Production of bacteriophage-encoded endolysin, LysP11, in Nicotiana benthamiana and its activity as a potent antimicrobial agent against Erysipelothrix rhusiopathiae
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We produced a biologically active phage-encoded endolysin, LysP11, in N. benthamiana. Plant-produced LysP11 exhibited robust antimicrobial activity against E. rhusiopathiae, and C-terminal domain of LysP11 bound specifically to E. rhusiopathiae.
Bacterial resistance to antibiotics, a serious issue in terms of global public health, is one of the leading causes of death today. Thus, new antimicrobial agents are needed to combat pathogens. Recent research suggests that bacteriophages and endolysins derived from bacteriophages are potential alternatives to traditional antibiotics. Here, we examined the antimicrobial activity of LysP11, which is encoded by Propionibacterium phage P1.1 and comprises an N-terminal amidase-2 domain and a C-terminal domain with no homology to other bacteriophage endolysins. LysP11 was produced in Nicotiana benthamiana (N. benthamiana) using an Agrobacterium-mediated transient expression strategy. LysP11 was purified on microcrystalline cellulose-binding resin after attachment of the Clostridium thermocellum-derived family 3 cellulose-binding domain as an affinity tag. The affinity tag was removed using the small ubiquitin-related modifier (SUMO) domain and SUMO-specific protease. Plant-produced LysP11 showed strong antimicrobial activity toward Erysipelothrix rhusiopathiae (E. rhusiopathiae), mediated via lysis of the cell wall. Lytic activity was optimal at pH 8.0–9.0 (37 °C) and increased at higher concentrations of NaCl up to 400 mM. Furthermore, the C-terminal domain of LysP11 bound specifically to the E. rhusiopathiae cell wall. Based on these results, we propose that LysP11 is a potential candidate antimicrobial agent against E. rhusiopathiae.
KeywordsPlant-based expression systems Nicotiana benthamiana Endolysin LysP11 Cellulose-binding domain Erysipelothrix rhusiopathiae
This work was supported by grants from the Ministry of Trade, Industry and Energy (MOTIE, Korea) under Industrial Technology Innovation Program (no. 10063301), Cooperative Research Program for Agriculture Science and Technology Development (no. PJ010953012019), Republic of Korea. Junho Lee and Dong Wook Lee supported by grants from the Next-Generation BioGreen 21 Program (nos. PJ01365001 and SSAC: PJ01335801 respectively), Rural Development Administration, Republic of Korea.
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Conflict of interest
The authors declare that they have no competing interests to report.
- Carrick BH, Hao L, Smaldino PJ, Engelke DR (2016) A novel recombinant DNA system for high efficiency affinity purification of proteins in Saccharomyces cerevisiae. G3 (Bethesda) 6:573–578Google Scholar
- Clark AE (2015) The occupational opportunist: an update on Erysipelothrix rhusiopathiae infection, disease pathogenesis, and microbiology. Clin Microbiol Newsl 37:143–151Google Scholar
- Gomord V, Denmat LA, Fitchette-Lainé AC, Satiat-Jeunemaitre B, Hawes C, Faye L (1997) The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER. Plant J 11:313–325PubMedGoogle Scholar
- Kashani HH, Fahimi H, Dasteh Goli Y, Moniri R (2017) A novel chimeric endolysin with antibacterial activity against methicillin-resistant Staphylococcus aureus. Front Cell Infect Microbiol 7:290Google Scholar
- Kashani HH, Schmelcher M, Sabzalipoor H, Hosseini ES, Moniri R (2018) Recombinant endolysins as potential therapeutics against antibiotic-resistant Staphylococcus aureus: current status of research and novel delivery strategies. Clin Microbiol Rev 31:e00071-17Google Scholar
- Kovalskaya N, Foster-Frey J, Donovan DM, Bauchan G, Hammond RW (2015) Antimicrobial activity of bacteriophage endolysin produced in Nicotiana benthamiana plants. J Microbiol Biotechnol 26:160–170Google Scholar
- Krishnan A, Woodard SL (2014) TrypZean™: an animal-free alternative to bovine trypsin. In: Howard J, Hood E (eds) Commercial plant-produced recombinant protein products. Springer, Berlin, pp 43–63Google Scholar
- Marinelli LJ, Fitz-Gibbon S, Hayes C, Bowman C, Inkeles M, Loncaric A, Russell DA, Jacobs-Sera D, Cokus S, Pellegrini M, Kim J (2012) Propionibacterium acnes bacteriophages display limited genetic diversity and broad killing activity against bacterial skin isolates. MBio 3:e00279-12PubMedPubMedCentralGoogle Scholar
- Sohn EJ, Lee Y, Park N, Park M, Kim NH, Park S, Min K, Gu S, Park Y, Song J, An DJ (2018) Development of plant-produced E2 protein for use as a green vaccine against classical swine fever virus. J Plant Biol 61:241–252Google Scholar
- Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72:211Google Scholar