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Research article

Biotechnology and Bioengineering (BB)  |  Antibiotics, Antifungals, and Antiviral compounds

Deleterious Effects of Histidine Tagging to the SH3b Cell Wall-Binding Domain on Recombinant Endolysin Activity

Jin-Mi Park1,3,5, Jun-Hyun Kim1,3,5, Kang-Seuk Choi1,3*, and Hyuk-Joon Kwon2,3,4,5*

1Laboratory of Avian Diseases, Department of Farm Animal Medicine, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul 08826, Republic of Korea
2Laboratory of Poultry Medicine, Department of Farm Animal Medicine, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul 08826, Republic of Korea
3Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul 08826, Republic of Korea
4Farm Animal Clinical Training and Research Center (FACTRC), GBST, Seoul National University, Gangwon-do 25354, Republic of Korea
5GeNiner Inc., Seoul 08826, Republic of Korea

Correspondence to:
Kang-Seuk Choi ,       kchoi0608@snu.ac.kr
Hyuk-Joon Kwon,       kwonhj01@snu.ac.kr
Received: August 2, 2024; Revised: September 27, 2024; Accepted: September 28, 2024

J. Microbiol. Biotechnol. 2024; 34(11): 2331-2337

Published November 28, 2024 https://doi.org/10.4014/jmb.2408.08003

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Natural and artificial endolysins exhibit bactericidal effects by destroying peptidoglycans in the cell wall of gram-positive bacteria and are usually composed of an N-terminal catalytic domain (CTD) and a C-terminal cell wall-binding domain (CBD). The structures and receptors of CBDs are variable, but bacterial Src homology 3 (SH3b) CBDs are prevalent among the natural endolysins of Staphylococcus aureus. Moreover, although recombinant endolysins with a C-terminal 6x histidine tag (His-tag) are often produced and convenient to purify, the deleterious effects of His-tags on antibacterial activity have not been evaluated thoroughly. Recently, we reported that the antibacterial activity of a commercial lysostaphin without a His-tag differed from that of cell-free lysostaphin with a C-terminal His-tag, and lysostaphin also contains a C-terminal SH3b CBD. In this study, we directly compared the effects of His-tags on the antibacterial activities of lysostaphin and several chimeric lysins possessing different SH3b CBDs. We confirmed that antibacterial activity decreased 16.0-32.0-fold after a His-tag was added to the SH3b CBD.

Keywords

Antibacterial activity, cell-free expression system, chimeric lysins, Staphylococcus aureus, histidine tag, SH3b cell wall-binding domain

