Ultrasonic Inducement, Purification And Antibacterial Activity In Vitro And In Vivo Of An Antibacterial Peptide From The Housefly Larvae

BACKGROUNDAntibacterial peptides are the effector molecules of innate immunity and a kind of resistance-infection-pathogen polypeptides. Cecropins, the first peptide antibiotics were found in 1981 in the pupae of Cecropia(a giant silk moth)and two years later defensins were identified in rabbit phagocytes. By now,antibacterial peptide have been known to exist broadly in insects, plants, animals and human body in vivo. They were mostly cationic, small-sized peptides with a broad spectrum of activity against bacteria. And they could resist not only bacteria, fungi, and virus but also tumor cells in some way. Especially they could kill the multi-drug resistance bacterial and tumor cells without affecting human normal cells. Therefore, it is a novel kind of drug with tremendous developing potential, and a hot focus in the exploitation of antibacterial biological drugs presently.OBJECTIVESTo induce, isolate and purify antibacterial peptides from hemolymph of immunized larvae of Musca domestica and study the antibacterial characters, then to investigate the antibacterial effect of the antibacterial substances on Pseudomonas aeruginosa infection of wound in mice and on Enterobacter cloacae infection of wound in mice. To provid the experiment foundation for production and application of the antibacterial peptide.METHODS1. The stable cultivation method in house was established with repeat experiments because the cultivation of Musca domestica and larvae is the premise condition for extracting antibacterial peptides.2. Antibacterial substances of the larvae of housefly were induced by ultrasonic treatment. A kind of protein with antibacterial activity was isolated and purified by means of boiling water bath, high speed centrifugation, two steps of CM-Sepharose ion exchange chromatography and vacuum freeze-dried. The purified protein was identified by Tricine SDS PAGE, and its antibacterial activity was investigated with inhibition experiment.3. Forty ICR mice were enrolled in the study, and the Pseudomonas aeruginosa infection model was reproduced by excision of the full layer of dorsal skin with an area of 1cm×1cm. Then they were randomly divided into4 groups: C (control, n=10, after bacterial inoculation treated with wet compress of isotonic saline), M (after bacterial inoculation treated with hydropathic compress of 100 g/L mafenide), A (after bacterial inoculation treated with wet compress of 0.1 g/L antibacterial peptide), W(without bacterial inoculation) group. The changes of white blood cell in each group were determined before and day 3 after injury. Body weight and the survival of the mice were observed for 14 days post-injury.4. Thirty ICR mice were enrolled in the study, and the Enterobacter cloacae infection model was reproduced by excision of the full layer of dorsal skin with an area of 1cm×1cm. Then they were randomly divided into 3 groups: C (control, n=10, after bacterial inoculation treated with wet compress of isotonic saline), M (after bacterial inoculation treated with hydropathic compress of 100 g/L mafenide) and A (after bacterial inoculation treated with wet compress of 0.1 g/L antibacterial peptide) group. The changes of white blood cell counts in each group were determined before and day 3 after injury. Bacterial growth in muscular tissue of the wounds and the survival of the mice were observed on 3 days post-injury.RESULTS1. The optimal feeding conditions were 25℃～35℃room temperature, 60%～70% air humidity, ample nutriment and reasonable light for normal growth of Musca domestica, and the feeding conditions for larvae growth were 22℃～36℃room temperature, ample nutriment, 50%～60% humidity and a little light.2. The ultrasonic treatment with 300 W, 50 Hz for 60 s could effectively induce the production of antibacterial peptide of housefly larvae. A purified approximate 5.8 kD antibacterial peptide was obtained, which was identified by chromatography and Tricine SDS PAGE. The purified peptide is highly potent against some gram-negative bacteria such as Enterobacter cloacae and Pseudomonas aeruginosa but not against gram-positive bacteria such as Staphylococcus aureus.3. In Pseudomonas aeruginosa infection study, C group showed significant higher mortality than the other groups. The number of leucocytes in each group was decreased after operation. The survival after 14 days in C group (30%) was evidently lower than that in A (80%) and M (90%) groups (P<0.05). Body weights of animals demonstrated a difference between treated and C group on day 6 (P< 0.05), and W group demonstrated no mortality.4. In Enterobacter cloacae infection study, the number of leucocytes in each group was decreased after operation and bacterial inoculation, especially C group, was significant less than that of M and A group (P< 0.05). Bacterial growth in muscle demonstrated a difference between treated and C group on day 3 [M group: (87±31)×102 CFU/g; A group: (99±32)×102 CFU/g; C group: (504±338)×103 CFU/g](P< 0.01). The survival on day 3 in C group (60%) was evidently lower than that in A (90%) and M (90%) groups (P< 0.05).CONCLUSIONS 1. Established cultivation method and conditions could obtain normal growth of Musca domestica and larvae.2. An antibacterial peptide of housefly larvae is induced and purified, which provides an effective way to produce, isolate and purify.3. The antibacterial peptide which was induced by ultrasonic treatment possesses obvious anti-bacterial effect on the Pseudomonas Aeruginasa infected wounds of ICR mice, and it could markedly increase the survival.4. The antibacterial peptide of housefly which was induced by ultrasonic treatment possesses obvious anti-bacterial effect on the Enterobacter cloacae infected wounds of ICR mice.