Purification And Characterization Of Antimicrobial Peptides From Skin Secretion Of Fujian Hylarana Guentheri

Hylarana guentheri is a kind of amphibian and widely distributes in south China and some Southeast Asian countries. They live in plain and hilly area below 1000 m elevation. There are large amount of antimicrobial peptides in their skin secretions in order to resist the harm of external microorganisms in their warm and wet living environment. So far, only 9 antimicrobial peptides from skin of Hylarana guentheri have been reported and little research focuse on the antimicrobial mechanism of these peptides have been published. This thesis focuses on the purification and characterization of antimicrobial peptides from the Fujian Hylarana guentheri skin secretions. The secondary structures of these peptides were studied by circular dichriosm and nuclear magnetic resonance, and the possible antimicrobial mechanism of these peptides was deduced.The skin secretions were obtained by 9V electrical stimulation of the dorsal skin glands and purified by Sephadex G-50 gel chromatography and C18 RP-HPLC. The antimicrobial activity against E. coli and S. aureus was evaluated using the agar disc diffusion method and six fractions with antimicrobial activity were obtained. Four new antimicrobial peptides were identified by the Edman degradation and designated as AMP-11(GFSSLFKAGAKYFLKQVGKAGAQQLACKAANNC), AMP-4(FLGALFKVASKLVPAAIRSISKK), AMP-5 (FLQHIIGALSKIFLVSIDK-VRCKVAGGCN) and AMP-7 (FFPLIFGALSKILPKIFL-NH2). The sequence alignment of each peptide was analyzed using protein blast item in NCBI. According to the sequence comparasion in the database, the peptides were assigned into specific AMPs families and named using traditional method. AMP-1 and AMP-5 belong to the brevinin-2 family and were named as brevinin-2GHal and brevinin-2GHd, respectively. AMP-7 with typical characteristics of temporin family was named as temporin-GHa. AMP-4 could not be assigned to any AMPs family because of the low sequence homology and was named as gylarana-GH.The minimum inhibitory concentrations (MICs) of the four peptides against E. coli, S. aureus, B. subtilis and Salmonella were determined. The results showed that brevinin-2GHal, brevinin-2GHd and gylarana-GH possessed broad-spectrum antimicrobial activity and could inhibit the growth of both Gram-positive and Gram-negative bacteria. Temporin-GHa exhibited strong activity against B. subtilis and S. aureus, but had no effect on E. coli. Hemolytic activities of the four antimicrobial peptides were also evaluated. The result indicated that both brevinin-2GHal and brevinin-2GHal had extremely low haemolytic activity, and gylarana-GH and temporin-GHa showed moderate hemolytic activity to rat blood.The secondary structures of the antimicrobial peptides in different solutions were determined by circular dichroism spectroscopy. The results showed that the antimicrobial peptides folded into a typical amphiphilic a-helix in cell membrane-mimic environment. The structure of previously reported brevinin-2GHb in 60%TFE was studied using nuclear magnetic resonance and the result indicated that I3-R20 folded into an amphiphilic a-helix. The structure plays an important role in the antimicrobial effect. Taken together, the study suggests that the antimicrobial peptides with negative charges could interact with cell membrane in terms of electrostatic interaction, and fold into helix in the hydrophobic environment of membrane. The interaction between peptide and bacterial cell membrane could interupt the stability and integrity of membrane and lead to the death of bacteria.