Characterization of Endolysin LysECP26 Derived from rV5-like Phage vB_EcoM-ECP26 for Inactivation of Escherichia coli O157:H7
Department of Food Science and Biotechnology, Gachon University, Seongnam 13120, Republic of KoreaCorrespondence to:
J. Microbiol. Biotechnol. 2020; 30(10): 1552-1558
Published October 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
Bacteriophages (phages) are host specific bacteria-infecting viruses that are non-toxic in humans and effective tools for combatting antibiotic-resistant pathogens [4, 5]. Because of these advantages, the process of isolating and identifying phages to control STEC has been steadily progressing, and the application of phages to various foods has shown significant results [6-9]. In particular, the recently classified and identified “rV5-like phage,” a member of the myovirus genus that shows strong antibacterial activity against
Phage-derived peptidoglycan hydrolase “endolysin” has also been suggested as an effective antimicrobial agent . Phages are clearly powerful antimicrobial agents; however, some pathogens can resist phages through restriction enzymes or the CRISPR-Cas system . In contrast, endolysin is considered to be more efficient than phages as it exerts its bactericidal effect by simply degrading peptidoglycan linkages . Therefore, direct treatment with purified endolysin has been known to be highly effective. However, this strategy is only effective on gram-positive pathogens; its effects on gram-negative pathogens are limited because of the protective outer membrane (OM) . To overcome this problem, strategies such as combining the hydrophobic peptide with endolysin or treatment with OM-permeabilizers (OMPs) such as ethylenediaminetetraacetic acid (EDTA) and organic acid have been attempted .
There have been several annotation reports of
Materials and Methods
LysECP26 has been previously identified as a phage endolysin. BLASTP analysis was performed to determine homologous proteins within the LysECP26 sequence . A domain search was performed using the NCBI’s Conserved Domain Database (CDD) . Amino acid sequences of LysECP26 and several known endolysins were aligned using Clustal Omega . The GenBank accession numbers of phage endolysins used in this study are as follows: LysECP26 (QDB73524.1), lambda (AAA96598.1), T4 (AAD42568.1), rV5 (YP_009177530.1), FV3 (YP_007006253.1), 203 (ATW61430.1).
Bacterial Strains and Growth Conditions
The bacterial strains used in this study are described in Table S1. All strains were cultured in Luria Bertani (LB) broth (Difco, USA) at 37°C and 150 rpm. When needed, 50 μg/ml of ampicillin or 12.5 μg/ml of chloramphenicol was added to the growth medium.
Plasmid Construction and Purification of Endolysin LysECP26
Purified genomic DNA of phage vB_EcoM-ECP26 was used as a template for PCR, and the LysECP26 gene was amplified using the following primers: LysECP26_F (5'-GGAATTC
Antimicrobial Activity of LysECP26
To confirm the bactericidal activity of LysECP26, exponentially grown
Antimicrobial Spectrum of LysECP26
The antimicrobial spectrum of LysECP26 was tested against six gram-positive strains and 29 gram-negative strains. All exponentially grown bacteria were pretreated in the same manner as in the antimicrobial activity assay. LysECP26 (final conc. 1,000 ng/ml) or the reaction buffer was added to the washed bacteria culture for 30 min at 37°C. The experiments were replicated three times. Comparison of the O.D600 values for the reaction-buffer-only treatment group and the LysECP26 treatment group revealed that lysis occurred when statistically significant results appeared (
Characterization of LysECP26
To evaluate the temperature stability of LysECP26, samples of LysECP26 (final conc. 1,000 ng/ml) were incubated at different temperatures (4°C, 25°C, 37°C, 42°C, 55°C, and 72°C) for 30 min . Non-incubated endolysin served as a positive control. After each treatment, endolysin was incubated with EDTA-treated
The effects of temperature, pH, and NaCl on the antimicrobial activity of LysECP26 were assessed by a turbidity reduction assay [26, 27]. LysECP26 was inoculated into the EDTA-treated
STEC Inactivation by LysECP26 with OMPs
Evaluation of STEC inactivation was carried out using a modified method from previous reports [17, 27]. EDTA, citric acid, and lactic acid were used as OMPs. Exponentially grown
The experiments were replicated three times, and the experimental results are expressed as means ± standard deviations (SD). Data were evaluated using a one-tailed t-test. The data were analyzed using SPSS ver. 25 (SPSS Inc., USA).