Graphical Abstract


References

  1. Sakwinska O, Giddey M, Moreillon M, Morisset D, Waldvogel A, Moreillon P. 2011. Staphylococcus aureus host range and humanbovine host shift. Appl. Environ. Microbiol. 77: 5908-5915.
    Pubmed PMC
  2. Price LB, Stegger M, Hasman H, Aziz M, Larsen J, Andersen PS, et al. 2012. Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock. mBio 3: e00305-11.
    Pubmed PMC
  3. Broendum SS, Buckle AM, McGowan S. 2018. Catalytic diversity and cell wall binding repeats in the phage-encoded endolysins. Mol. Microbiol. 110: 879-896.
    Pubmed
  4. Fischetti VA. 2008. Bacteriophage lysins as effective antibacterials. Curr. Opin. Microbiol. 11: 393-400.
    Pubmed PMC
  5. Kurochkina N, Guha U. 2013. SH3 domains: modules of protein-protein interactions. Biophys. Rev. 5: 29-39.
    Pubmed PMC
  6. Liu H, Hu Z, Li M, Yang Y, Lu S, Rao X. 2023. Therapeutic potential of bacteriophage endolysins for infections caused by Grampositive bacteria. J. Biomed. Sci. 30: 29.
    Pubmed PMC
  7. Schmelcher M, Shen Y, Nelson DC, Eugster MR, Eichenseher F, Hanke DC, et al. 2015. Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection. J. Antimicrob. Chemother. 70: 14531465.
    Pubmed PMC
  8. Lee C, Kim J, Son B, Ryu S. 2021. Development of advanced chimeric endolysin to control multidrug-resistant Staphylococcus aureus through domain shuffling. ACS Infect. Dis. 7: 2081-2092.
    Pubmed
  9. Amarasinghe C, Jin J-P. 2015. The use of affinity tags to overcome obstacles in recombinant protein expression and purification. Protein Peptide Lett. 22: 885-892.
    Pubmed
  10. Roshanak S, Yarabbi H, Shahidi F, Tabatabaei Yazdi F, Movaffagh J, Javadmanesh A. 2023. Effects of adding poly-histidine tag on stability, antimicrobial activity and safety of recombinant buforin I expressed in periplasmic space of Escherichia coli. Sci. Rep. 13: 5508.
    Pubmed PMC
  11. Chaga GS. 2001. Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. J. Biochem. Biophys. Methods 49: 313-334.
    Pubmed
  12. Boksha IS, Lavrova NV, Grishin AV, Demidenko AV, Lyashchuk AM, Galushkina ZM, et al. 2016. Staphylococcus simulans recombinant lysostaphin: production, purification, and determination of antistaphylococcal activity. Biochemistry. 81: 502-510.
    Pubmed
  13. Park J-M, Kim J-H, Kim G, Sim H-J, Ahn S-M, Choi K-S, et al. 2024. Rapid antibacterial activity assessment of chimeric lysins. Int. J. Mol. Sci. 25: 2430.
    Pubmed PMC
  14. Mitkowski P, Jagielska E, Nowak E, Bujnicki JM, Stefaniak F, Niedziałek D, et al. 2019. Structural bases of peptidoglycan recognition by lysostaphin SH3b domain. Sci. Rep. 9: 5965.
    Pubmed PMC
  15. Park J-M, Ko D-S, Kim H-S, Kim N-H, Kim E-K, Roh Y-H, et al. 2023. Rapid screening and comparison of chimeric lysins for antibacterial activity against Staphylococcus aureus strains. Antibiotics 12: 667.
    Pubmed PMC
  16. Wei H, Yang, Hang, YU, Junping. 2018. Staphylococcus lyase and Use thereof. PCT/CN2016/079044.
  17. Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, et al. 2022. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50: D439-d444.
    Pubmed PMC
  18. Pokhrel R, Shakya R, Baral P, Chapagain P. 2022. Molecular modeling and simulation of the peptidoglycan layer of Gram-positive bacteria Staphylococcus aureus. J. Chem. Inform. Model. 62: 4955-4962.
    Pubmed
  19. Heckman KL, Pease LR. 2007. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat. Protoc. 2: 924-932.
    Pubmed
  20. Fall RR, Vagelos PR. 1972. Acetyl coenzyme A carboxylase. Molecular forms and subunit composition of biotin carboxyl carrier protein. J. Biol. Chem. 247: 8005-8015.
    Pubmed
  21. Tossavainen H, Raulinaitis V, Kauppinen L, Pentikainen U, Maaheimo H, Permi P. 2018. Structural and functional insights into lysostaphin-substrate interaction. Front. Mol. Biosci. 5: 60.
    Pubmed PMC
  22. Pokhrel R, Shakya R, Baral P, Chapagain P. 2022. Molecular modeling and simulation of the peptidoglycan layer of gram-positive bacteria Staphylococcus aureus. J. Chem. Inf. Model 62: 4955-4962.
    Pubmed
  23. Vollmer W, Blanot D, de Pedro MA. 2008. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32: 149-167.
    Pubmed
  24. Oliveira H, Melo LD, Santos SB, Nobrega FL, Ferreira EC, Cerca N, et al. 2013. Molecular aspects and comparative genomics of bacteriophage endolysins. J. Virol. 87: 4558-4570.
    Pubmed PMC
  25. Kim H, Seo J. 2023. A novel strategy to identify endolysins with lytic activity against methicillin-resistant Staphylococcus aureus. Int. J. Mol. Sci. 24: 5772.
    Pubmed PMC
  26. Yang H, Zhang Y, Yu J, Huang Y, Zhang X-E, Wei H. 2014. Novel chimeric lysin with high-level antimicrobial activity against methicillin-resistant Staphylococcus aureus in vitro and in vivo. Antimicrob. Agents Chemother. 58: 536-542.
    Pubmed PMC
  27. Eichenseher F, Herpers Bjorn L, Badoux P, Leyva-Castillo Juan M, Geha Raif S, van der Zwart M, et al. 2022. Linker-improved chimeric endolysin selectively kills Staphylococcus aureus in vitro, on reconstituted human epidermis, and in a murine model of skin infection. Antimicrob. Agents Chemother. 66: e02273-02221.
    Pubmed PMC
  28. Shen W, Yang N, Teng D, Hao Y, Ma X, Mao R, et al. 2021. Design and high expression of non-glycosylated lysostaphins in Pichia pastoris and their pharmacodynamic study. Front. Microbiol. 12: 637662.
    Pubmed PMC
  29. Zhao H, Verma D, Li W, Choi Y, Ndong C, Fiering SN, et al. 2015. Depletion of T cell epitopes in lysostaphin mitigates anti-drug antibody response and enhances antibacterial efficacy in vivo. Chem. Biol. 22: 629-639.
    Pubmed PMC

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Article

Research article

J. Microbiol. Biotechnol. 2024; 34(11): 2331-2337

Published online November 28, 2024 https://doi.org/10.4014/jmb.2408.08003

Copyright © The Korean Society for Microbiology and Biotechnology.

