Pseudomonas Phage ZCPS1 Endolysin as a Potential Therapeutic Agent

Abstract

The challenge of antibiotic resistance has gained much attention in recent years due to the rapid emergence of resistant bacteria infecting humans and risking industries. Thus, alternatives to antibiotics are being actively searched for. In this regard, bacteriophages and their enzymes, such as endolysins, are a very attractive alternative. Endolysins are the lytic enzymes, which are produced during the late phase of the lytic bacteriophage replication cycle to target the bacterial cell walls for progeny release. Here, we cloned, expressed, and purified LysZC1 endolysin from Pseudomonas phage ZCPS1. The structural alignment, molecular dynamic simulation, and CD studies suggested LysZC1 to be majorly helical, which is highly similar to various phage-encoded lysozymes with glycoside hydrolase activity. Our endpoint turbidity reduction assay displayed the lytic activity against various Gram-positive and Gram-negative pathogens. Although in synergism with EDTA, LysZC1 demonstrated significant activity against Gram-negative pathogens, it demonstrated the highest activity against Bacillus cereus. Moreover, LysZC1 was able to reduce the numbers of logarithmic-phase B. cereus by more than 2 log₁₀ CFU/mL in 1 h and also acted on the stationary-phase culture. Remarkably, LysZC1 presented exceptional thermal stability, pH tolerance, and storage conditions, as it maintained the antibacterial activity against its host after nearly one year of storage at 4 °C and after being heated at temperatures as high as 100 °C for 10 min. Our data suggest that LysZC1 is a potential candidate as a therapeutic agent against bacterial infection and an antibacterial bio-control tool in food preservation technology.

Keywords: antibacterial tool; antibiotic resistance; bacteriophages; foodborne pathogens; phage-encoded enzymes; thermo-stable.

Figure 1

LysZC1 protein functions as a lysozyme. (A) SDS-PAGE and western blotting analysis for LysZC1. Lane 1, protein marker; Lane 2, Purified Lysin LysZC1 loaded on an SDS-PAGE gel (15%). Lane 3, Western blotting for LysZC1 protein expression; (B) Zymography of the LysZC1 protein. The left panel shows SDS-PAGE of various proteins used for zymography and right panel shows zymography of the same proteins against Micrococcus lysodeikticus with their respective zone of clearance. The zone of clearance due to the activity of the enzyme (LysZC1) is clearly visible. Lysozyme and BSA were used as positive and negative controls, respectively.

Figure 2

MD simulation of LysZC1 protein. The protein structure post MD simulation was visualized with PyMOL. Alpha helix, beta-sheet and loops are shown with cyan, magenta and salmon red colour respectively.

Figure 3

Structural analysis of LysZC1 protein. (A) Phyre2 server generated LysZC1 protein structure (grey colour). Structural alignment of predicted LysZC1 protein structure with (B) Lysozyme 056 from Deep neural language modeling (deep teal colour), (C) Crystal structure of a pesticin and T4-lysozyme chimera (olive colour), (D) Muramidase domain of SpmX from Asticaccaulis excentricus (deep blue colour), (E) Endolysin from Escherichia coli O157:H7 phage FAHEc1 (forest green colour), (F) Crystal structure of muramidase from Acinetobacter baumannii AB 5075UW prophage (deep purple colour).

Figure 4

Circular Dichroism spectroscopic analysis of LysZC1. (A) Plot shows the CD spectrum of LysZC1. (B) Thermal stability analysis of the protein at indicated temperatures. Only 222 nm data are plotted. In both panels (A,B), the data are plotted as molar ellipticity.

Figure 5

Activity of LysZC1 on Gram-positive and Gram-negative foodborne pathogens. (A) Turbidity reduction assay of LysZC1 against Bacillus cereus and Staphylococcus aureus (OD₆₀₀ = 1). Lysozyme and BSA were used as controls. (B) The plot shows the lytic activity of 100 µg/mL LysZC1 with and without 3 mM EDTA on different Gram-negative bacterial strains as indicated. Tris-buffer was used as negative control. Data are shown as Mean ± SD.

Figure 6

The lytic activity of LysZC1 on Bacillus cereus. (A) Estimation of Minimum Inhibitory Concentration of LysZC1 against logarithmic phase of B. cereus. (B) CFU reduction assay of LysZC1 against Logarithmic phase of B. cereus. Data is shown as Mean ± SD. The data is statistically significant considering p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.0001 (****); (C) SEM micrograph of untreated (left panel) and LysZC1-treated (right panel) B. cereus strains; (Assessment of the antibacterial activity of LysZC1 against B. cereus at both logarithmic and stationary phases by turbidity reduction assay. The line graphs represent the activity of LysZC1 against B. cereus with OD₆₀₀ = 1 (D), OD₆₀₀ = 2 (E), and OD₆₀₀ = 3 (F). (G) CFU reduction of logarithmic and stationary phase B. cereus. Bacteria at both phases were treated individually with 100 μg/mL of LysZC1 for 1 h at 37 °C. Data are shown as Mean ± SD.

Figure 7

LysZC1 is active at different temperatures and after prolonged storage. A-F show the bactericidal activity of LysZC1 against B. cereus after incubating the enzyme at different temperatures ((A)—10 °C, (B)—25 °C, (C)—45 °C, (D)—60 °C, (E)—80 °C, and (F)—100 °C). Lysozyme and BSA were used as positive and negative controls and the data was plotted as Mean ± SD. In panels (D–F), LysZC1 was first incubated at the indicated temperature for 10 min, followed by cooling to room temperature, before proceeding for activity assay. Panel (G) shows the same effect on a TSA plate covered with Bacillus lawn, treated with LysZC1 after heating at different temperatures as indicated. Panel (H) depicts the effect of different pH on the activity of LysZC1. Panel (I) shows the same plate assay but after storing the enzyme at 4 °C for 11 months. The zone of clearance in both the panels represents bacterial killing.