Results and Discussion
Bioinformatic Analysis of Endolysin LysECP26
With a few exceptions, most phage endolysins from gram-negative bacteria are composed of a globular protein with a single domain . The endolysin of the phage vB_EcoM-ECP26, named LysECP26, is a 156-amino-acid protein with a molecular mass of 17.5 kDa and a lysozyme-like (
Bioinformatic analysis of endolysin LysECP26.( A) NCBI conserved domain database (CDD)-based annotation of the endolysin LysECP26 ( B) Sequence alignment of various E. coliphage endolysin; vB_EcoM-ECP26 endolysin, rV5 phage endolysin, phage FV3 endolysin, phage 203 endolysin, phage Lambda endolysin. Red box means common catalytic residues.
Purification and evaluation of lytic activity of endolysin LysECP26.( A) SDS-PAGE of the samples from different purification steps. M; standard molecular wight marker, P; purified remcombinant endolysin. ( B) Antimicrobial activity of purified LysECP26. Different concentration of LysECP26 (1, 10, 100, 1,000 mg/ml) were added to E. coliDH5α cell pellets. The data were presented as mean ± SD ( n= 3). The asterisk indicate significant differences; * p< 0.01.
Antimicrobial Activity and Spectrum of LysECP26
To assess the bactericidal activity of LysECP26, purified LysECP26 was serially diluted and treated with OM-removed
To identify the antimicrobial spectrum of LysECP26, a turbidity reduction assay was performed against six gram-positive strains and 29 gram-negative strains (Table 1). The gram-negative strains were all dissolved by LysECP26 with in 30 min. However, all six gram-positive strains were not lysed (Table S1). The lysis spectrum of LysECP26 resembled previous reports of
Biochemical Properties of LysECP26
The thermal stability of LysECP26 was assessed under various temperatures from 4°C to 72°C for 1 h (Fig. 3A). LysECP26 remained stable without losing activity from 25°C to 42°C and maintained 80% residual activity at 4°C. However, when heated to 55°C, LysECP26 activity dropped below 55%, and it completely disappeared when heated to 72°C. These results resemble the thermal stability tendency of T4 phage endolysin. T4 phage endolysin also maintained 100% residual activity at around 37°C, but activity decreased as the temperature increased above 50°C .
Characterization of LysECP26 at different conditions.( A) Thermal stability of LysECP26. The effects at various pH ( B), temperatures ( C), and NaCl concentrations ( D) on the lytic activity of LysECP26. The data were presented as mean ± SD ( n= 3). The asterisk indicate significant differences; * p< 0.01.
LysECP26 activity was also determined at the various temperatures (4°C–72°C), pH (2.0–9.0), and NaCl (0–1,000 mM) conditions. At 37°C and 42°C, it showed normal activity as well as temperature stability, whereas at 4°C, 25°C, and 72°C, the activity dropped sharply by 8%, 37%, and 8%, respectively (Fig. 2C). Interestingly, it showed 78% activity at 55°C, which was in contrast to the low enzyme stability at 55°C. Based on these results, the thermal stability and optimal temperature of LysECP26 are distinguished, and a temperature of 37°C–42°C is optimal for the enzyme. In pH 7.0–8.0 conditions, LysECP26 showed normal activity without any decrease, but in acidic condition below pH 5.6, activity decreased in proportion to the change in pH, and was completely lost at pH 2.0. Under basic conditions at pH 9.0, it showed 69% activity, which was higher than that under acidic conditions (Fig. 3B). The addition of NaCl did not affect the enzyme efficacy, and the addition of over 750 mM of NaCl decreased the activity (Fig. 3D). LysPA26 and SPN9CC endolysin, two previously studied endolysins, showed the highest activity at neutral pH and around 37°C, and decreased activity at low pH and high temperature [25, 27]. While it was common for NaCl supplementation to increase endolysin activity, LysECP26 experienced no effects, proving to be different from other endolysins in this regard. LysPA26, an endolysin derived from
STEC Inactivation by LysECP26 with OMPs
Gram-negative bacteria, including
Inactivation of( E. coliO157:H7 by LysECP26 with outermembrane permeabilizers (OMPs). A) Reduction of E. coliO157:H7 after treatment of LysECP26 with DW, lactate, citrate, and EDTA at 37°C for 30 min. The data were presented as mean ± SD ( n= 3). The asterisk indicate significant differences; * p< 0.01. ( B) Visualization of the combinational effect of LysECP26 and OMPs against E. coliO157:H7. After the reaction, 10 μl of sequential dilutions were spotted on the SMAC plates. The numbers under the colony represent the dilution ratio.
