Bacillus phage endolysin, lys46, bactericidal properties against Gram-negative bacteria
-
Mohammad Reza Sarjoughian
,
Fereshte Rahmani
,
Shamsozoha Abolmaali
,
Shakiba Darvish Alipour Astaneh
Abstract
Background and objective: The great potential of bacteriophage for removing pathogen bacteria via targeting the cell wall is highly concerned. With a priority for drug-resistant, we screened endolysins targeting Gram-negative bacteria to introduce a new antibacterial agent. The study was aimed to identify endolysins from the lysogenic phage of the Siphoviridea family in Bacillus subtilis.
Materials and Methods: The Bacillus subtilis strain DDBCC46 was isolated from a preliminary antibacterial screening program. The endolysin(s) was extracted, concentrated with ammonium sulfate saturation, and their activity evaluated against the indicator bacteria. The phage particles was extracted from the bacteria using the minimum inhibition concentration of mitomycin C, followed by testing the phage inhibitory effect on the growth of indicator bacteria. The NCBI, Virus-Host DB, and EXPASY databases were used to obtain and confirm the sequences of the genes encoding PG hydrolases in Siphoviridea phages hosted in B. subtilis.
Results: An 816 bp gene encoding an endolysin enzyme, was approved in the B. subtilis, DDBCC 46, with specific primers of Bacillus phage SPP1. The purified-endolysin indicated antibacterial activity against Klebsiella pneumoniae, Salmonella typhimurium, Proteus (sp), and Escherichia coli. SDS-PAGE profiling followed by silica gel purification, led to introduce lys4630 as a therapeutic product and food preservative.
Conclusion: lys4630 showed specific effects on the very known gram-negative pathogens in clinics and food industries; E. coli, P. aeruginosa, Salmonella (sp).
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26. Oliveira H, Vilas Boas D, Mesnage S, Kluskens LD, Lavigne R, Sillankorva S, et al. Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-gram-negative bacterial activity. Front Microbiol 2016; 7: 208.
27. Lai WCB, Chen X, Ho MKY, Xia J, Leung SSY. Bacteriophage-derived Endolysins to Target Gram-Negative Bacteria. Int J Pharm 2020: 589:119833.
28. Salazar F, Ortiz A, Sansinenea E. Characterisation of two novel bacteriocin-like substances produced by Bacillus amyloliquefaciens ELI149 with broad-spectrum antimicrobial activity. J Glob Antimicrob Resist 2017; 11: 177-182.
29. Yadav N, Gupta MN, Khare SK. Three phase partitioning and spectroscopic characterization of bioactive constituent from halophilic Bacillus subtilis EMB M15. Bioresour Technol 2017; 242: 283-286.
30. Hyman P. Phages for phage therapy: isolation, characterization, and host range breadth. Pharmaceuticals (Basel) 2019; 12: 35.
31. Doria F, Napoli C, Costantini A, Berta G, Saiz J-C, Garcia-Moruno E. Development of a new method for detection and identification of Oenococcus oeni bacteriophages based on endolysin gene sequence and randomly amplified polymorphic DNA. Appl Environ Microbiol 2013; 79: 4799-4805.
32. Langlois C, Ramboarina S, Cukkemane A, Auzat I, Chagot B, Gilquin B, et al. Bacteriophage SPP1 tail tube protein self-assembles into β-structure-rich tubes. J Biol Chem 2015; 290: 3836-3849.
33. Barth A. Infrared spectroscopy of proteins. Biochim Biophys Acta 2007; 1767:1073-1101.
34. Flayhan A, Wien F, Paternostre M, Boulanger P, Breyton C. New insights into pb5, the receptor binding protein of bacteriophage T5, and its interaction with its Escherichia coli receptor FhuA. Biochimie 2012; 94: 1982-1989.
35. Dziewit L, Oscik K, Bartosik D, Radlinska M. Molecular characterization of a novel temperate Sinorhizobium bacteriophage, ФLM21, encoding DNA methyltransferase with CcrM-like specificity. J Virol 2014; 88: 13111-13124.
2. Schmelcher M, Donovan DM, Loessner MJ. Bacteriophage endolysins as novel antimicrobials. Future Microbiol 2012; 7:1147-1171.
3. Lim J-A, Shin H, Heu S, Ryu S. Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria. J Microbiol Biotechnol 2014; 24: 803-811.
4. Tisáková L, Godány A. Bacteriophage endolysins and their use in biotechnological processes. J Microbiol Biotechnol Food Sci 2014; 3: 164.
5. Shi Y, Yan Y, Ji W, Du B, Meng X, Wang H, et al. Characterization and determination of holin protein of Streptococcus suis bacteriophage SMP in heterologous host. Virol J 2012; 9: 70.
6. Briers Y, Lavigne R. Breaking barriers: expansion of the use of endolysins as novel antibacterials against Gram-negative bacteria. Future Microbiol 2015; 10: 377-390.
7. Gondil VS, Harjai K, Chhibber S. Endolysins as emerging alternative therapeutic agents to counter drug-resistant infections. Int J Antimicrob Agents 2020; 55: 105844.
8. Shannon R, Radford DR, Balamurugan S. Impacts of food matrix on bacteriophage and endolysin antimicrobial efficacy and performance. Crit Rev Food Sci Nutr 2020; 60: 1631-1640.
9. Fernández-Ruiz I, Coutinho FH, Rodriguez-Valera F. Thousands of novel endolysins discovered in uncultured phage genomes. Front Microbiol 2018; 9: 1033.
10. Noormohammadi H, Abolmaali S, Astaneh SDA. Identification and characterization of an endolysin–Like from Bacillus subtilis. Microb Pathog 2018; 119: 221-224.
11. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal 2016; 6: 71-79.
