Multiple-site Mutations of Phage Bp7 Endolysin Improves Its Activities Against Target Bacteria

Overview

Journal Virol Sin

Specialty Microbiology

Date 2015 Oct 23

PMID 26490045

Citations 7

Authors

Can Zhang

Yuanchao Wang

Huzhi Sun

Huiying Ren

Affiliations

Soon will be listed here.

Abstract

The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7 (Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a pET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated (L99A, M102E) to construct pET28a-Bp7Δe, with pET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.

Citing Articles

Phage-derived proteins: Advancing food safety through biocontrol and detection of foodborne pathogens.

Choi D, Ryu S, Kong M Compr Rev Food Sci Food Saf. 2025; 24(2):e70124.

PMID: 39898971 PMC: 11891642. DOI: 10.1111/1541-4337.70124.

Bacteriophage as a potential biotherapeutics to combat present-day crisis of multi-drug resistant pathogens.

Pattnaik A, Pati S, Samal S Heliyon. 2024; 10(18):e37489.

PMID: 39309956 PMC: 11416503. DOI: 10.1016/j.heliyon.2024.e37489.

Engineering strategies and challenges of endolysin as an antibacterial agent against Gram-negative bacteria.

Zheng T, Zhang C Microb Biotechnol. 2024; 17(4):e14465.

PMID: 38593316 PMC: 11003714. DOI: 10.1111/1751-7915.14465.

Design, Screening, and Characterization of Engineered Phage Endolysins with Extracellular Antibacterial Activity against Gram-Negative Bacteria.

Sui B, Wang X, Zhao T, Zhen J, Ren H, Liu W Appl Environ Microbiol. 2023; 89(7):e0058123.

PMID: 37338346 PMC: 10370328. DOI: 10.1128/aem.00581-23.

Endolysins of bacteriophage vB_Sal-S-S10 can naturally lyse Salmonella enteritidis.

Wang X, Han L, Rong J, Ren H, Liu W, Zhang C BMC Vet Res. 2022; 18(1):410.

PMID: 36411417 PMC: 9677904. DOI: 10.1186/s12917-022-03514-y.

References

Borysowski J, Weber-Dabrowska B, Gorski A . Bacteriophage endolysins as a novel class of antibacterial agents. Exp Biol Med (Maywood). 2006; 231(4):366-77. DOI: 10.1177/153537020623100402. View

Legotsky S, Vlasova K, Priyma A, Shneider M, Pugachev V, Totmenina O . Peptidoglycan degrading activity of the broad-range Salmonella bacteriophage S-394 recombinant endolysin. Biochimie. 2014; 107 Pt B:293-9. DOI: 10.1016/j.biochi.2014.09.017. View

Lim J, Shin H, Heu S, Ryu S . Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria. J Microbiol Biotechnol. 2014; 24(6):803-11. DOI: 10.4014/jmb.1403.03035. View

Zhang T, Wang C, Lv J, Wang R, Zhong X . Survey on tetracycline resistance and antibiotic-resistant genotype of avian Escherichia coli in North China. Poult Sci. 2012; 91(11):2774-7. DOI: 10.3382/ps.2012-02453. View

Xu J, Baase W, Baldwin E, Matthews B . The response of T4 lysozyme to large-to-small substitutions within the core and its relation to the hydrophobic effect. Protein Sci. 1998; 7(1):158-77. PMC: 2143816. DOI: 10.1002/pro.5560070117. View

Liu L, Baase W, Michael M, Matthews B . Use of stabilizing mutations to engineer a charged group within a ligand-binding hydrophobic cavity in T4 lysozyme. Biochemistry. 2009; 48(37):8842-51. DOI: 10.1021/bi900685j. View

Lukacik P, Barnard T, Hinnebusch B, Buchanan S . Specific targeting and killing of Gram-negative pathogens with an engineered phage lytic enzyme. Virulence. 2013; 4(1):90-1. PMC: 3544754. DOI: 10.4161/viru.22683. View

Burrowes B, Harper D, Anderson J, McConville M, Enright M . Bacteriophage therapy: potential uses in the control of antibiotic-resistant pathogens. Expert Rev Anti Infect Ther. 2011; 9(9):775-85. DOI: 10.1586/eri.11.90. View

Fischetti V . Bacteriophage lysins as effective antibacterials. Curr Opin Microbiol. 2008; 11(5):393-400. PMC: 2597892. DOI: 10.1016/j.mib.2008.09.012. View

10.

DuPont H, Jiang Z, Belkind-Gerson J, Okhuysen P, Ericsson C, Ke S . Treatment of travelers' diarrhea: randomized trial comparing rifaximin, rifaximin plus loperamide, and loperamide alone. Clin Gastroenterol Hepatol. 2007; 5(4):451-6. DOI: 10.1016/j.cgh.2007.02.004. View

11.

Eriksson A, Baase W, Wozniak J, Matthews B . A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene. Nature. 1992; 355(6358):371-3. DOI: 10.1038/355371a0. View

12.

Xu Y, Liu Y, Liu Y, Pei J, Yao S, Cheng C . Bacteriophage therapy against Enterobacteriaceae. Virol Sin. 2015; 30(1):11-8. PMC: 8200896. DOI: 10.1007/s12250-014-3543-6. View

13.

Baase W, Liu L, Tronrud D, Matthews B . Lessons from the lysozyme of phage T4. Protein Sci. 2010; 19(4):631-41. PMC: 2867005. DOI: 10.1002/pro.344. View

14.

Lopez C, Yang Z, Altenbach C, Hubbell W . Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants. Proc Natl Acad Sci U S A. 2013; 110(46):E4306-15. PMC: 3832024. DOI: 10.1073/pnas.1318754110. View

15.

Olsen R, Frantzen C, Christensen H, Bisgaard M . An investigation on first-week mortality in layers. Avian Dis. 2012; 56(1):51-7. DOI: 10.1637/9777-051011-Reg.1. View

16.

Zhang C, Li W, Liu W, Zou L, Yan C, Lu K . T4-like phage Bp7, a potential antimicrobial agent for controlling drug-resistant Escherichia coli in chickens. Appl Environ Microbiol. 2013; 79(18):5559-65. PMC: 3754183. DOI: 10.1128/AEM.01505-13. View

17.

Fischetti V . Bacteriophage endolysins: a novel anti-infective to control Gram-positive pathogens. Int J Med Microbiol. 2010; 300(6):357-62. PMC: 3666336. DOI: 10.1016/j.ijmm.2010.04.002. View

18.

Li H, Ma M, Xie H, Kong J . Biosafety evaluation of bacteriophages for treatment of diarrhea due to intestinal pathogen Escherichia coli 3-2 infection of chickens. World J Microbiol Biotechnol. 2012; 28(1):1-6. DOI: 10.1007/s11274-011-0784-5. View

19.

Liu L, Quillin M, Matthews B . Use of experimental crystallographic phases to examine the hydration of polar and nonpolar cavities in T4 lysozyme. Proc Natl Acad Sci U S A. 2008; 105(38):14406-11. PMC: 2567155. DOI: 10.1073/pnas.0806307105. View

20.

Nelson D, Schmelcher M, Rodriguez-Rubio L, Klumpp J, Pritchard D, Dong S . Endolysins as antimicrobials. Adv Virus Res. 2012; 83:299-365. DOI: 10.1016/B978-0-12-394438-2.00007-4. View