Effect of alanine, leucine, and arginine substitution on antimicrobial activity against candida albicans and action mechanism of a cationic octadecapeptide derived from α-amylase of rice.

AmyI-1-18, an antimicrobial peptide, is a cationic α-helical octadecapeptide derived from α-amylase of rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antifungal activity of AmyI-1-18 against Candida albicans, 11 analogs bearing substitutions with alanine, leucine, and/or arginine, which were designed on the basis of the helical wheel projection of AmyI-1-18, were synthesized, and their antifungal activity was investigated. The antifungal activities of four analogs obtained by replacing arginine or lysine with alanine were significantly reduced. The results suggested that the cationic arginine and lysine residues in AmyI-1-18 are important for its antifungal activity. The antifungal activities of two single leucine-substituted analogs were not improved, but among three single arginine-substituted analogs, AmyI-1-18(D15R) had approximately a twofold higher antifungal activity [50% growth-inhibitory concentration (IC50 ): 31 µM] than AmyI-1-18 (IC50 : 64 µM) and exhibited low hemolytic activity (4% at 100 µM). Flow cytometric analysis using propidium iodide revealed that the antifungal activity of AmyI-1-18(D15R) was dependent on its membrane-disrupting activity in a manner different from that of AmyI-1-18. Further enhancement of the cationicity and hydrophobicity of AmyI-1-18(D15R) resulted in no improvement in antifungal activity and a significant increase in hemolytic activity. In this study, the results demonstrated that the antifungal activity of AmyI-1-18 against C. albicans was enhanced through increasing its membrane-disrupting activity by replacing aspartic acid at position 15 with arginine without a significant increase in hemolytic activity. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 219-229, 2016.

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

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.

Antimicrobial activity against Porphyromonas gingivalis and mechanism of action of the cationic octadecapeptide AmyI-1-18 and its amino acid-substituted analogs.

The antimicrobial peptide AmyI-1-18 is a cationic α-helical octadecapeptide derived from α-amylase in rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antibacterial activity of AmyI-1-18 against Porphyromonas gingivalis (a bacterium associated with periodontal disease), we synthesized 12 analogs bearing substitutions with alanine, leucine, and/or arginine that were designed based on helical wheel projections and investigated their antibacterial properties. The antibacterial properties of four analogs bearing substitution of a single arginine or lysine with alanine were almost similar to those of AmyI-1-18, suggesting that the antibacterial properties depend on the presence of three cationic amino acid residues. Of three single arginine-substituted analogs, AmyI-1-18(G12R) exhibited an antibacterial activity 2.8-fold higher [50% growth-inhibitory concentration (IC50): 4.6 µM] than that of AmyI-1-18 (IC50: 13 µM). Likewise, the antibacterial properties of two single leucine-substituted analogs were significantly enhanced; in particular, AmyI-1-18(N3L) exhibited an antibacterial activity (IC50: 2.5 µM) 5.2-fold higher than that of AmyI-1-18. The hemolytic activity of AmyI-1-18(N3L) against mammalian red blood cells was low (2% at 50 µM). A membrane-depolarization assay using a membrane potential-sensitive fluorescent dye revealed that, similar to AmyI-1-18, the antibacterial activity of AmyI-1-18(N3L) was not dependent on its membrane-disrupting activity. Our results demonstrate that the antibacterial properties of AmyI-1-18 against P. gingivalis are significantly improved, without a significant increase in hemolytic activity, by replacing asparagine with leucine at position 3.

Antimicrobial activity and mechanism of action of a novel cationic α-helical octadecapeptide derived from heat shock protein 70 of rice.

Hsp70(241-258), an octadecapeptide derived from the heat shock protein 70 (Hsp70) of rice (Oryza sativa L. japonica), is a novel cationic α-helical antimicrobial peptide (AMP) that contains four lysine, two arginine, and two histidine residues. The antimicrobial activity of Hsp70(241-258) against Porphyromonas gingivalis, a periodontal pathogen, and Candida albicans, an opportunistic fungal pathogen, was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentrations of Hsp70(241-258) against P. gingivalis and C. albicans cells were 63 µM and 70 µM, respectively. Hsp70(241-258) had little or no hemolytic activity even at 1mM, and showed negligible cytotoxicity up to 300 µM. The degrees of calcein leakage from large unilamellar vesicles, which mimic the membranes of Gram-negative bacteria, and 3,3'-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of Hsp70(241-258) increased in a concentration-dependent manner. When Hsp70(241-258) was added to calcein-acetoxymethyl ester-loaded C. albicans cells, calcein release from the cells increased in a concentration-dependent manner. Flow cytometric analysis also showed that the percentages of C. albicans cells stained with propidium iodide, a DNA-intercalating dye, increased as the concentration of Hsp70(241-258) added was increased. Therefore, Hsp70(241-258) appears to exhibit antimicrobial activity against P. gingivalis and C. albicans through membrane disruption. These results suggest that Hsp70(241-258) could be useful as a safe and potent AMP against P. gingivalis and C. albicans in many fields of health care, especially in the control of oral infections.