Deleterious Effects of Histidine Tagging to the SH3b Cell Wall-Binding Domain on Recombinant Endolysin Activity

Jin-Mi Park1,3,5, Jun-Hyun Kim1,3,5, Kang-Seuk Choi1,3*, and Hyuk-Joon Kwon2,3,4,5*

1Laboratory of Avian Diseases, Department of Farm Animal Medicine, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul 08826, Republic of Korea
2Laboratory of Poultry Medicine, Department of Farm Animal Medicine, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul 08826, Republic of Korea
3Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul 08826, Republic of Korea
4Farm Animal Clinical Training and Research Center (FACTRC), GBST, Seoul National University, Gangwon-do 25354, Republic of Korea
5GeNiner Inc., Seoul 08826, Republic of Korea

Correspondence to:Kang-Seuk Choi ,       kchoi0608@snu.ac.kr
Hyuk-Joon Kwon,       kwonhj01@snu.ac.kr

Received: August 2, 2024; Revised: September 27, 2024; Accepted: September 28, 2024

Abstract

Natural and artificial endolysins exhibit bactericidal effects by destroying peptidoglycans in the cell wall of gram-positive bacteria and are usually composed of an N-terminal catalytic domain (CTD) and a C-terminal cell wall-binding domain (CBD). The structures and receptors of CBDs are variable, but bacterial Src homology 3 (SH3b) CBDs are prevalent among the natural endolysins of Staphylococcus aureus. Moreover, although recombinant endolysins with a C-terminal 6x histidine tag (His-tag) are often produced and convenient to purify, the deleterious effects of His-tags on antibacterial activity have not been evaluated thoroughly. Recently, we reported that the antibacterial activity of a commercial lysostaphin without a His-tag differed from that of cell-free lysostaphin with a C-terminal His-tag, and lysostaphin also contains a C-terminal SH3b CBD. In this study, we directly compared the effects of His-tags on the antibacterial activities of lysostaphin and several chimeric lysins possessing different SH3b CBDs. We confirmed that antibacterial activity decreased 16.0-32.0-fold after a His-tag was added to the SH3b CBD.

Keywords: Antibacterial activity, cell-free expression system, chimeric lysins, Staphylococcus aureus, histidine tag, SH3b cell wall-binding domain

