Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Bactericidal Effect of Cecropin A Fused Endolysin on Drug-Resistant Gram-Negative Pathogens

  • Lim, Jeonghyun (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Hong, Juyeon (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Jung, Yongwon (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Ha, Jaewon (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Kim, Hwan (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Myung, Heejoon (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Song, Miryoung (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
  • Received : 2021.05.09
  • Accepted : 2021.05.16
  • Published : 2022.06.28
Download PDF
⟨ Previous Next ⟩

Abstract

The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 µM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 µM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.

Keywords

Acknowledgement

This work was supported by the National Research Foundation of Korea grant funded by the Korean government (2021R1F1A1060072 and 2019M3E5D5066666), and by the Hankuk University of Foreign Studies Research Fund (of 2022).

References

  1. Ligon BL. 2004. Penicillin: its discovery and early development. Semin. Pediatr. Infect. Dis 15: 52-57. https://doi.org/10.1053/j.spid.2004.02.001
  2. Lewis K. 2012. Antibiotics: recover the lost art of drug discovery. Nature 485: 439-440. https://doi.org/10.1038/485439a
  3. Martinez JL, Baquero F. 2014. Emergence and spread of antibiotic resistance: setting a parameter space. Ups. J. Med. Sci. 119: 68-77. https://doi.org/10.3109/03009734.2014.901444
  4. Jim O'Neill. 2016. Nat. Rev. Drug Discov. 15: 526. https://doi.org/10.1038/nrd.2016.160
  5. Magiorakos A-P, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18: 268-281. https://doi.org/10.1111/j.1469-0691.2011.03570.x
  6. Bassetti M, Righi E, Vena A, Graziano E, Russo A, Peghin M. 2018. Risk stratification and treatment of ICU-acquired pneumonia caused by multidrug- resistant/extensively drug-resistant/pandrug-resistant bacteria. Curr. Opin. Crit. Care 24: 385-393. https://doi.org/10.1097/MCC.0000000000000534
  7. Chegini Z, Khoshbayan A, Vesal S, Moradabadi A, Hashemi A, Shariati A. 2021. Bacteriophage therapy for inhibition of multi drug-resistant uropathogenic bacteria: a narrative review. Ann. Clin. Microbiol. Antimicrob. 20: 30. https://doi.org/10.1186/s12941-021-00433-y
  8. Bhargava K, Nath G, Bhargava A, Aseri GK, Jain N. 2021. Phage therapeutics: from promises to practices and prospectives. Appl. Microbiol. Biotechnol. 105: 9047-9067. https://doi.org/10.1007/s00253-021-11695-z
  9. Mousavi SM, Babakhani S, Moradi L, Karami S, Shahbandeh M, Mirshekar M, et al. 2021. Bacteriophage as a novel therapeutic weapon for killing colistin-resistant multi-drug-resistant and extensively drug-resistant Gram-negative bacteria. Curr. Microbiol. 78: 4023-4036. https://doi.org/10.1007/s00284-021-02662-y
  10. Labrie SJ, Samson JE, Moineau S. 2010. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 8: 317-327. https://doi.org/10.1038/nrmicro2315
  11. Meile S, Du J, Dunne M, Kilcher S, Loessner MJ. 2021. Engineering therapeutic phages for enhanced antibacterial efficacy. Curr. Opin. Virol. 52: 182-191.
  12. Samson JE, Magadan AH, Sabri M, Moineau S. 2013. Revenge of the phages: defeating bacterial defences. Nat. Rev. Microbiol. 11: 675-687. https://doi.org/10.1038/nrmicro3096
  13. Young I, Wang I, Roof WD. 2000. Phages will out: strategies of host cell lysis. Trends Microbiol. 8: 120-128. https://doi.org/10.1016/S0966-842X(00)01705-4
  14. Nelson D, Loomis L, Fischetti VA. 2001. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc. Natl. Acad. Sci. USA 98: 4107-4112. https://doi.org/10.1073/pnas.061038398
