Studies On The Antimicrobial Peptides With Anti-Toxoplasma Gondii Activity Purified From The Housefly (Musca Domestica) Larvae

Objectives:1.To isolate and purified the antimicrobial peptide with anti-Toxoplasma gondii activity form haemolymph of housefly (Musca domestica) larvae using grinding, high speed centrifugation and ATKA purifier chromatographic system..2.To observe the anti-Toxoplasma activity of purified antimcirobial peptide with MTT chromatometry and Giemsa -Wright's staining.3.and to study the toxicity and stability with MTT chromatometry.4.To investigate the mechanism of action initially with transmission electron microscope (TEM) and flow cytometry (FCM).Methods:1.The antimicrobial peptides were induced largely from the third instar housefly by puncture and infection. The haemolymph was obtained and purified by using Resource S cation column chromatography and Superdex G75 gel column chromatography sequencely. The anti-T. gondii activity was detected by MTT chromatometry. The purified antimcirobial peptide was identified with Tricine-SDS-PAGE.2. AMP-1 was adjusted to 12.5μg/ml, 25μg/ml and 50μg/ml with PBS, and added to free and intracellular T.gondii tachyzoites. The inhibition of T.gondii was detected at 24h, 48h and 72h repectively by MTT chromatometry and Giemsa-Wright's staining. And the toxocity of AMP-1was detected by MTT chromatometry.3. AMP-1 of 50μg/ml was treated by different pH(2, 4, 6, 8,10)and different temperature (20℃, 40℃, 60℃, 80℃, 100℃) for 60min, and heated at 100℃for 1min, 5min, 15min, 30min and 60min. The inhibition ratio of T.gondii tachyzoite was observed by MTT chromatometry.4.The free T.gondii tachyzoites were treated by AMP-1 of 50μg/ml, which ultrastructural change and DNA injury were observed by transmission electron microscope (TEM) and flow cytometry (FCM) respectively after 24h.Results:1.It was found that there was 2 ultraviolet absorption peaks at 280nm when retention volume was 14ml and 16ml through Resource S cationic exchange column chromatography which the later had anti-Toxoplasma activity. This protein was collected and purified again through Superdex G75 gel column chromatography. And there were six ultraviolet absorption peaks at 280nm through Superdex G75 gel column chromatography, which the peaks collected at 19ml and 25ml had anti-toxoplasma activity, and the later had a better activity. It's molecular weight was 5.0kD, which verificated by Tricine-SDS-PAGE. It was temporarily named as AMP-1.2. It was shown that anit-free Toxoplasma activity of AMP-1 increased along with the increase of concentration of AMP-1(P<0.05), and increased along with time. From Giemsa-Wright's staining, it was shown that the injury of Vero cells incubated with T.gondii tachyzoites treated with AMP-1 was more slight than that treated with PBS at each time. Because Vero cells were destroyed seriously in the control group of 72h and the AMP-1 group of 12.5μg/ml, we only analyzed the invasion of T.gondii in Vero cells at 24h and 48h. The invasion rate of T.gondii decreased with the increase of the concentration of AMP-1 (P<0.05). The survival rate of Vero cells were 94.04%, 90.70% and 81.94% respectively when treated with AMP-1 from low concentration to high concentraion. The differernce of A value between the AMP-1 group of 50μg/ml and the control group had statistical significance (P<0.05).3. The anti-Toxoplasma activity of AMP-1 decreased with the increase of temperature when treated with different temprture for 60 min, which the difference had statistical significance (P<0.05). When treated with 100℃, AMP-1 was inactive on T.gondii. Through further study, it was found that the activity of AMP-1 was decreased rapidly when heated with 100℃over 5 min, and no activity when heated for 15 min.The anti-Toxoplasma activity of AMP-1 first increased and then decreased with the increase of temperature when treated with different temprture for 60 min, which the difference had statistical significance (P<0.05).When treated with pH 4, 6 and 8, the activity of AMP-1 was good. When treated with pH 2 and 10, there was no activity. of AMP-1 (P<0.05).4. It was observed that the longitudinal section of T.gondii tachyzoites in the control group were crescent-shaped. The pellice consisted two membranes, which were smooth and continuous. The nucelus was spherical or elliptic, which karyolemma was clear, and the chromatin was spread in the karyoplasm. The organella were around nucelus, including mitochondria, endoplasmic reticulum, Golgi complex, etc. The mitochondria had many cristae of tubiform and scrotiform. In the AMP-1 group, some tachyzoites were destroyed. The morphous was round or elliptic; the plasmalemma was protrudent and ruptured; the cytoplasmic structure was no clear and homogenized. The mitochondria was swelling, which cristae were chaotic and decreased partly. The dense granule was decreased, the vacuolus was increased in the cytoplasm. The nucleus was swelling, the perinuclear cisterna was augmented, the chromatic was agglutinated.From the bar chart of DNA contents, it was shown the difference between the control group and AMP-1 group. The tachyzoites of the M1 phase were more than that of the M2 phase in the control group, but the condition was oppsite in the AMP-1 group. Furthermore, M1 peak had a obvious antedisplacement (P<0.05). Conclusions:1.There is an antimicrobial peptide with anti-Toxoplasma in the haemolymph of housefly larvae, which is about 5ku.2. AMP-1 can damage T.gondii tachyzoites of free and incubated with Vero cells effctively, and no harm to Vero cells.3.AMP-1 have good stability when heated from 20℃to 80℃and treated from pH2-4.4.AMP-1 can destroy the membrane, cytoplasmic, nucleolus and nucleinic acid of T.gondii tachyzoites.