In vitro PCR verification that lysozyme inhibits nucleic acid replication and transcription

Abstract

Lysozyme can kill bacteria by its enzymatic activity or through a mechanism involving its cationic nature, which can facilitate electrostatic interactions with the viral capsid, the negatively charged parts of nucleic acids, and polymerase, so binding to nucleic acids may be another biological function of lysozyme. Here, PCR was used as a research tool to detect the effects of lysozyme on the replication and transcription of nucleic acids after treatment in different ways. We found that lysozyme and its hydrolysate can enter cells and inhibit PCR to varying degrees in vitro, and degraded lysozyme inhibited nucleic acid replication more effectively than intact lysozyme. The inhibition of lysozyme may be related to polymerase binding, and the sensitivity of different polymerases to lysozyme is inconsistent. Our findings provide a theoretical basis for further explaining the pharmacological effects of lysozyme, such as antibacterial, antiviral, anticancer, and immune regulatory activities, and directions for the development of new pharmacological effects of lysozyme and its metabolites.

Figure 1

Lysozyme blocks EmGfp, Amp and Grp78 DNA replication, transcription and reverse transcription. (A)–(C) Gel electrophoresis of PCR products. Lysozyme at the corresponding working concentration was added to each reaction system. (A) Replication, (B) transcription and (C) reverse transcription. Intensity analysis of target bands in gel electrophoresis is shown at the top of the blot, original blots/gels are presented in Supplementary data (PCR-full-length immunoblots). Data are representative of 2 independent experiments and presented as the mean ± SD; *p < 0.05, ***p < 0.001. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).

Figure 2

Cell lysate-treated lysozyme blocks EmGfp, Amp and Grp78 DNA replication, transcription and reverse transcription. (A)–(C) Gel electrophoresis of PCR products. Cell lysate-treated lysozyme at the corresponding working concentration was added to the reaction system: (A) replication, (B) transcription and (C) reverse transcription. Intensity analysis of target bands in gel electrophoresis is shown at the top of the blot. Data are representative of 2 independent experiments and presented as the mean ± SD; *p < 0.05, ***p < 0.001. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).

Figure 3

After coculture of HepG2 cells with lysozyme for 24 h, the cell lysate and supernatant of HepG2 cells showed different effects on the PCR process in vitro. (A)–(F) Gel electrophoresis of PCR products. The supernatant (A)–(C) and cell lysate (D)–(F) of HepG2 cells were added to the reaction system: (A/D) replication, (B/E) transcription and (C/F) reverse transcription. Intensity analysis of target bands in gel electrophoresis results shown to the top of the blot. Data are representative of 2 independent experiments and presented as the mean ± SD; *p < 0.05, ***p < 0.001. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).

Figure 4

Artificial gastric juice and rabbit liver tissue lysate-treated lysozyme blocks EmGfp, Amp and Grp78 DNA replication, transcription and reverse transcription in vitro. (A)–(F) Gel electrophoresis of PCR products. Artificial gastric juice-treated lysozyme (A)–(C) and rabbit liver tissue lysate-treated lysozyme (D)–(F) were added to the reaction system: (A/D) replication, (B/E) transcription and (C/F) reverse transcription. Intensity analysis of target bands in gel electrophoresis is shown at the top of the blot. Data are representative of 2 independent experiments and presented as the mean ± SD; *p < 0.05, ***p < 0.001. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).

Figure 5

Rabbit jejunum lavage fluid-treated lysozyme partially blocks EmGfp, Amp and Grp78 DNA replication, transcription and reverse transcription in vitro. (A)–(F) Gel electrophoresis of PCR products. Lysozyme treated with lavage fluid of the upper (A)–(C) and lower (D)–(F) jejunum was added to the reaction system: (A/D) replication, (B/E) transcription and (C/F) reverse transcription. Intensity analysis of target bands in gel electrophoresis is shown at the top of the blot. Data are representative of 2 independent experiments and presented as the mean ± SD; *p < 0.05, ***p < 0.001. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).

Figure 6

After lysozyme API gavage, rabbit gastric juice and jejunum fluid alter EmGfp, Amp and Grp78 DNA replication, transcription and reverse transcription in vitro. (A)–(I) Gel electrophoresis of PCR products. Lavage fluid of the gastric juices (A)–(C) and the upper (D)–(F) and lower (G)–(I) jejunum were added to the reaction system: (A/D/G) replication, (B/E/H) transcription and (C/F/I) reverse transcription. Intensity analysis of target bands in gel electrophoresis is shown at the top of the blot. Data are representative of 2 independent experiments and presented as the mean ± SD; *p < 0.05, ***p < 0.001. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).

Figure 7

After lysozyme ECT gavage, rabbit gastric juice and jejunum fluid showed different effects on EmGfp, Amp and Grp78 DNA replication, transcription and reverse transcription in vitro. (A)–(I) Gel electrophoresis of PCR products. Lavage fluid of the gastric juice (A)–(C) and the upper (D)–(F) and lower (G)–(I) jejunum were added to the reaction system: (A/D/G) replication, (B/E/H) transcription and (C/F/I) reverse transcription. Intensity analysis of target bands in gel electrophoresis is shown at the top of the blot. Data are representative of 2 independent experiments and presented as the mean ± SD; *p < 0.05, ***p < 0.001. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).

Figure 7

After lysozyme ECT gavage, rabbit gastric juice and jejunum fluid showed different effects on EmGfp, Amp and Grp78 DNA replication, transcription and reverse transcription in vitro. (A)–(I) Gel electrophoresis of PCR products. Lavage fluid of the gastric juice (A)–(C) and the upper (D)–(F) and lower (G)–(I) jejunum were added to the reaction system: (A/D/G) replication, (B/E/H) transcription and (C/F/I) reverse transcription. Intensity analysis of target bands in gel electrophoresis is shown at the top of the blot. Data are representative of 2 independent experiments and presented as the mean ± SD; *p < 0.05, ***p < 0.001. Significant differences were evaluated using one-way ANOVA with a post hoc test (Fisher’s least significant difference).