  15. Jado I, Lopez R, Garcia E, Fenoll A, Casal J, Garcia P, et al. 2003. Phage lytic enzymes as therapy for antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model. J. Antimicrob. Chemother. 52: 967-973. https://doi.org/10.1093/jac/dkg485
  16. Traczewski MM, Ambler JE, Schuch R. 2021. Determination of MIC quality control parameters for exebacase, a novel lysin with antistaphylococcal activity. J. Clin. Microbiol. 59: e0311720. https://doi.org/10.1128/JCM.03117-20
  17. Nelson DC, Schmelcher M, Rodriguez-Rubio L, Klumpp J, Pritchard DG, Dong S, et al. 2012. Endolysins as antimicrobials. Adv. Virus Res. 83: 299-365. https://doi.org/10.1016/B978-0-12-394438-2.00007-4
  18. Briers Y, Walmagh M, Van Puyenbroeck V, Cornelissen A, Cenens W, Aertsen A, et al. 2014. Engineered endolysin-based 'Artilysins' to combat multidrug-resistant gram-negative pathogens. mBio 5: e01379-01314.
  19. Briers Y, Walmagh M, Grymonprez B, Biebl M, Pirnay J-P, Defraine V, et al. 2014. Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 58: 3774-3784. https://doi.org/10.1128/AAC.02668-14
  20. Saeed SI, Mergani A, Aklilu E, Kamaruzzman NF. 2022. Antimicrobial peptides: bringing solution to the rising threats of antimicrobial resistance in livestock. Front. Vet. Sci. 9: 851052. https://doi.org/10.3389/fvets.2022.851052
  21. Moore AJ, Beazley WD, Bibby MC, Devine DA. 1996. Antimicrobial activity of cecropins. J. Antimicrob. Chemother. 37: 1077-1089. https://doi.org/10.1093/jac/37.6.1077
  22. Abdelkader K, Gutierrez D, Tames-Caunedo H, Ruas-Madiedo P, Safaan A, Khairalla AS, et al. 2022. Engineering a lysin with intrinsic antibacterial activity (LysMK34) by cecropin A fusion enhances its antibacterial properties against Acinetobacter baumannii. Appl. Environ. Microbiol. 88: e0151521. https://doi.org/10.1128/AEM.01515-21
  23. Hong H-W, Kim YD, Jang J, Kim MS, Song M, Myung H. 2022. Combination effect of engineered endolysin EC340 with antibiotics. Front. Microbiol. 13: 821936. https://doi.org/10.3389/fmicb.2022.821936
  24. Hockett KL, Baltrus DA. 2017. Use of the soft-agar overlay technique to screen for bacterially produced inhibitory compounds. J. Vis. Exp. 14: 55064.
  25. Kim MS, Kim YD, Hong SS, Park K, Ko KS, Myung H. 2015. Phage-encoded colanic acid-degrading enzyme permits lytic phage infection of a capsule-forming resistant mutant Escherichia coli strain. Appl. Environ. Microbiol. 81: 900-909. https://doi.org/10.1128/AEM.02606-14
  26. Gouet P, Courcelle E, Stuart DI, Metoz F. 1999. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15: 305-308. https://doi.org/10.1093/bioinformatics/15.4.305
  27. Sievers F, Higgins DG. 2018. Clustal Omega for making accurate alignments of many protein sequences. Protein Sci. 27: 135-145. https://doi.org/10.1002/pro.3290
  28. Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, et al. 2005. InterProScan: protein domains identifier. Nucleic Acids Res. 33: W116-120. https://doi.org/10.1093/nar/gki442
  29. Blasco L, Ambroa A, Trastoy R, Bleriot I, Moscoso M, Fernandez-Garcia L, et al. 2020. In vitro and in vivo efficacy of combinations of colistin and different endolysins against clinical strains of multi-drug resistant pathogens. Sci. Rep. 10: 7163. https://doi.org/10.1038/s41598-020-64145-7
  30. Murray E, Draper LA, Ross RP, Hill C. 2021. The advantages and challenges of using endolysins in a clinical setting. Viruses 13: 680. https://doi.org/10.3390/v13040680
  31. Gerstmans H, Rodriguez-Rubio L, Lavigne R, Briers Y. 2016. From endolysins to Artilysin®s: novel enzyme-based approaches to kill drug-resistant bacteria. Biochem. Soc. Trans 44: 123-128. https://doi.org/10.1042/BST20150192
  32. Briers Y, Lavigne R. 2015. Breaking barriers: expansion of the use of endolysins as novel antibacterials against Gram-negative bacteria. Future Microbiol. 10: 377-390. https://doi.org/10.2217/fmb.15.8