Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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Development of a novel endolysin, PanLys.1, for the specific inhibition of Peptostreptococcus anaerobius

  • Joonbeom Moon (Department of Animal Science, Life and Industry Convergence Research Institute, College of Natural Resources and Life Sciences, Pusan National University) ;
  • Hanbeen Kim (Department of Animal Science, Life and Industry Convergence Research Institute, College of Natural Resources and Life Sciences, Pusan National University) ;
  • Dongseok Lee (Department of Animal Science, Life and Industry Convergence Research Institute, College of Natural Resources and Life Sciences, Pusan National University) ;
  • Jakyeom Seo (Department of Animal Science, Life and Industry Convergence Research Institute, College of Natural Resources and Life Sciences, Pusan National University)
  • Received : 2022.12.01
  • Accepted : 2023.03.08
  • Published : 2023.08.01
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Abstract

Objective: The objective of this study was to develop a novel endolysin (PanLys.1) for the specific killing of the ruminal hyper-ammonia-producing bacterium Peptostreptococcus anaerobius (P. anaerobius). Methods: Whole genome sequences of P. anaerobius strains and related bacteriophages were collected from the National Center for Biotechnology Information database, and the candidate gene for PanLys.1 was isolated based on amino acid sequences and conserved domain database (CDD) analysis. The gene was overexpressed using a pET system in Escherichia coli BL21 (DE3). The lytic activity of PanLys.1 was evaluated under various conditions (dosage, pH, temperature, NaCl, and metal ions) to determine the optimal lytic activity conditions. Finally, the killing activity of PanLys.1 against P. anaerobius was confirmed using an in vitro rumen fermentation system. Results: CDD analysis showed that PanLys.1 has a modular design with a catalytic domain, amidase-2, at the N-terminal, and a cell wall binding domain, from the CW-7 superfamily, at the C-terminal. The lytic activity of PanLys.1 against P. anaerobius was the highest at pH 8.0 (p<0.05) and was maintained at 37℃ to 45℃, and 0 to 250 mM NaCl. The activity of PanLys.1 significantly decreased (p<0.05) after Mn2+ or Zn2+ treatment. The relative abundance of P. anaerobius did not decrease after administration PanLys.1 under in vitro rumen conditions. Conclusion: The application of PanLys.1 to modulate P. anaerobius in the rumen might not be feasible because its lytic activity was not observed in in vitro rumen system.

Keywords

Acknowledgement

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (NRF-2019R1F1A1056904 and NRF-2022R1A2C1006958).

