The Compact Engineered Peptide Targeting Staphylococcus Aureus

Colicin Ia belongs to the family of bactericidal proteins, the channel-forming domain of which can form lethal ion channel in the membrane of Escherichia coli and related strains such as Shigella sonnei. However, the receptor and translocation domains of the Colicin Ia can only recognize the inner and outer membrane receptors of the Escherichia coli, therefore the wild type Colicin Ia can not be developed into antibiotics against other types of bacteria. In the 1990s, the pheromones of gram-positive bacteria, which regulates the growth and toxin secretion of the same type bacteria, were identified they were peptides consisted by dozens of amino acids. The pheromones can auto-recognize membrane receptor of the identical types of bacteria. We had constructed a fusion protein named Pheromonicin by introducing a staphylococcal pheromone AgrD at the C-terminal of the Colicin Ia. The fusion protein was bactericidal active against Staphylococcus aureus. In present study, we will truncate the none channel-forming domain, then attach the AgrD to the pore-forming region(K544-I626) to construct a new engineered multidqmain protein machine-compact engineered peptide targeting Staphylococcus aureus.Such engineered peptide was constructed by linking the gene ofStaphylococcal AgrD pheromone with the gene of C-terminal (1626) of Colicin la pore-forming region(K544-I626) with site-directed mutation. Mutated plasmid was transformed into E.coli TG1 cells to produce engineered peptide, then the peptide was purified by CM sepharose ion-exchange column. In vitro bactericidal assay and drug withdrawal were used to identify the bioactivity of the engineered peptide. The planar lipid bilayer membrane was used to assay the electrophysiology of the engineered peptide .Toxicity studies on mammalian cells were used to assay the toxicity of the engineered peptide.The results obtained were as follows: the MIC of engineered peptide against Staphylococcus aureus ATCC 25923 (penicillin sensitive strain) , ATCC 29213 ( penicillinase-producing strain ) and ATCC BAA-42 (methicillin resistant strain) were 1g/ml, 2g/ml, 4g/nil respectively. PI staining showed the Staphylococcus aureus treated with the engineered peptide were dead. And the engineered peptide can form a brand new voltage-dependent ion channel on the planar lipid bilayer membrane which were different from wild type Colicin Ia and Pheromonicin. Toxicity studies on mammalian cells showed the engineered peptide had insignificant toxicity to the mammalian cells.In conclusion, the compact engineered peptide can form lethal ion channel in the membrane of Staphylococcus aureus guided by Staphylococcal AgrD pheromone, therefore the engineered peptide had bactericidal activity against Staphylococcus aureus, especially against the drug-resistant strains. Because of the properties mentioned above, the engineered peptide showed great potential in the future. The engineered peptide was an example ofbactericidal protein machine combining two minidomains with different bioactivities and different protein origins.