Inhibiting Effect Of Melittin On Pathogens And Its Expression In E. Coli

The inhibiting effect and possible effective factors of melittin were investigated on several pathogens and engineered bacteria, such as Staphylococcus aureus, GS115 and Fulvia fulva, by means of cylinder- plate on series dilutions and growth curve monitoring. We explored the mechanisms by examining protein expression level, cell cycle and cell ultrastructural alterations, using SDS-PAGE, flow cytometry and electron microscopy. Promelittin geen was inserted into expression vector pET-42a(+) and transformed into E.coli BL21(DE3) after identified by PCR and sequencing.The experimental results show that the inhibitory activity of melittin was insensitive to temperature change within room range, which could be applied in each season. At 37℃, bacteriostatic activity of melittin began to decline after 5 d and dropped precipitously after 10 d. The inhibition curve suggests that pathogens went into decline phase directly instead of entering the normal logarithmic phase. From flow cytometry analysis, after several hours treatment with melittin, the percentage of cells in phase I (or in phase G1) increased, while those in phase R(or in phase S) decreased. This indicates that melittin disrupted phase I (or phase G1) rather than phase R (or phase S), causing most cells to remain in phase I (or in phase G1). Most protein expression in pathogens declined quickly with melettin treatment, some bands on SDS-PAGE gels almost vanished especially. These results suggest that melittin inhibited gene expression quickly, blocking cell division after 4 h. Electron microscopy revealed that melittin interacted with and disrupted the cytoplasmic membrane and caused the dissolution of proton motive force and leakage of essential molecules, resulting in cell death.In summary, it was confirmed that three pathways could be involved in inhibition: melittin integrated with the cell membrane causing cell bursting and cytoplasm release, inhibited the synthesis of proteins and caused the cytoplasm to condense, and delayed pathogens in phase I (or phase G1) so that they could not complete the cell cycle. These results suggest that melittin could serve as a broad-spectrum biological pesticide with fast-action and high-efficiency.In order to solve the problem of lacking natural resource, the fusion protein of promelittin and GST was expressed in E.coli under IPTG inducing.