The activity of LysECP26 was stronger when mixed with citric acid (pKa 3.13) than that with lactic acid (pKa 3.86). These synergistic effects suggested that the combination of organic acid and LysECP26 could be an excellent
In this study, an endolysin of
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
This research was supported by the National Research Foundation Korea (Grant #2013R1A1A2062065).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
- Conway T, Krogfelt KA, Cohen PS. 2004. The life of commensal Escherichia coli in the mammalian intestine. EcoSal Plus. 1. doi:10.1128/ecosalplus.126.96.36.199.
- Hunt JM. 2010. Shiga toxin-producing Escherichia coli (STEC). Clin. Lab. Med. 30: 21-45.
- Yang SC, Lin CH, Aljuffali IA, Fang JY. 2017. Current pathogenic Escherichia coli foodborne outbreak cases and therapy development. Arch. Microbiol. 199: 811-825.
- Pirnay JP, Verbeken G, Ceyssens PJ, Huys I, De Vos D, Ameloot C, et al. 2018. The magistral phage. Viruses 10: 64.
- Lin DM, Koskella B, Lin HC. 2017. Phage therapy: an alternative to antibiotics in the age of multi-drug resistance. World J. Gastrointest. Pharmacol. Ther. 8: 162-173.
- Viazis S, Akhtar M, Feirtag J, Brabban AD, Diez-Gonzalez F. 2011. Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli. J. Appl. Microbiol. 110: 1323-1331.
- Ferguson S, Roberts C, Handy E, Sharma M. 2013. Lytic bacteriophages reduce Escherichia coli O157: H7 on fresh cut lettuce introduced through cross-contamination. Bacteriophage 3: e24323-e24323.
- McLean SK, Dunn LA, Palombo EA. 2013. Phage inhibition of Escherichia coli in ultrahigh-temperature-treated and raw milk. Foodborne Pathog. Dis. 10: 956-962.
- Magnone JP, Marek PJ, Sulakvelidze A, Senecal AG. 2013. Additive approach for inactivation of Escherichia coli O157:H7, Salmonella, and Shigella spp. on contaminated fresh fruits and vegetables using bacteriophage cocktail and produce wash. J. Food Prot. 76: 1336-1341.
- Kropinski AM, Waddell T, Meng J, Franklin K, Ackermann H-W, Ahmed R, et al. 2013. The host-range, genomics and proteomics of Escherichia coli O157:H7 bacteriophage rV5. Virol. J. 10: 76-76.
- Truncaite L, Šimoliūnas E, Zajančkauskaite A, Kaliniene L, Mankevičiūte R, Staniulis J, et al. 2012. Bacteriophage vB_EcoM_FV3: a new member of “rV5-like viruses”. Arch. Virol. 157: 2431-2435.
- Kim M, Heu S, Ryu S. 2014. Complete genome sequence of enterobacteria phage 4MG, a new member of the subgroup “PVP-SE1like phage” of the “rV5-like viruses”. Arch. Virol. 159: 3137-3140.
- Korf IHE, Meier-Kolthoff JP, Adriaenssens EM, Kropinski AM, Nimtz M, Rohde M, et al. 2019. Still something to discover: novel insights into Escherichia coli phage diversity and taxonomy. Viruses 11: 454.
- Schmelcher M, Loessner MJ. 2016. Bacteriophage endolysins: applications for food safety. Curr. Opin. Biotechnol. 37: 76-87.
- Shabbir MAB, Hao H, Shabbir MZ, Wu Q, Sattar A, Yuan Z. 2016. Bacteria vs. bacteriophages: parallel evolution of immune arsenals. Front. Microbiol. 7: 1292-1292.
