Characterization of a novel endolysin from bacteriophage infecting Vibrio parahaemolyticus, vB_VpaP_KF2

The antimicrobial resistance of food-borne pathogenic bacteria, including Vibrio parahaemolyticus, has been reported globally, warranting the need to identify promising alternative antibiotics such as endolysins that originate from bacteriophages. In our previous study, we characterized a bacteriophage infecting V. parahaemolyticus, vB_VpaP_KF2, at the molecular level. In this study, an open reading frame encoding putative endolysin was cloned from the complete genome data and expressed in the Escherichia coli expression system. The recombinant endolysin, vB_VpaP_KF2_Lys, exhibited a novel lytic property against Gram-negative bacteria regardless of pretreatment with an outer-membrane permeabilizer. It was also stable over a wide range of temperatures, pH, and NaCl concentrations, and its hydrolytic spectrum was broader than that of the parent bacteriophage. From the results, vB_VpaP_KF2_Lys could be used as a biocontrol agent against food-borne pathogens in the field of food safety.


Introduction
Antimicrobial resistance arising from antibiotic abuse has been recognized as a critical global issue that poses a threat to public health and food safety, and the incidence of multidrug-resistant food-borne bacteria has been increasing [1]. Bacteriophages have been proposed as natural antimicrobial agents not synthetic antibiotics, which have lytic properties against target bacterial hosts by invading them and disrupting the metabolism. Some phage formulations, including ListShield ™ and Sal-moFresh ™ for the control of Listeria monocytogenes and Salmonella enterica, respectively, have been given the Generally Recognized as Safe (GRAS) status for use in the food industry [2].
Endolysins, phage-encoded lytic enzymes produced during the late phase of gene expression in the lytic cycle, are responsible for the enzymatic cleavage of peptidoglycans [3]. Increasing attention is being directed to them as effective candidates for alternative antibiotics as they do not incur bacterial resistance and have a high host specificity without host natural microbial community interruption [4]. The use of external recombinant endolysins as antimicrobial materials against Gram-positive bacteria has been attempted in various applications, while its use against Gram-negative bacteria is limited, as the outer-membrane of the bacteria prevents endolysin from accessing the peptidoglycan [5].
Vibrio parahaemolyticus, a major Gram-negative foodborne pathogen that is widely distributed in marine and estuarine environments, leads to gastrointestinal infections on consumption of raw or undercooked seafood [6]. V. parahaemolyticus accounts for nearly 34,664 food poisoning incidents annually in the United State and 11 cases in South Korea in 2018 [7,8]. Continuous reports of antimicrobial resistance of V. parahaemolyticus as well as other food-borne pathogens have warranted the need to find promising antimicrobial agents and broaden the pool of compounds that can substitute antibiotics [6].

Open Access
*Correspondence: hjchang@kfri.re.kr 1 Research Group of Consumer Safety, Korea Food Research Institute, Wanju-gun 55365, Republic of Korea Full list of author information is available at the end of the article In our previous study, we isolated the bacteriophages infecting V. parahaemolyticus from the western and southern coastal areas of Korea, investigated their growth inhibitory effect against target host in manila clam, and analyzed the comparative genomic properties of six V. parahaemolyticus phages [9,10].
In this present study, we characterized a novel endolysin from V. parahaemolyticus phage, vB_VpaP_ KF2, and showed that its lytic activity against bacterial cells whose outer-membrane has not been treated with a permeabilizer, is effective. These results elucidate the fundamental properties of vB_VpaP_KF2 endolysin and its potential as an antibacterial agent that can be applied in the food chain.

Purification of recombinant vB_VpaP_KF2 endolysin
The endolysin (vB_VpaP_KF2_Lys, abbreviated as KF2_ Lys) gene was amplified using PCR and cloned into pET-28a at the NcoI and XhoI sites. This recombinant plasmid was transformed into Escherichia coli Rosetta 2 (DE3) pLysS. Expression of the endolysin was induced by adding 0.1 mM IPTG and incubating for 16 h at 18 °C. The KF2_Lys was then purified using Ni-NTA affinity chromatography under native conditions and stored at − 20 °C in storage buffer containing 50 mM Tris-HCl (pH 8.0), 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol [11,12].

Antimicrobial activity of KF2_Lys
The antimicrobial activity of KF2_Lys was determined by measuring the decrease in optical density (OD) of the bacterial cell suspension (V. parahaemolyticus isolate KF1) after the addition of endolysin [13]. The bacterial cells in the log phase were harvested and resuspended in 50 mM Tris-HCl (pH 8.0). Next, they were treated with 1, 10, and 100 mM EDTA for 5 min at 37 °C and washed four times with the buffer. Then, 100 μl of endolysin (20 μg/ml) was added to 900 μl of EDTA pre-treated cell suspension and incubated for 30 min at room temperature, following which, its optical density at 600 nm was measured. For the negative control, 100 μl of resuspension buffer was used instead of the endolysin. The results of the experiment performed in triplicate are presented as a representative or mean ± SD.
The lytic activitis of different concentrations of KF2_ Lys (1-30 μg/ml) were measured in the same way as the above experiment except for using the intact bacterial cell suspension instead of the EDTA pre-treated cell suspension as substrate.