12. Usmiati S, Marwati T. Selection and optimization process of bacteriocin production from Lactobacillus sp. Indones. Indones J Agric 2009; 2: 82-92.
13. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254.
14. Park S, Jun SY, Kim C-H, Jung GM, Son JS, Jeong ST, et al. Characterisation of the antibacterial properties of the recombinant phage endolysins AP50-31 and LysB4 as potent bactericidal agents against Bacillus anthracis. Sci Rep 2018; 8: 18.
15. Siddiqui A, Hart E, Mandagere U, Goldberg I. Rapid plate method for the isolation of lysogenic bacteriophages. Appl Microbiol 1974; 27: 278-280.
16. Sousa Dias R, Eiko Abe A, Santiago Lim H, Carneiro Fidélis Silv L, Oliveira de Paula S, Canêdo da Silva C. Viral concentration methods for diversity studies in soil samples. Appl Soil Ecol 2020; 155: 103666.
17. Abedon ST, Yin J (2009). Bacteriophage Plaques: Theory and Analysis. In: Bacteriophages. Eds, MR Clokie, AM Kropinski. Humana Press, pp. 161-74.
18. Lin NT, Chiou PY, Chang KC, Chen LK, Lai MJ. Isolation and characterization of ϕAB2: a novel bacteriophage of Acinetobacter baumannii. Res Microbiol 2010; 161: 308-314.
19. Mao DP, Zhou Q, Chen CY, Quan ZX. Coverage evaluation of universal bacterial primers using the metagenomic datasets. BMC Microbiol 2012; 12: 66.
20. Gupta R, Prasad Y. P-27/HP endolysin as antibacterial agent for antibiotic resistant Staphylococcus aureus of human infections. Curr Microbiol 2011; 63: 39-45.
21. Asenjo J, Andrews B. Protein purification using chromatography: selection of type, modelling and optimization of operating conditions. J Mol Recognit 2009; 22: 65-76.
22. Love MJ, Abeysekera GS, Muscroft-Taylor AC, Billington C, Dobson RC. On the catalytic mechanism of bacteriophage endolysins: Opportunities for engineering. Biochim Biophys Acta Proteins Proteom 2020; 1868: 140302.
23. Lood R, Raz A, Molina H, Euler CW, Fischetti VA. A highly active and negatively charged Streptococcus pyogenes lysin with a rare D-alanyl-L-alanine endopeptidase activity protects mice against streptococcal bacteremia. Antimicrob Agents Chemother 2014; 58: 3073-3084.
24. Mishra A, Sharma N, Kumar A, Kumar N, Bayyappa MG, Kumar S, et al. Isolation, characterization and therapeutic potential assessment of bacteriophages virulent to Staphylococcus aureus associated with goat mastitis. Iran J Vet Res 2014; 15: 320-325.
25. Oliveira H, Thiagarajan V, Walmagh M, Sillankorva S, Lavigne R, Neves-Petersen MT, et al. A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids. PLoS One 2014; 9(10):e108376.
26. Oliveira H, Vilas Boas D, Mesnage S, Kluskens LD, Lavigne R, Sillankorva S, et al. Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-gram-negative bacterial activity. Front Microbiol 2016; 7: 208.
27. Lai WCB, Chen X, Ho MKY, Xia J, Leung SSY. Bacteriophage-derived Endolysins to Target Gram-Negative Bacteria. Int J Pharm 2020: 589:119833.
28. Salazar F, Ortiz A, Sansinenea E. Characterisation of two novel bacteriocin-like substances produced by Bacillus amyloliquefaciens ELI149 with broad-spectrum antimicrobial activity. J Glob Antimicrob Resist 2017; 11: 177-182.
29. Yadav N, Gupta MN, Khare SK. Three phase partitioning and spectroscopic characterization of bioactive constituent from halophilic Bacillus subtilis EMB M15. Bioresour Technol 2017; 242: 283-286.
30. Hyman P. Phages for phage therapy: isolation, characterization, and host range breadth. Pharmaceuticals (Basel) 2019; 12: 35.
31. Doria F, Napoli C, Costantini A, Berta G, Saiz J-C, Garcia-Moruno E. Development of a new method for detection and identification of Oenococcus oeni bacteriophages based on endolysin gene sequence and randomly amplified polymorphic DNA. Appl Environ Microbiol 2013; 79: 4799-4805.
32. Langlois C, Ramboarina S, Cukkemane A, Auzat I, Chagot B, Gilquin B, et al. Bacteriophage SPP1 tail tube protein self-assembles into β-structure-rich tubes. J Biol Chem 2015; 290: 3836-3849.
33. Barth A. Infrared spectroscopy of proteins. Biochim Biophys Acta 2007; 1767:1073-1101.
34. Flayhan A, Wien F, Paternostre M, Boulanger P, Breyton C. New insights into pb5, the receptor binding protein of bacteriophage T5, and its interaction with its Escherichia coli receptor FhuA. Biochimie 2012; 94: 1982-1989.
35. Dziewit L, Oscik K, Bartosik D, Radlinska M. Molecular characterization of a novel temperate Sinorhizobium bacteriophage, ФLM21, encoding DNA methyltransferase with CcrM-like specificity. J Virol 2014; 88: 13111-13124.
| Files | ||
| Issue | Vol 12 No 6 (2020) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v12i6.5036 | |
| Keywords | ||
| Antibacterial activity Bacteriophage SPP1 Endolysin Siphoviridea | ||
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How to Cite
1.
Sarjoughian MR, Rahmani F, Abolmaali S, Darvish Alipour Astaneh S. Bacillus phage endolysin, lys46, bactericidal properties against Gram-negative bacteria. Iran J Microbiol. 2020;12(6):607-615.