References

  1. Bach A, Calsamiglia S, Stern MD. Nitrogen metabolism in the rumen. J Dairy Sci 2005;88:E9-E21. https://doi.org/10.3168/jds.S0022-0302(05)73133-7
  2. Tan P, Liu H, Zhao J, et al. Amino acids metabolism by rumen microorganisms: Nutrition and ecology strategies to reduce nitrogen emissions from the inside to the outside. Sci Total Environ 2021;800:149596. https://doi.org/10.1016/j.scitotenv.2021.149596
  3. Rychlik JL, Russell JB. Mathematical estimations of hyper-ammonia producing ruminal bacteria and evidence for bacterial antagonism that decreases ruminal ammonia production. FEMS Microbiol Ecol 2000;32:121-8. https://doi.org/10.1111/j.1574-6941.2000.tb00706.x
  4. Chen G, Russell JB. Fermentation of peptides and amino acids by a monensin-sensitive ruminal Peptostreptococcus. Appl Environ Microbiol 1988;54:2742-9. https://doi.org/10.1128/aem.54.11.2742-2749.1988
  5. Chen G, Russell JB. More monensin-sensitive, ammonia-producing bacteria from the rumen. Appl Environ Microbiol 1989;55:1052-7. https://doi.org/10.1128/aem.55.5.1052-1057.1989
  6. Houlihan AJ, Russell JB. The susceptibility of ionophore-resistant Clostridium aminophilum F to other antibiotics. J Antimicrob Chemother 2003;52:623-8. https://doi.org/10.1093/jac/dkg398
  7. Flythe MD, Andries K. The effects of monensin on amino acid catabolizing bacteria isolated from the Boer goat rumen. Small Rumin Res 2009;81:178-81. https://doi.org/10.1016/j.smallrumres.2008.12.004
  8. Wang ZB, Xin HS, Wang MJ, et al. Effects of dietary supplementation with hainanmycin on protein degradation and populations of ammonia-producing bacteria in vitro. Asian-Australas J Anim Sci 2013;26:668-74. https://doi.org/10.5713/ajas.2012.12589
  9. Flythe MD, Kagan I. Antimicrobial effect of red clover (Tri-folium pratense) phenolic extract on the ruminal hyper ammonia-producing bacterium, Clostridium sticklandii. Curr Microbiol 2010;61:125-31. https://doi.org/10.1007/s00284-010-9586-5
  10. Flythe MD, Harlow BE, Aiken GE, Gellin GL, Kagan IA, Pappas J. Inhibition of growth and ammonia production of ruminal hyper ammonia-producing bacteria by chinook or galena hops after long-term storage. Fermentation 2017;3:68. https://doi.org/10.3390/fermentation3040068
  11. Shen J, Yu Z, Zhu W. Insights into the populations of proteolytic and amino acid-fermenting bacteria from microbiota analysis using in vitro enrichment cultures. Curr Microbiol 2018;75:1543-50. https://doi.org/10.1007/s00284-018-1558-1
  12. Schmelcher M, Donovan DM, Loessner MJ. Bacteriophage endolysins as novel antimicrobials. Future Microbiol 2012;7:1147-71. https://doi.org/10.2217/fmb.12.97
  13. Gerstmans H, Criel B, Briers Y. Synthetic biology of modular endolysins. Biotechnol Adv 2018;36:624-40. https://doi.org/10.1016/j.biotechadv.2017.12.009
  14. Jiang Y, Xu D, Wang L, et al. Characterization of a broad-spectrum endolysin LysSP1 encoded by a Salmonella bacteriophage. Appl Microbiol Biotechnol 2021;105:5461-70. https://doi.org/10.1007/s00253-021-11366-z
  15. Lai MJ, Lin NT, Hu A, et al. Antibacterial activity of Acinetobacter baumannii phage ϕAB2 endolysin (LysAB2) against both gram-positive and gram-negative bacteria. Appl Microbiol Biotechnol 2011;90:529-39. https://doi.org/10.1007/s00253-011-3104-y
  16. Wang C, Shi S, Wei M, Luo Y. Characterization of a novel broad-spectrum endolysin PlyD4 encoded by a highly conserved prophage found in Aeromonas hydrophila ST251 strains. Appl Microbiol Biotechnol 2022;106:699-711. https://doi.org/10.1007/s00253-021-11752-7
  17. Kim HB, Lee HG, Kwon IH, Seo JK. Characterization of endolysin LyJH307 with antimicrobial activity against Streptococcus bovis. Animals 2020;10:963. https://doi.org/10.3390/ani10060963
  18. Akhter S, Aziz RK, Edwards RA. PhiSpy: a novel algorithm for finding prophages in bacterial genomes that combines similarity- and composition-based strategies. Nucleic acids Res 2012;40:e126. https://doi.org/10.1093/nar/gks406
  19. Bustamante N, Campillo NE, Garcia E, et al. Cpl-7, a lysozyme encoded by a pneumococcal bacteriophage with a novel cell wall-binding motif. J Biol Chem 2010;285:33184-96. https://doi.org/10.1074/jbc.M110.154559
  20. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 2013;30:772-80. https://doi.org/10.1093/molbev/mst010
  21. Zhou L, Feng T, Xu S, et al. ggmsa: a visual exploration tool for multiple sequence alignment and associated data. Brief Bioinform 2022;23:bbac222. https://doi.org/10.1093/bib/bbac222
  22. Mirdita M, Schutze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods 2022;19:679-82. https://doi.org/10.1038/s41592-022-01488-1
  23. Goddard TD, Huang CC, Meng EC, et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci 2018;27:14-25. https://doi.org/10.1002/pro.3235
  24. Goering HK, Van Soest PJ. Forage fiber analysis (apparatus, reagents, prcedures, and some applications). Agriculture Handbook No. 379. Washington, DC, USA: US Agriculture Research Service; 1970.
  25. Yu Z, Morrison M. Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques 2004;36:808-12. https://doi.org/10.2144/04365ST04
  26. Kim HB, Kim BW, Cho SK, Kwon IH, Seo JK. Dietary lysophospholipids supplementation inhibited the activity of lipolytic bacteria in forage with high oil diet: an in vitro study. Asian-Australas J Anim Sci 2020;33:1590-8. https://doi.org/10.5713/ajas.19.0850
  27. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc 2008;3:1101-8. https://doi.org/10.1038/nprot.2008.73
  28. Dziarski R, Gupta D. The peptidoglycan recognition proteins (PGRPs). Genome Biol 2006;7:232. https://doi.org/10.1186/gb-2006-7-8-232
  29. Bustamante N, Iglesias-Bexiga M, Bernardo-Garcia N, et al. Deciphering how Cpl-7 cell wall-binding repeats recognize the bacterial peptidoglycan. Sci Rep 2017;7:16494. https://doi.org/10.1038/s41598-017-16392-4
  30. Kikelomo AM. Preliminary physico-chemical investigation of local binding agents in mineral salt licks production for ruminants. Int J Environ Agric Biotechnol 2016;1:238626. http://doi.org/10.22161/ijeab/1.4.52
  31. Yang H, Wang M, Yu J, Wei H. Antibacterial activity of a novel peptide-modified lysin against Acinetobacter baumannii and Pseudomonas aeruginosa. Front Microbiol 2015;6:1471. https://doi.org/10.3389/fmicb.2015.01471
  32. Vouillamoz J, Entenza JM, Giddey M, Fischetti VA, Moreillon P, Resch G. Bactericidal synergism between daptomycin and the phage lysin Cpl-1 in a mouse model of pneumococcal bacteraemia. Int J Antimicrob Agents 2013;42:416-21. https://doi.org/10.1016/j.ijantimicag.2013.06.020
  33. Garcia JL, Garcia E, Arraras A, Garcia P, Ronda C, Lopez R. Cloning, purification, and biochemical characterization of the pneumococcal bacteriophage Cp-1 lysin. J Virol 1987;61:2573-80. https://doi.org/10.1128/jvi.61.8.2573-2580.1987
  34. Oliveira H, Sao-Jose C, Azeredo J. Phage-derived peptidoglycan degrading enzymes: challenges and future prospects for in vivo therapy. Viruses 2018;10:292. https://doi.org/10.3390/v10060292