학술/공연/행사

• 일시 : 2013.12.02

Antimicrobial peptides (AMPs) are considered as promising alternatives to conventional antibiotics for future generation. Four novel hexapeptides were selected that had antimicrobial activity; KCM11:TWWRWW-NH2, KCM12: KWRWIW-NH2, KCM21: KWWWRW-NH2, and KRS22: WRWFIH-NH2 through positional scanning of synthetic peptide combinational library (PS-SCL). All four hexapeptides showed difference level of antimicrobial activities against various phytopathogenic bacteria as well as uni-cell fungal strains; KCM11 and KRS22 preferentially inhibit the normal growth of fungal strains, while KCM12 and KCM21 were more active against bacterial strains. Eventually, the symptom development in detached cabbage leave inoculated with Pectobacterium carotovorum subsp. carotovorum, could be completely abolished or delayed by pretreatment of KCM21. There was no indication of cytotocxicity of the four hexapeptides when they were tested in mouse as well as human epithelial cell lines by using MTT test. The mode of killing action was studied with KCM21 in detail. The killing process occurred rapidly (less than 10 min) and addition of divalent cations such as Ca+2 or Mg+2 drastically reduced the bactericidal activity in dose-dependent manner. The reduction of killing by adding divalent cations was more stressed in Gram-negative bacteria (Pseudomonas syringae pv. tomato DC3000 (DC3000)) comparing to Gram-negative bacteria (Clavibacter michiganensis supsp. michiganensis (CM)). When all the L-form amino acid residues of KCM21 were converted into D-form enantiomers, resulting in DKCM21, The MBC (minimal bactericidal concentration) and MIC (minimum inhibition concentration) of DKCM21 was almost same as that of KCM21 indicating that the short hexapeptide was tolerable against the major proteolytic enzymes of testing organisms. Autonomous internalization of KCM21 was confirmed by construction of 5FKCM21, which conjugated FITC at N-termianl of KCM21. Interestingly, 5FKCM21 lost its growth inhibition activity about 50% and the killing activity was almost nullified. Confocal laser scanning microscopy (CLSM) and fluorescence microscopy were employed to see if the hexapeptides were able to permeabilize the membrane resulting in internalization FITC. In fact, FITC was internalized into the bacterial cytosole only when the hexapeptides were pretreated. These results indicate that perturbation of bacterial membrane could be one of the main mechanisms of killing. However the fact that KCM21 could be internalized also indicates that metabolic alteration or degradation of macromolecules such as nucleotides inside the cell could be another target of KCM21. Electron microscopic analyses (SEM and TEM) were performed to see any alterations on cell structure. Treatment of KCM21 or KCM11 caused severe structural damages such as rough surface and collapse of cell envelops or shrunken cells. The cell surface damage was also observed even in sub-lethal concentration (6.25 µM). Similar results were observed with AFM (Atomic force microscopy) analysis. Treatment of KCM21 drastically increased the roughness of cell surface in both tested strains. In the Gram-positive strain CM, cells were swollen, while in the Gram-negative strain DC3000, various sizes of micelles were observed around the cells. The formation of micelles strongly suggests that the mode of killing action of KCM21 follows the detergent like mechanism. The different damage pattern between the two strains indicates that the mode of action of KCM21 may differ between Gram-positive and Gram-negative strains. The increase of roughness could be nullified by CaCl2 (1mM). Above results suggested that the bacterial membrane would be the main target site of the hexapeptides. In order to dissect the membrane damage, three additional experiments were performed; the outer membrane perturbation was assayed by NPN assay, and the inner membrane damage was accessed by β-galactosidase assay, and overall membrane disintegration was measured by monitoring the increase of OD at 260 nm which correlated with the release of macromolecules such as DNA or RNA. Treatment of KCM11 or KCM21 damaged outer membrane as well as cytoplasmic membrane in dose dependent manner. The bactericidal activity of KCM21 was more closely involved in the disintegration of cytoplasmic membrane and the integration on the cytoplasmic membrane was partially nullified by adding Ca+2 ions.  It is logical to expect that the negatively charged DNA would be interacted with positively charged antimicrobial peptides resulting in band shift in gel retardation assay. There are a few reports showing the gel retardation phenomenon suggesting the simple interaction between DNA and AMPs. However the selected peptides in this study showed that they not only interact with DNA but also degrade the DNA. The nucleaseactivity of KCM21 was stronger than KRS22. It is not clear yet if there is correlation between bactericidal activity and nuclease activity, however it suggests that there are certain possibility that these peptides may get inside of cytoplasm and inhibit the normal metabolism or degrade DNA/RNA or other macromolecules as an alternative killing mechanism besides of damaging membrane.     Overall this study provided a way to develop a new generation peptide antibiotics with new mode of action. The selected antimicrobial peptides here showed the actual potential to control the plant disease.