Bacteriophage Lytic Enzyme P9ly as an Alternative Antibacterial Agent Against Antibiotic-Resistant Shigella dysenteriae and Staphylococcus aureus

Abstract

Developing new strategies to replace or supplement antibiotics to combat bacterial infection is a pressing task in the field of microbiological research. In this study, we report a lytic enzyme named P9ly deriving from the bacteriophage PSD9 that could infect multidrug-resistant Shigella. This enzyme was identified through whole-genome sequencing of PSD9. The results show that P9ly contains a conserved T4-like_lys domain and belongs to the phage lysozyme family. Recombinant P9ly obtained from protein purification presented biological activity and could digest bacterial cell walls (CW), resulting in the destruction of cell structure and leakage of intracellular components. Furthermore, P9ly exhibited bacteriolytic and bactericidal activity on different strains, especially multidrug-resistant Gram-negative Shigella dysenteriae and Gram-positive Staphylococcus aureus. Additionally, combined use of P9ly with ceftriaxone sodium (CRO) could decrease necessary dose of the antibiotic used and improve the antibacterial effect. In summary, under the current backdrop of extensive antibiotic usage and the continuous emergence of bacterial resistance, this study provides an insight into developing bacteriophage-based antibacterial agents against both Gram-negative and Gram-positive pathogens.

Keywords: antibiotic resistance; bacteriolytic activity; bacteriophage; bioengineering; endolysin.

Figure 1

Whole-genome sequencing of bacteriophage PSD9 was carried out to identify the lytic enzyme P9ly encoded by it. (A) Bacteriophage PSD9 genome map. The inner to outer circles are as: GC skew [(G − C)/(G + C)]; GC content percentage; negative strand open reading frames (ORFs; shown in green); positive strand ORFs (shown in red); and genome size (with an interval of 10 kb). (B) Conserved domain analysis of P9ly at nucleic acid and protein levels.

Figure 2

Multiple sequence alignment of lytic enzyme P9ly and other seven representative members of the lyz-like family. This analysis was performed using the EBI tool CLUSTAL Omega 1.2.4. In the figure, a star denotes conserved amino acid residues among all the sequences, while a black circle indicates critical residues that built the enzyme’s catalytic center. Bold underlined regions show the position of the T4-like_lys domain in the lytic enzyme P9ly. The accession numbers of NP_944846, BAB90980, 1K28, 2LZM, 4PK0, P62692, and P07540 represent protein sequences of the lysozyme from Salmonella phage FelixO1 (GenBank ID: 38707815), tail lysozyme from Escherichia phage RB49 (GenBank ID: 20218971), chain A, tail-associated lysozyme from Escherichia virus T4 (GenBank ID: 18655470), chain A, T4 lysozyme from Escherichia virus T4 (GenBank ID: 157835331), chain A, lysozyme from Actinoplanes teichomyceticus (GenBank ID: 683437436), endolysin from Lactococcus virus c2 (GenBank ID: 50402201), and endolysin from Bacillus phage PZA (GenBank ID: 126604), respectively.

Figure 3

Gene cloning, recombinant protein expression, and purification of the lytic enzyme P9ly. (A) The bacteriophage PSD9 genome was used as a template for PCR amplification of the P9ly gene. (B) A final concentration of 1 g/L lactose was used to induce overexpression of recombinant P9ly, and the final purified recombinant lytic enzyme P9ly was analyzed using 15% SDS-PAGE. Black arrows indicate the protein band of the recombinant P9ly.

Figure 4

Validation of the biological activity of recombinant P9ly, which manifests as bacterial cell wall (CW) digestion and the exertion of inhibitory effects toward Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. (A) P9ly can digest the CW obtained from Shigella dysenteriae KUST9, the host bacterium for phage PSD9. (B) Inhibitory zones of recombinant P9ly against Gram-negative E. coli and Gram-positive S. aureus. Phosphate-buffered saline (PBS) buffer and 100 μg/ml kanamycin were used as negative and positive controls, respectively. *** indicates p < 0.001; NS, not significant.

Figure 5

Effects of different experimental conditions on lytic enzyme P9ly activity. (A) pH; (B) NaCl concentration; and (C) Temperature. Shigella dysenteriae KUST9, the host bacterium for bacteriophage PSD9, was used as an effector substrate for lytic enzyme P9ly. In addition, the maximum activity of P9ly under various test conditions was set as 100% to facilitate comparison. Every experiment was repeated in triplicate. The error bars represent the SD.

Figure 6

Dependence of P9ly bacteriolytic activity on its concentration. Standard turbidity reduction assays were used to measure the effects of lytic enzyme P9ly under different treatment durations and different concentrations. The various panels in the figure show that P9ly acts on different strains. (A) Multidrug-resistant Shigella dysenteriae (KUST9); (B) Shigella flexneri [CMCC(B)51572]; (C) Shigella sonnei [CMCC(B)51592]; (D) Escherichia coli [CMCC(B)44102]; (E) Multidrug-resistant E. coli O157 (KUST401); and (F) Multidrug-resistant Staphylococcus aureus (1606BL1486). It should be noted that there was a blank measurement subtracted from the assayed values in the experiment. For all strains, OD600 reduction was calculated in comparison with PBS-treated control at each time point. Values are expressed as mean ± SD. The experiment was repeated in triplicate.

Figure 7

Bactericidal activity of P9ly against different bacterial strains. Among them, Staphylococcus aureus (1606BL1486) and Staphylococcus argenteus (KUST101) are Gram-positive bacteria. Approximately 10⁷ CFU/ml of different strains were treated with 75 μg/ml (4.05 μM) of P9ly at 37°C for 60 min, log kills were evaluated as the relative decrease in the viable counts of bacterial cell suspensions. All data were expressed as mean ± SD of three biological replicates.

Figure 8

Scanning electron microscopy was used to observe lysis in multidrug-resistant Gram-negative bacteria (Escherichia coli O157 and Shigella dysenteriae KUST9) and Gram-positive bacteria (Staphylococcus aureus 1606BL1486) after P9ly treatment. In all experiments, approximately 10⁵ CFU/ml of different bacterial strains were incubated with 75 μg/ml (4.05 μM) P9ly or PBS control at 37°C for 60 min. In the figure, black arrows indicate the bacteriolytic effects of P9ly on bacterial cells. The scale bar for each bacterial strain is 10 μm.