LytU-SH3b fusion protein as a novel and efficient enzybiotic against methicillin-resistant Staphylococcus aureus

Document Type : Original article

Authors

1 Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran

2 Recombinant Protein Laboratory, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

3 Cardiovascular Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

4 Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran

5 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

6 Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

7 Maternal-Fetal Medicine Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a challenging infectious agent worldwide. The ever growing antibiotic resistance has made the researchers to look for new anti-staphylococcal agents. Autolysins are staphylococcal enzymes that lyse bacterial cell wall for cell division. Autolysins can be used as novel enzybiotics (enzymes have antibiotic effects) for staphylococcal infections. LytU is a newly explored autolysin. SH3b is a potent cell wall binding domain that can be fused to lytic enzymes to increase their activity. The aim of this study was to design a novel and efficient fusion enzybiotic that could lyse staphylococcal cell wall peptidoglycan by disrupting the bacteria. LytU-SH3b fusion construct was synthesized and LytU was amplified through the construct, using overhang PCR. The fusion and native forms that had his-tag were synthesized by recombinant technology in Escherichia coli BL21 (DE3) strain and purified utilizing Ni-NTA agarose beads. LytU and LytU-SH3b activity and potency were assessed using plate lysis assay, turbidity reduction assay and minimal inhibitory concentration (MIC) tests. All these tests showed that LytU-SH3b has more activity and potency than LytU. LytU-SH3b has MIC 421 fold lesser than LytU. Finally, LytU-SH3b is a novel and efficient recombinant enzybiotic that can lyse MRSA as an alternative to chemical small molecule antibiotics.