References

  1. Sakwinska O, Giddey M, Moreillon M, Morisset D, Waldvogel A, Moreillon P. 2011. Staphylococcus aureus host range and humanbovine host shift. Appl. Environ. Microbiol. 77: 5908-5915.
    Pubmed KoreaMed
  2. Price LB, Stegger M, Hasman H, Aziz M, Larsen J, Andersen PS, et al. 2012. Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock. mBio 3: e00305-11.
    Pubmed KoreaMed
  3. Broendum SS, Buckle AM, McGowan S. 2018. Catalytic diversity and cell wall binding repeats in the phage-encoded endolysins. Mol. Microbiol. 110: 879-896.
    Pubmed
  4. Fischetti VA. 2008. Bacteriophage lysins as effective antibacterials. Curr. Opin. Microbiol. 11: 393-400.
    Pubmed KoreaMed
  5. Kurochkina N, Guha U. 2013. SH3 domains: modules of protein-protein interactions. Biophys. Rev. 5: 29-39.
    Pubmed KoreaMed
  6. Liu H, Hu Z, Li M, Yang Y, Lu S, Rao X. 2023. Therapeutic potential of bacteriophage endolysins for infections caused by Grampositive bacteria. J. Biomed. Sci. 30: 29.
    Pubmed KoreaMed
  7. Schmelcher M, Shen Y, Nelson DC, Eugster MR, Eichenseher F, Hanke DC, et al. 2015. Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection. J. Antimicrob. Chemother. 70: 14531465.
    Pubmed KoreaMed
  8. Lee C, Kim J, Son B, Ryu S. 2021. Development of advanced chimeric endolysin to control multidrug-resistant Staphylococcus aureus through domain shuffling. ACS Infect. Dis. 7: 2081-2092.
    Pubmed
  9. Amarasinghe C, Jin J-P. 2015. The use of affinity tags to overcome obstacles in recombinant protein expression and purification. Protein Peptide Lett. 22: 885-892.
    Pubmed
  10. Roshanak S, Yarabbi H, Shahidi F, Tabatabaei Yazdi F, Movaffagh J, Javadmanesh A. 2023. Effects of adding poly-histidine tag on stability, antimicrobial activity and safety of recombinant buforin I expressed in periplasmic space of Escherichia coli. Sci. Rep. 13: 5508.
    Pubmed KoreaMed
  11. Chaga GS. 2001. Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. J. Biochem. Biophys. Methods 49: 313-334.
    Pubmed
  12. Boksha IS, Lavrova NV, Grishin AV, Demidenko AV, Lyashchuk AM, Galushkina ZM, et al. 2016. Staphylococcus simulans recombinant lysostaphin: production, purification, and determination of antistaphylococcal activity. Biochemistry. 81: 502-510.
    Pubmed
  13. Park J-M, Kim J-H, Kim G, Sim H-J, Ahn S-M, Choi K-S, et al. 2024. Rapid antibacterial activity assessment of chimeric lysins. Int. J. Mol. Sci. 25: 2430.
    Pubmed KoreaMed
  14. Mitkowski P, Jagielska E, Nowak E, Bujnicki JM, Stefaniak F, Niedziałek D, et al. 2019. Structural bases of peptidoglycan recognition by lysostaphin SH3b domain. Sci. Rep. 9: 5965.
    Pubmed KoreaMed
  15. Park J-M, Ko D-S, Kim H-S, Kim N-H, Kim E-K, Roh Y-H, et al. 2023. Rapid screening and comparison of chimeric lysins for antibacterial activity against Staphylococcus aureus strains. Antibiotics 12: 667.
    Pubmed KoreaMed
  16. Wei H, Yang, Hang, YU, Junping. 2018. Staphylococcus lyase and Use thereof. PCT/CN2016/079044.
  17. Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, et al. 2022. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50: D439-d444.
    Pubmed KoreaMed
  18. Pokhrel R, Shakya R, Baral P, Chapagain P. 2022. Molecular modeling and simulation of the peptidoglycan layer of Gram-positive bacteria Staphylococcus aureus. J. Chem. Inform. Model. 62: 4955-4962.
    Pubmed
  19. Heckman KL, Pease LR. 2007. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat. Protoc. 2: 924-932.
    Pubmed
  20. Fall RR, Vagelos PR. 1972. Acetyl coenzyme A carboxylase. Molecular forms and subunit composition of biotin carboxyl carrier protein. J. Biol. Chem. 247: 8005-8015.
    Pubmed
  21. Tossavainen H, Raulinaitis V, Kauppinen L, Pentikainen U, Maaheimo H, Permi P. 2018. Structural and functional insights into lysostaphin-substrate interaction. Front. Mol. Biosci. 5: 60.
    Pubmed KoreaMed
  22. Pokhrel R, Shakya R, Baral P, Chapagain P. 2022. Molecular modeling and simulation of the peptidoglycan layer of gram-positive bacteria Staphylococcus aureus. J. Chem. Inf. Model 62: 4955-4962.
    Pubmed
  23. Vollmer W, Blanot D, de Pedro MA. 2008. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32: 149-167.
    Pubmed
  24. Oliveira H, Melo LD, Santos SB, Nobrega FL, Ferreira EC, Cerca N, et al. 2013. Molecular aspects and comparative genomics of bacteriophage endolysins. J. Virol. 87: 4558-4570.
    Pubmed KoreaMed
  25. Kim H, Seo J. 2023. A novel strategy to identify endolysins with lytic activity against methicillin-resistant Staphylococcus aureus. Int. J. Mol. Sci. 24: 5772.
    Pubmed KoreaMed
  26. Yang H, Zhang Y, Yu J, Huang Y, Zhang X-E, Wei H. 2014. Novel chimeric lysin with high-level antimicrobial activity against methicillin-resistant Staphylococcus aureus in vitro and in vivo. Antimicrob. Agents Chemother. 58: 536-542.
    Pubmed KoreaMed
  27. Eichenseher F, Herpers Bjorn L, Badoux P, Leyva-Castillo Juan M, Geha Raif S, van der Zwart M, et al. 2022. Linker-improved chimeric endolysin selectively kills Staphylococcus aureus in vitro, on reconstituted human epidermis, and in a murine model of skin infection. Antimicrob. Agents Chemother. 66: e02273-02221.
    Pubmed KoreaMed
  28. Shen W, Yang N, Teng D, Hao Y, Ma X, Mao R, et al. 2021. Design and high expression of non-glycosylated lysostaphins in Pichia pastoris and their pharmacodynamic study. Front. Microbiol. 12: 637662.
    Pubmed KoreaMed
  29. Zhao H, Verma D, Li W, Choi Y, Ndong C, Fiering SN, et al. 2015. Depletion of T cell epitopes in lysostaphin mitigates anti-drug antibody response and enhances antibacterial efficacy in vivo. Chem. Biol. 22: 629-639.
    Pubmed KoreaMed