Antimicrobial activity and mechanism of action of a novel cationic α‐helical octadecapeptide derived from α‐amylase of rice

Abstract

AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice (Oryza sativa L. japonica), is a novel cationic α-helical antimicrobial peptide (AMP) that contains two lysine and two arginine residues. The antimicrobial activity of AmyI-1-18 against human pathogens was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. Of the ten kinds of human pathogens, AmyI-1-18 exhibited antimicrobial activity against nine. Its 50% growth-inhibitory concentrations (ICs50 ) against Porphyromonas gingivalis, Propionibacterium acnes, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans were 13, 19, 50, 64, and 77 μM, respectively. AmyI-1-18 had little or no hemolytic activity even at 500 μM, and showed negligible cytotoxicity up to 1200 μM. The degree of 3,3'-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of AmyI-1-18 was significantly lower than that induced by the addition of melittin. Flow cytometric analysis showed that the percentages of P. aeruginosa, S. mutans, and C. albicans cells stained with propidium iodide (PI), a DNA-intercalating dye, were 89%, 43%, and 3%, respectively, when AmyI-1-18 was added at a concentration equal to its 4×IC50 . Therefore, the antimicrobial activity of AmyI-1-18 against P. aeruginosa and S. mutans appears to be mainly attributable to its membrane-disrupting activity. In contrast, its antimicrobial activity against P. gingivalis and C. albicans most likely depends upon interactions with intracellular targets other than their cell membranes. Collectively, these results indicate that AmyI-1-18 is useful as a safe and potent AMP against the pathogens described above in many fields of healthcare.

Endotoxin-neutralizing activity and mechanism of action of a cationic α-helical antimicrobial octadecapeptide derived from α-amylase of rice

“…4). The almost negligible differences in both the EC 50 and K D of AmyI-1-18 between LPS and lipid A strongly supported the assertion that AmyI-1-18 specifically binds to the lipid A moiety of LPS through electrostatic and hydrophobic interactions and thereby is capable of detoxifying these endotoxins. In our previous study [45], we reported the ability of CL (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) to inhibit NO production in RAW264 cells stimulated with LPS or lipid A from E. coli CL(14-25) significantly repressed endotoxin-induced NO production in mouse microphage cells.…”

“…In our previous studies, we reported that AmyI-1-18 is a cationic and amphipathic AMP derived from ␣-amylase (AmyI-1) of rice and that this peptide exhibited antimicrobial activity against several human pathogens [50]. In this study, as another host defense function of AmyI-1-18, we investigated its ability to inhibit NO induction in RAW264 cells stimulated with endotoxins (LPSs or lipid A from E. coli) because NO is believed to have an important role in the pathogenesis of inflammatory diseases.…”

“…The amino acid sequence of AmyI-1-18 used in this study is HLNKRVQRELIGWLDWLK as reported previously [50]. Its calculated molecular weight, isoelectric point, and net charge are 2304.72 Da, 9.99, and +2, respectively [50].…”

“…In a previous study [50], we reported that the peptide inhibited the growth of human pathogenic microorganisms, including P. gingivalis, Pseudomonas aeruginosa, Propionibacterium acnes, Streptococcus mutans, and Candida albicans. In addition, AmyI-1-18 had little or no hemolytic activity and exhibited negligible cytotoxicity in the range of concentrations at which it exhibits antimicrobial activity against human pathogens.…”

“…Its calculated molecular weight, isoelectric point, and net charge are 2304.72 Da, 9.99, and +2, respectively [50]. In addition, AmyI-1-18 conjugated with cysteine at the N-terminus (Cys-AmyI-1-18) was used for quantitatively determining its ability to bind to LPS or lipid A in SPR analysis, in which Cys-AmyI-1-18 was immobilized on a sensor chip.…”