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KR-20260065047-A - Bacteriophage-derived recombinant depolymerase enzyme protein as a bacterial infection treatment agent and production method thereof

KR20260065047AKR 20260065047 AKR20260065047 AKR 20260065047AKR-20260065047-A

Abstract

The present invention aims to provide a protein capable of eliminating E. coli O157:H7 based on biofilm removal ability and serum-mediated bacterial killing ability of E. coli O157:H7 , by utilizing the depolymerase enzyme activity of the tail spike protein (ORF161) of the bacteriophage SFP10 (GenBank ID: HQ259103) that infects E. coli O157:H7 .

Inventors

  • 공민석
  • 서진

Assignees

  • 서울과학기술대학교 산학협력단

Dates

Publication Date
20260508
Application Date
20241031

Claims (5)

  1. Recombinant dipolymerase enzyme protein having the ability to remove bacterial biofilms and kill bacteria.
  2. In claim 1, The above recombinant depolymerase enzyme protein is a recombinant depolymerase enzyme protein obtained by removing the N-terminal of the bacteriophage tail spike protein (TSP).
  3. In claim 2, The above recombinant depolymerase enzyme protein is a recombinant depolymerase enzyme protein that further comprises histidine.
  4. In claim 1, The above bacteria is pathogenic E. coli, Recombinant dipolymerase enzyme protein.
  5. Antimicrobial composition comprising the recombinant dipolymerase enzyme protein of claim 1.

Description

Bacteriophage-derived recombinant depolymerase enzyme protein as a bacterial infection treatment agent and production method thereof The present invention developed a treatment for bacterial infection based on biofilm removal ability and serum-mediated bacterial killing ability by utilizing the depolymerase enzyme activity of the tail spike protein (ORF161, TSP) of a bacteriophage that infects bacteria. Shiga toxin-producing bacteria, particularly Escherichia coli (STEC), are representative pathogenic bacteria that cause gastroenteritis due to toxin formation or acute renal failure due to hemolytic uremic syndrome (HUS). In particular, among STECs , the E. coli O157:H7 serotype is a representative enterohemorrhagic E. coli and accounts for the highest proportion of STEC-induced infections. While antibiotics are generally used to treat HUS caused by STEC infections, reports suggest that this approach may increase the risk of HUS; therefore, there is a need for the development of specific and effective antibiotic alternative therapies for the treatment of STEC infections. Recently, various methods utilizing bacteriophages are being developed to create antimicrobial agents that can replace antibiotics. Bacteriophages are viruses that infect bacteria and possess highly specific characteristics. They operate on a mechanism in which they recognize and infect a host, replicate within the host through the expression of their own DNA, and ultimately kill the host, releasing numerous virions. During this process, the Tail Spike Protein (TSP), which is involved in host recognition and attachment, possesses depolymerase activity—specifically, the ability to degrade a portion of LPS—to facilitate DNA injection into the host. Furthermore, research suggests that this enzyme can remove biofilms to access bacteria forming biofilms. Therefore, it was predicted that if such a depolymerase is used, bacteria in which specific parts of the outer membrane of Gram-negative bacteria are degraded will become more sensitive to the antimicrobial activity of antibiotics, other antimicrobial substances, or complements in human serum, and will exhibit enhanced antimicrobial activity. Therefore, the inventors identified TSPs possessing the corresponding activity, constructed a recombinant protein, expressed and purified it, and conducted an activity evaluation. The potential of the depolymerase as an antimicrobial agent was confirmed by evaluating its ability to remove biofilms formed by bacteria and the degree to which bacteria with partially degraded LPS were killed by human serum. Furthermore, the safety and potential for practical application of the depolymerase were demonstrated through cytotoxicity evaluations, hemolytic activity evaluations, and in vivo efficacy tests using larvae. Figure 1 lists the sequence of SFP10_Dpo with the N-terminal head-binding domain removed, specifically the result of amino acid sequence alignment between ORF161 of phage SFP10 for LPS degradation activity site analysis and the tail spike proteins of other E. coli O157:H7 phages (CBA120 and PhiV10). Figure 2 shows the protein structure and size of SFP10_Dpo with the N-terminal head-binding domain removed. Specifically, (A) is a schematic diagram of the predicted homotrimeric structure of the recombinant SFP10_Dpo protein using the Swiss-Model, and (B) is the SDS-PAGE result of SFP10_Dpo (72 kDa) (M: protein marker). Figure 3 shows the results of confirming the activity of SFP10_Dpo with the N-terminal head-binding domain removed, specifically (A) is the result of confirming the activity of purified SFP10_Dpo through a Halo assay, and (B) is the SDS-PAGE result confirming LPS with the O-antigen of E. coli O157:H7 removed after SFP10_Dpo treatment (EC : E. coli O157:H7 OE50; ST : Salmonella Typhimurium SL1344; M : marker, E. coli O55:B5). Figure 4 shows the results of the stability evaluation of SFP10_Dpo, specifically (A) is the stability evaluation of SFP10_Dpo according to temperature, and (B) is the results of the stability evaluation of SFP10_Dpo according to pH. Figure 5 shows the results of confirming the biofilm removal ability of SFP10_Dpo, where (A) is E. coli O157:H7 OE50 and (B) is the biofilm removal activity result of SFP10_Dpo against a biofilm formed by E. coli O157:H7 ATCC 43890 for 24 hours. Figure 6 shows the results of the evaluation of the human serum-mediated antimicrobial activity of SFP10_Dpo, where (A) is the result of confirming the antimicrobial activity of SFP10_Dpo in the presence of 25% human serum, and (B) is the result showing the change in activity of SFP10_Dpo according to serum concentration. Figure 7 shows the results of toxicity and hemolytic evaluations in intestinal epithelial cells. Specifically, (A) is the result of toxicity evaluation using an MTS assay after treating human intestinal epithelial cells Caco-2 with a high concentration of SFP10_Dpo for 24 hours. (B) is the result of hemolytic evaluation of SFP10_Dpo