Effects of temperature, pH, and ionic strength on the lytic activity of KF2_Lys
The lytic activities of the KF2_Lys (10 μg/ml) were compared at different temperatures (4-65 °C) and NaCl concentrations (0-200 mM) [13]. The lytic activity under different pH conditions was measured using the following pH buffers instead of the resuspension buffer: 0.1% trifluoroacetic acid (TFA) for pH 2.0; 50 mM sodium acetate for pH 4.3; 50 mM 2-(N-morpholino) ethanesulfonic acid (MES hydrate) for pH 6.0; 50 mM potassium phosphate for pH 7.0; 50 mM Tris-HCl for pH 8.0 and 8.5; 50 mM glycine for pH 9.0 and 9.5; and 50 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) for pH 10.0 and 10.5 [13]. The mean values of the experiment performed in triplicate are presented as mean ± SD of relative lytic activity (%), calculated as 100-(OD after 30 min × 100/ OD at 0 time).

Antimicrobial spectrum of KF2_Lys
One hundred microliters of endolysin (10 μg/ml) was added to 900 μl of bacterial cell suspension and incubated for 30 min at room temperature before measuring OD 600 . The bacterial strains used are shown in Table 1. The mean values of the experiment performed in triplicate are presented as mean ± SD of the relative lytic activity (%) [13].

Results and discussion
From the complete genome sequence of V. parahaemolyticus phage, vB_VpaP_KF2, it was found that the phage has two putative endolysin genes encoding a zinc peptidase and a glycosyl hydrolase, respectively, [10]. We focused on the putative peptidase (KF2_Lys) because it had a higher lytic activity than the glycosyl hydrolase (data not shown). Purified KF2_Lys revealed a band of 42.5 kDa by SDS-PAGE, which correlated with the predicted mass of the KF2_Lys protein (Fig. 1a). The yield of KF2_Lys was 10.22 mg from 1 L of culture.
The antimicrobial activity of KF2_Lys endolysin was verified via the decrease in optical density of the target bacterial suspension. The optical density of the V. parahaemolyticus suspension decreased from 1.01 to 0.313 within 30 min after adding KF2_Lys (20 μg/ml) (Fig. 1b), and the lytic activity was dependent on the concentration of KF2_Lys (1-20 μg/ml) (Fig. 1c). The activity of KF2_Lys was slightly higher than that of other reported endolysins obtained from V. parahaemolyticus bacteriophage (400 μg/ml or 1 mg/ml) [11,14]. Generally, to verify the lytic activity of endolysin against Gram-negative bacteria, outer-membrane permeabilizers such as EDTA, are often used to destabilize the outer-membrane. For example, for endolysin qdvp001 and LysVPp1 to lyse their target bacteria V. parahaemolyticus, EDTA pretreatment is essential [11,14]. Thus, the necessity of pretreatment with outer-membrane permeabilizers can act as a hurdle in the application of endolysin as a biocontrol agent against Gram-negative bacteria. Hence, studies are being carried out to overcome this limitation [15,16]. KF2_Lys showed lytic activity against V. parahaemolyticus without EDTA pretreatment, and it showed the highest activity compared to the OD change of groups treated with different concentrations of EDTA (Fig. 1b). A few studies have shown the lytic activity of endolysin against Gram-negative bacteria without an outer-membrane permeabilizer such as SPN9CC endolysin against E. coli and Bacillus amyloliquefaciens phage endolysin against P. aeruginosa [17,18]. These endolysins have hydrophobic transmembrane regions that may allow them to pass through the outer-membrane. However, unlike them, KF2_Lys does not possess a predicted transmembrane domain (data not shown). Further study is needed to analyze the lytic mechanism of KF2_Lys.
The antimicrobial activity of KF2_Lys endolysin was similar at temperatures ranging from 4 to 50 °C but decreased at temperatures above 55 °C (Fig. 2a). KF2_Lys was relatively stable under a wide pH range (2.0-10.5) and had the highest lytic activity at pH 8.5-10.5 (Fig. 2b). NaCl concentrations did not significantly affect lytic activity until 200 mM although activity slightly decreased as concentration of NaCl increased (Fig. 2c). These results show that KF2_Lys is stable at a broad range of temperatures, pH, and NaCl concentrations, which is furthers its potential as an antimicrobial agent.
The antimicrobial spectrum of KF2_Lys against several Gram-positive and Gram-negative bacteria In conclusion, the recombinant endolysin KF2_Lys was overexpressed and purified to identify its lytic characteristics. As the endolysin has a broader lytic spectrum than its original vB_VpaP_KF2 phage and independently exhibits high lytic activities without the requirement of an outer-membrane permeabilizer, it