- Nelson DC, Schmelcher M, Rodriguez-Rubio L, Klumpp J, Pritchard DG, Dong S, et al. 2012. Endolysins as antimicrobials. Adv. Virus Res. 83: 299-365.
- Oliveira H, Thiagarajan V, Walmagh M, Sillankorva S, Lavigne R, Neves-Petersen MT, et al. 2014. A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids. PLoS One 9: e108376.
- Park DW, Lim GY, Lee YD, Park JH. 2020. Characteristics of lytic phage vB_EcoM-ECP26 and reduction of shiga-toxin producing Escherichia coli on produce romaine. Appl. Biol. Chem. 63: 19.
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410.
- Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. 2020. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 48: D265-D268.
- Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega. Mol. Syst. Biol. 7: 539-539.
- Dumon-Seignovert L, Cariot G, Vuillard L. 2004. The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). Protein Exp. Purif. 37: 203-206.
- Wu M, Hu K, Xie Y, Liu Y, Mu D, Guo H, et al. 2018. A novel phage PD-6A3, and its endolysin Ply6A3, with extended lytic activity against Acinetobacter baumannii. Front. Microbiol. 9: 3302.
- Lim JA, Shin H, Kang DH, Ryu S. 2012. Characterization of endolysin from a Salmonella Typhimurium-infecting bacteriophage SPN1S. Res. Microbiol. 163: 233-241.
- Guo M, Feng C, Ren J, Zhuang X, Zhang Y, Zhu Y, et al. 2017. A novel antimicrobial endolysin, LysPA26, against Pseudomonas aeruginosa. Front. Microbiol. 8: 293.
- Yu JH, Lim JA, Chang HJ, Park JH. 2019. Characteristics and lytic activity of phage-derived peptidoglycan hydrolase, LysSAP8, as a potent alternative biocontrol agent for Staphylococcus aureus. J. Microbiol. Biotechnol. 29: 1916-1924.
- Lim JA, Shin H, Heu S, Ryu S. 2014. Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria. J. Microbiol. Biotechnol. 24: 803-811.
- Schmelcher M, Donovan DM, Loessner MJ. 2012. Bacteriophage endolysins as novel antimicrobials. Future Microbiol. 7: 1147-1171.
- Briers Y, Lavigne R, Volckaert G, Hertveldt K. 2007. A standardized approach for accurate quantification of murein hydrolase activity in high-throughput assays. J. Biochem. Biophys. Methods 70: 531-533.
- Tsugita A, Inouye M. 1968. Purification of bacteriophage T4 lysozyme. J. Biol. Chem. 243: 391-397.
- Briers Y, Lavigne R. 2015. Breaking barriers: expansion of the use of endolysins as novel antibacterials against Gram-negative bacteria. Future Microbiol. 10: 377-390.
- Heimbach J, Rieth S, Mohamedshah F, Slesinski R, Samuel-Fernando P, Sheehan T, et al. 2000. Safety assessment of iron EDTA [sodium iron (Fe3+) ethylenediaminetetraacetic acid]: summary of toxicological, fortification and exposure data. Food Chem. Toxicol. 38: 99-111.
- Oliveira H, Vilas Boas D, Mesnage S, Kluskens LD, Lavigne R, Sillankorva S, et al. 2016. Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-gram-negative bacterial activity. Front. Microbiol. 7: 208.
- Alakomi HL, Skyttä E, Saarela M, Mattila-Sandholm T, Latva-Kala K, Helander IM. 2000. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Appl. Environ. Microbiol. 66: 2001-2005.
- Lu HJ, Breidt F, Jr., Pérez-Díaz IM, Osborne JA. 2011. Antimicrobial effects of weak acids on the survival of Escherichia coli O157:H7 under anaerobic conditions. J. Food Prot. 74: 893-898.
- Hyldgaard M, Mygind T, Meyer RL. 2012. Essential oils in food preservation: mode of action, synergies, and interactions with food matrix components. Front. Microbiol. 3: 12.
- Díez-Martínez R, de Paz H, Bustamante N, García E, Menéndez M, García P. 2013. Improving the lethal effect of Cpl-7, a Pneumococcal phage lysozyme with broad bactericidal activity, by inverting the net charge of its cell wall-binding module. Antimicrob. Agents Chemother. 57: 5355-5365.