKNLQGVNPDLVKVTRRALEISEVDFGITEGLRSRYRQKQLVATGKSQTMNSRHLTGHAVDVVAYIGSQVSWEWPLYEKIAAAFRQASRELNIPVEWGGDWKTLKDGPHFQLPHGAYPA.

[0013] SEQ ID No. 4 is as follows: MMRISEKGITLIKEFEGCSLTAYPDPGTGGDPWTIGYGWTHSVDGKPVKPGMMIDEATAERLLKTGLVGYENDVSRLVKVKLTQGQFDALVSFAYNLGARTLSTSTLLRKLNAGDYAGAADEFLRWNKAGGKALNGLTRRREAERALFLS.

[0014] This invention provides four recombinant Escherichia coli strains, which can efficiently express SMLYS-1, SMLYS-2, SMLYS-3 and SMLYS-4, which can inhibit Salmonella.

[0015] The method for preparing recombinant Escherichia coli according to 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 transformed into Escherichia coli BL21 (Arctic-Express).

[0016] The method for preparing the antibacterial endolysin described in this invention involves expressing it in recombinant Escherichia coli and then isolating and purifying it from cell lysates.

[0017] The endolysins SMLYS-1, SMLYS-2, SMLYS-3 and SMLYS-4 of the present invention have significant bactericidal effects against Salmonella. Attached Figure Description

[0018] Figure 1 It involves the expression and purification of endolysin proteins.

[0019] Figure 2 The in vitro bactericidal activity of endolysin protein was measured. Four Salmonella strains 0 (ATCC14028), 51 (CP091558.1), 52 (ATCC9150), and 62 (ATCC8759) were treated with SMLYS-1, SMLYS-2, SMLYS-3, and SMLYS-4, respectively, and incubated at 37°C for 8 hours. The control group was treated with EDTA alone.

[0020] Figure 3 The MICs (ug / ml) of endolysins SMLYS-1, SMLYS-2, SMLYS-3, and SMLYS-4, as well as 50 mM EDTA, against four Salmonella strains are given. Detailed Implementation

[0021] The following embodiments further illustrate the above-described content of the present invention in detail. Obviously, the described embodiments are merely a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] Example 1: Mining of endolysins derived from prophages, which are the most frequently occurring prophages in Salmonella. Prephage and endolysin-derived prophage sequences were predicted from 38,390 Salmonella genomes obtained from the NCBI database, identifying 68,604 prophage sequences and 84,458 endolysin sequences. To improve the reliability of predicted sequences as endolysins, only protein sequences containing the holin gene upstream or downstream of the endolysin gene were retained. Cluster analysis was performed on protein sequences with a similarity greater than or equal to 90% using the ClustalW tool. Finally, the obtained protein sequences were mapped to bacterial genome IDs to obtain the frequency of different types of endolysin sequences in the Salmonella genome. The top four most frequent endolysin sequences were selected as follows: SEQ ID No. 1 is as follows: MNQQQFQQAAGISAGLSARWYPYITAAMSEFGITAPLDQAMFIAQAGHESAGFTVLKESFNYSVEALKKTFGKRLTPYQCEMLGRIDGRQVAHQPQIANLVYGGRMGNKDAGDGWKYRGRGLIQITGLENYTRCGVALKLDLVANPGQLELERHAARSAAWFFVTKGCLKYSGDLVRVTQIINGGQNGFGDRRERFEKAKSVLV.

[0023] SEQ ID No. 2 is as follows: MQISSNGITRLKREEGERLKAYPDSRGIPTIGVGHTGKVDGNPVVSGMTITSEKSSELLKEDLQWVEDAISSLVRVTLNQNQNQYDALCSLIFNIGKSAFAGSTVLRQLNLKNYQAAADAFLLWKKAGKDPDILLPRRRRRERALFLS.

[0024] SEQ ID No. 3 is as follows: MSNTFKFSSRSEKNLQGVNPDLVKVTRRALEISEVDFGITEGLRSRYRQKQLVATGKSQTMNSRHLTGHAVDVVAYIGSQVSWEWPLYEKIAAAFRQASRELNIPVEWGGDWKTLKDGPHFQLPHGAYPA.

[0025] SEQ ID No. 4 is as follows: MMRISEKGITLIKEFEGCSLTAYPDPGTGGDPWTIGYGWTHSVDGKPVKPGMMIDEATAERLLKTGLVGYENDVSRLVKVKLTQGQFDALVSFAYNLGARTLSTSTLLRKLNAGDYAGAADEFLRWNKAGGKALNGLTRRREAERALFLS.

