M13 phage peptide ZL4 exerts its targeted binding effect on schistosoma japonicum via alkaline phosphatase

Abstract

The present study was to determine the targeting effect of M13 phage peptide ZL4 (MppZL4) on Schistosoma japonicum (S.j). Mice infected with S.j were injected with MppZL4. Real-time PCR was used to detect the distribution and metabolism of MppZL4 in the livers and lungs of mice. In vivo refusion test was performed to detect the targeting of MppZL4. Western blotting was employed to determine the expression of MppZL4. Live imaging was used to detect the distribution of oligopeptide MppZL4. Immunohistochemistry was employed to determine MppZL4 location on adult S.j body surface. Gomori method was employed to detect the influence of oligopeptide MppZL4 on alkaline phosphatase activity. The distribution and metabolism of MppZL4 and M13KE are not significantly different from each other at each time point. The abundance of MppZL4 is changed as S.j migrates in mice. The targeted binding effect of MppZL4 varies at different stages. ZL4 oligopeptide targets S.j in mice. The specific binding sites of MppZL4 on S.j body are mainly located in syncytial cells. The binding sites of MppZL4 on S.j body surface might be ALP or ALP-related proteins. MppZL4 had targeted binding effect on S.j with its binding site being associated with proteins related to S.j alkaline phosphatase. S.j tegument had a specifically binding site with exogenous peptides, offering new means to explore the interactions between hosts and parasites. Additionally, MppZL4 can possibly be used as targeting molecules in worm-resistant drugs or as tracing molecules in imaging diagnosis technologies.

Keywords: MppZL4; Schitosoma japonicum; alkaline phosphatase.

Figure 1

Distribution and metabolism of M13 phages in the liver and lungs of normal mice. The amount of phages in the liver and the lungs at different time points after phage injection were measured and plotted.

Figure 2

The levels of M13KE and MppZL4 protein expression in the liver and lungs of mice at different time points after infection by Schistosoma japonicum. Protein (10 μg) underwent electrophoresis using 20 mA for 4 hours at 4°C. Then, the samples were electrotransferred to nitrocellulose membrane. After blocking for 1 h at room temperature, the nitrocellulose membrane was kept in horseradish-peroxidase/mouse anti-M13 antibody (1:1000) and 4°C overnight. Enhanced chemiluminescence was used to visualize the reactions. Each sample was probed with a rabbit anti-β-tubulin antibody as a loading control.

Figure 3

Fluorescence images of mice infected with Schistosoma japonicum for 3 days. In vivo fluorescence imaging of mice infected with Schistosoma japonicum 3 days after intravenous injection of 1.0 nmol Cy5.5-MppZL4. The fluorescence in the lungs can be clearly visualized as indicated from 20 min to 72 h. The fluorescence intensity was recorded as per second per centimeter squared per steradian (p/s/cm²/sr).

Figure 4

Fluorescence images of mice infected with Schistosoma japonicum for 10 days. In vivo fluorescence imaging of mice infected with Schistosoma japonicum 10 days after intravenous injection of 1.0 nmol Cy5.5-MppZL4. The fluorescence in livers can be clearly visualized as indicated from 20 min to 72 h. The fluorescence intensity was recorded as per second per centimeter squared per steradian (p/s/cm²/sr).

Figure 5

MppZL4 binding to Schistosoma japonicum adults. Mice were sacrificed to collect S.j cercaria. Afterwards, agar-paraffin double embedding and slicing was performed. Immunohistochemical staining was carried out before positively stained worms were observed and counted under a microscope (× 100). A. Strong reactivity on the tegumental ectoplasts of the worms 20 minutes after injection as indicated by red arrows. B. Positive particles observed in syncytium cells 6 h after injection as indicated by green arrows.

Figure 6

The effect of MppZL4 on alkaline phosphates activity in adult Schistosoma japonicum. A. Hematoxylin and eosin staining of Schistosoma japonicum from mice injected with MppZL4 (× 100). B. Gomori’s staining of Schistosoma japonicum from mice injected with MppZL4 (× 100). C. Hematoxylin and eosin staining of Schistosoma japonicum from mice injected with random 12 peptides (× 100). D. Gomori’s staining of Schistosoma japonicum from mice injected with random 12 peptides (× 100). Black precipitates observed in the body wall of S.j worms indicate alkaline phosphatase.