Keywords

References

1. Okwu MU, Olley M, Akpoka AO, Izevbuwa OE. Methicillin-resistant Staphylococcus aureus (MRSA) and anti-MRSA activities of extracts of some medicinal plants: A brief review. AIMS Microbiol 2019;5:117-137.
2. Borrell S, Trauner A. Strain Diversity and the Evolution of Antibiotic Resistance. Adv Exp Med Biol 2017;1019:263-279.
3. Osipovitch DC, Griswold KE. Fusion with a cell wall binding domain renders autolysin LytM a potent anti-Staphylococcus aureus agent. FEMS Microbiol Lett 2015;362:1-7.
4. Tafvizizavareh S, Shariati P, Sharifirad A, Maleki B, Aliakbari F, Christiansen G, Morshedi D. Antibiotic hypersensitivity in MRSA induced by special protein aggregates. Int J Biol Macromol 2019;137:528-536.
5. Szweda P, Schielmann M, Kotlowski R, Gorczyca G, Zalewska M, Milewski S. Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus. Appl Microbiol Biotechnol 2012;96:1157-1174.
6. Jagielska E, Chojnacka O, Sabała I. LytM Fusion with SH3b-Like Domain Expands Its Activity to Physiological Conditions. Microb Drug Resist 2016;22:461-469.
7. Raulinaitis V, Tossavainen H, Aitio O, Juuti JT, Hiramatsu K, Kontinen V, Permi P. Identification and structural characterization of LytU, a unique peptidoglycan endopeptidase from the lysostaphin family. Sci Rep 2017;7:6020.
8. Climo MW, Patron RL, Goldstein BP, Archer GL. Lysostaphin treatment of experimental methicillin-resistant Staphylococcus aureus aortic valve endocarditis. Antimicrob Agents Chemother 1998;42:1355-1360.
9. Kokai-Kun JF, Walsh SM, Chanturiya T, Mond JJ. Lysostaphin cream eradicates Staphylococcus aureus nasal colonization in a cotton rat model. Antimicrob Agents Chemother 2003;47:1589-1597.
10. Kokai-Kun JF, Chanturiya T, Mond JJ. Lysostaphin as a treatment for systemic Staphylococcus aureus infection in a mouse model. J Antimicrob Chemother 2007;60:1051-1059.
11. Kokai-Kun JF, Chanturiya T, Mond JJ. Lysostaphin eradicates established Staphylococcus aureus biofilms in jugular vein catheterized mice. J Antimicrob Chemother 2009;64:94-100.
12. Placencia FX, Kong L, Weisman LE. Treatment of methicillin-resistant Staphylococcus aureus in neonatal mice: lysostaphin versus vancomycin. Pediatr Res 2009;65:420-424.
13. Frankel MB, Schneewind O. Determinants of murein hydrolase targeting to cross-wall of Staphylococcus aureus peptidoglycan. J Biol Chem 2012;287:10460-10471.
14. Baba T, Schneewind O. Target cell specificity of a bacteriocin molecule: a C‐terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus. EMBO J 1996;15:4789-4797.
15. Tossavainen H, Raulinaitis V, Kauppinen L, Pentikäinen U, Maaheimo H, Permi P. Structural and functional insights into lysostaphin–substrate interaction. Front Mol Biosci 2018;5:60.
16. Gründling A, Schneewind O. Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus. J Bacteriol 2006;188:2463-2472.
17. Becker SC, Swift S, Korobova O, Schischkova N, Kopylov P, Donovan DM, Abaev I.  Lytic activity of the staphylolytic Twort phage endolysin CHAP domain is enhanced by the SH3b cell wall binding domain. FEMS Microbiol Lett 2015;362:1-8.
18. Idelevich EA, von Eiff C, Friedrich AW, Iannelli D, Xia G, Peters G, Peschel A, Wanninger I, Becker K. In vitro activity against Staphylococcus aureus of a novel antimicrobial agent, PRF-119, a recombinant chimeric bacteriophage endolysin. Antimicrob Agents Chemother 2011;55:4416-4419.
19. Mousavi P, Mostafavi-Pour Z, Morowvat MH, Nezafat N, Zamani M, Berenjian A, Ghasemi Y. In silico analysis of several signal peptides for the excretory production of reteplase in Escherichia coli. Curr Proteomics 2017;14:326-335.
20.Taheri-Anganeh M, Khatami SH, Jamali Z, Savardashtaki A, Ghasemi Y, Mostafavi-pour Z. In silico analysis of suitable signal peptides for secretion of a recombinant alcohol dehydrogenase with a key role in atorvastatin enzymatic synthesis. Mol Biol Res Commun 2019;8:17-26.
21. Savardashtaki A, Sarkari B, Arianfar F, Mostafavi-Pour Z. Immunodiagnostic value of Echinococcus granulosus recombinant B8/1 subunit of antigen B. Iran J Immunol 2017;14:111-122.
23. Lee AS, de Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Peschel A, Harbarth S. Methicillin-resistant Staphylococcus aureus. Nat Rev Dis Primers 2018;4:18033.
24. Mitkowski P, Jagielska E, Nowak E, Bujnicki JM, Stefaniak F, Niedziałek D, Bochtler M, Sabala I. Structural bases of peptidoglycan recognition by lysostaphin SH3b domain. Sci Rep 2019;9:5965.
25. Becker SC, Foster-Frey J, Stodola AJ, Anacker D, Donovan DM. Differentially conserved staphylococcal SH3b_5 cell wall binding domains confer increased staphylolytic and streptolytic activity to a streptococcal prophage endolysin domain. Gene 2009;443:32-41.
26. Mao J, Schmelcher M, Harty WJ, Foster-Frey J, Donovan DM. Chimeric Ply187 endolysin kills Staphylococcus aureus more effectively than the parental enzyme. FEMS Microbiol Lett 2013;342:30-36.
27. Lechner S, Lewis K, Bertram R. Staphylococcus aureus persisters tolerant to bactericidal antibiotics. J Mol Microbiol Biotechnol 2012;22:235-244.
28.Yamakawa T, Mitsuyama J, Hayashi K. In vitro and in vivo antibacterial activity of T-3912, a novel non-fluorinated topical quinolone. J Antimicrob Chemother 2002;49:455-465.
29. Peschel A, Vuong C, Otto M, Götz F. The D-alanine residues of Staphylococcus aureus teichoic acids alter the susceptibility to vancomycin and the activity of autolytic enzymes. Antimicrob Agents Chemother 2000;44:2845-2847.

Files

Share

How to cite

Statistics