[0026] Example 2: Expression and purification of Salmonella prophage in vivo lysin The predicted endosomal gene was seamlessly cloned into the pSumo-mut vector. The plasmid was transformed into *E. coli* BL21 (Arctic-Express) using CaCl2. Fusion protein expression was induced for 18 hours at 15°C with 0.2 mM IPTG. The bacteria were centrifuged at 8000 rpm for 20 minutes, the bacterial pellet was resuspended in PBS, sonicated, and then centrifuged at 12000 rpm for 30 minutes at 4°C. The supernatant was collected. The supernatant was loaded into a pre-equilibrated 5 mL HisTrap™ FFcrud at a flow rate of 0.5 mL / min, 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, and then eluted with Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min. The eluent was collected. The collected protein solution was added to a dialysis bag, dialyzed overnight with PBS, and concentrated via ultrafiltration centrifuge tubes. The concentrations of proteins SMLYS-1, SMLYS-2, SMLYS-3, and SMLYS-4 were determined using the BCA Protein Assay Kit.

[0027] Example 3: Analysis of the antibacterial activity of endolysins derived from Salmonella prophages 1. In vitro bactericidal activity assay Salmonella strains were grown in LB broth at 37°C until OD600 = 0.6. The bacterial cells were collected and resuspended in PBS. The turbidity of the bacterial suspension was adjusted to 0.5 McFarland turbidity (approximately 1 × 10⁻⁶). 8 (CFU / mL) Use a cotton swab to apply an appropriate amount of bacterial suspension evenly to an LB solid culture plate. After the bacterial suspension has dried, add 10µL of endosomalin with a final concentration of 600 µg / mL and 10µL of EDTA solution with a concentration of 50mM. Add the same concentration of EDTA as a negative control.

[0028] 2. Determination of Minimum Inhibitory Concentration (MIC) Pick a single colony of Salmonella and inoculate it into 5 mL LB broth. Incubate at 37°C with shaking for 16–18 hours until the logarithmic growth phase. Dilute with physiological saline to 0.5 McFarland turbidity (approximately 1 × 10⁻⁶). 8 (CFU / mL), then diluted with MH to a final concentration of 1×10⁻⁶. 6 Prepare CFU / mL for later use. Add 100 µL of the highest test concentration of endolysin to column 1 (row AH) of a 96-well plate. Perform serial dilutions of 2-fold from column 2 to column 10. Add 80 µL of diluted bacterial culture and 20 µL of 50 mM EDTA to columns 1-9. Column 10 serves as a blank control. Add 80 µL of diluted bacterial culture and 20 µL of 50 mM EDTA to column 11 as a negative control. After incubating overnight at 37°C, visually observe the turbidity. The lowest concentration in wells that does not become turbid is considered the MIC of the endolysin.

Claims

1. An anti-Salmonella endosomalin, characterized in that: Endolysin SMLYS-1, whose amino acid sequence is shown in SEQ ID No. 1, or a similar sequence as shown in SEQ ID No. 1 obtained by substitution, insertion, or deletion of one or more amino acids, 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. 1 and still has the same or similar function; Endolysin SMLYS-2, whose amino acid sequence is shown in SEQ ID No. 2, or a similar sequence as shown in SEQ ID No. 2 obtained by substitution, insertion, or deletion of one or more amino acids that still has the same or similar function; or a sequence that has more than 90% similarity to the amino acid sequence shown in SEQ ID No. 2 and still has the same or similar function; Endolysin SMLYS-3, whose amino acid sequence is shown in SEQ ID No. 3, or a similar sequence as shown in SEQ ID No. 3 obtained by substitution, insertion, or deletion of one or more amino acids, 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; Endolysin SMLYS-4, whose amino acid sequence is shown in SEQ ID No. 4, or a similar sequence as shown in SEQ ID No. 4 obtained by substitution, insertion, or deletion of one or more amino acids, 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. 4 and still has the same or similar function.

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

3. The method for preparing recombinant Escherichia coli according to claim 2, characterized in that: The endolysins SMLYS-1, SMLYS-2, SMLYS-3, or SMLYS-4 were obtained by lysing recombinant E. coli cells and then purifying them from the lysate.

4. The use of the endolysin according to claim 1 in the preparation of products against Salmonella.

5. The application according to claim 4, characterized in that: The application of any one or more endosomalins in the preparation of anti-Salmonella products.

6. The use of the endolysin according to claim 1 in the preparation of products for treating Salmonella infection.

7. The application according to claim 6, characterized in that: The use of any one or more endolysins in the preparation of products for treating Salmonella infections.

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