Abstract
The activity of neuropeptide Y (NPY) against Candida albicans, which was revealed to be fungicidal, was enhanced significantly by the truncation of amino acid residues at the N terminus. The most active peptides (MICs, approximately 1 μM) were about 10-fold more potent than the intact NPY (MIC, approximately 10 μM). The enhancement was weakened by the replacement of the N terminus by negatively charged residues and/or acylation of the α-amino group. These results suggest that only the α-helical region of NPY is necessary for the antimicrobial activity and that the net charge of the peptide is important for the activity.
Neuropeptide Y (NPY) is an amidated peptide of 36 amino acids and is known to regulate physiological functions, such as food intake, learning behavior, vasoconstriction, and neurotransmitter release (10). In addition to these functions, NPY has been recently reported to have activity against Cryptococcus neoformans, Candida albicans, and Arthroderma simii (8). NPY is suggested to belong to a group of antimicrobial peptides such as dermaseptins, cecropins, or magainins (8), which are helical and devoid of cysteine. The antimicrobial mechanism of this group is considered to involve initial electrostatic interactions between the positive charges of the peptides and the negative charges of the cell membrane, following penetration of the peptides into the membrane, and then formation of lipid bilayer-spanning pores which results in osmolysis (1). It is generally thought that a length of approximately 20 residues is necessary to provide an α-helix capable of spanning the bilayer. The three-dimensional structure of NPY is considered to consist of an amphipathic α-helix (residues 14 to 32) that corresponds to this length, a polyproline helix (residues 1 to 8), a β-turn (residues 9 to 13), and a flexible tail (residues 33 to 36) (3, 6). In this study, to determine whether the α-helical region is responsible for the antimicrobial activity of NPY, we have investigated the effects of truncation at the N terminus on the activity and found that removal of 10 to 12 amino acids from the N terminus caused a significant increase in the activity. Then, to clarify the involvement of electrostatic interactions in the antimicrobial mechanism of NPY, we have synthesized peptides in which net charges were changed by replacement of the N-terminal residues and/or acylation of the α-amino groups.
Porcine NPY (YPSKPDNPGEDAPAEDLARYYSALRHYINLITRQRY-NH2) and its analogs were synthesized on an automated solid-phase peptide synthesizer (PSSM-8; Shimadzu, Kyoto, Japan), using Tenta Gel TG-RAM resin and Fmoc (9-fluorenylmethoxycarbonyl) chemistry as previously de-scribed (5). Acetylation and succinylation of the α-amino groups of the NPY analogs were performed before cleavage from the resin as previously described (5). C. albicans ATCC 885-653, which was originally from the Institute of Virology and Microbiology of the Ukraine Academy of Sciences, was a generous gift of Elena Ivanova of the Pacific Institution of Bioorganic Chemistry, Vladivostok, Russia. Activity against C. albicans was measured in sterilized 96-well plates (ICN Biomedicals, Aurora, Ohio) according to the method of Mor et al. (4). The concentrations of the synthetic peptide solutions were determined by measuring the optical density at 275 nm due to tyrosine, because the peptides did not include any other aromatic amino acids and the acylation of the α-amino groups of the peptides did not affect the absorbance. Different concentrations of peptides were added to suspensions containing 106 spores/ml in Sabouraud dextrose broth. The inhibition of growth was determined by measuring the optical density at 492 nm after incubation for 24 h at 30°C with an MTP-22 microplate photometer (Corona, Ibaraki, Japan). The effect of the peptides on cell viability was determined according to the method of Langner et al. (2).
We have synthesized porcine NPY and its truncated analogs, in which 10 to 12 amino acids were eliminated and the N termini were replaced. The MICs of all synthesized peptides are summarized in Table 1. Porcine NPY had a MIC (10 to 12 μM) for C. albicans similar to that reported for human NPY (30 μg/ml, 7 μM) (8). This result was expected due to the fact that porcine NPY differs from human NPY only in a single amino acid residue—position 17 has a leucine in porcine NPY and a methionine in human NPY. All truncated and N-terminally substituted analogs showed enhanced antimicrobial activity. Especially the activities of [Lys13]NPY(13–36), [Asn12]NPY(12–36), and [Lys11]NPY(11–36) were about 10-fold higher than that of the intact NPY. A recent nuclear magnetic resonance study by members of our group revealed that in such truncated analogs of NPY, the α-helical region spans almost the entire length of the peptide (7). These results suggest that only the α-helical region of NPY is necessary for the antimicrobial activity.
TABLE 1.
MICs of NPY and its analogs for C. albicans
| Peptide | MIC (μM)a |
|---|---|
| NPY | 10–12 |
| NPY(13–36) | 1.5–2.0 |
| [Lys13]NPY(13–36) | 1.0–1.5 |
| [Thr13]NPY(13–36) | 1.5–2.0 |
| [Asp13]NPY(13–36) | 3.0–4.0 |
| [Asn12]NPY(12–36) | 1.0–1.5 |
| [Lys12]NPY(12–36) | 1.5 |
| [Thr12]NPY(12–36) | 1.5 |
| [Asp12]NPY(12–36) | 3.0–4.0 |
| [Glu12]NPY(12–36) | 6.0–7.0 |
| [Arg11]NPY(11–36) | 1.5–2.0 |
| [Asn11]NPY(11–36) | 1.5–2.0 |
| [Lys11]NPY(11–36) | 1.0–1.5 |
| [Glu11]NPY(11–36) | 5.0–6.0 |
The MICs were determined from at least three independent experiments.
The MICs tended to increase when acidic amino acids were located at the N termini {[Asp13]NPY(13–36), [Asp12]NPY(12–36), [Glu12]NPY(12–36), and [Glu11]NPY(11–36)}, suggesting that the negative charges at the N terminus have a repressive effect on the antimicrobial activity. This explanation is supported by the results of the acylation of the α-amino groups in the analogs except for [Lys12]NPY(12–36) (Table 2). Acetylation of [Asn12]NPY(12–36), [Thr12]NPY(12–36), and [Asp12]NPY(12–36) increased the MIC, with a change in the net charge at the N terminus from +1 to 0. Succinylation of these analogs, in which the N-terminal charge was changed to −1, led to more increased MICs.
TABLE 2.
MICs of the acylated analogs of NPY for C. albicans
| Peptide | MIC (μM)a
|
||
|---|---|---|---|
| Unmodified | Acetylatedb | Succinylatedb | |
| [Asn12]NPY(12–36) | 1.0–1.5 | 3.0–3.5 | 14–16 |
| [Thr12]NPY(12–36) | 1.5 | 7.0–8.0 | 12–14 |
| [Asp12]NPY(12–36) | 3.0–4.0 | 20–25 | 40–60 |
| [Lys12]NPY(12–36) | 1.5 | 1.5–2.0 | 1.0–1.5 |
The MICs were determined from at least three independent experiments.
Acetylation and succinylation of the α-amino groups of the NPY analogs were performed before cleavage from the resin by the addition of acetic anhydride and succinic anhydride to the suspension of the resin-bound peptides, respectively.
In order to determine whether the activity of the peptides is fungistatic or fungicidal, the viable cells were counted after incubation of the strain in the presence of NPY or [Lys13]NPY(13–36), one of the most active analogs, at concentrations which were 5- and 10-fold higher than their MICs. The results shown in Table 3 indicate that the peptides were fungicidal for C. albicans. The kinetic kill assay also indicated that the N-terminal truncation enhanced the antimicrobial activity of NPY.
TABLE 3.
Fungicidal effects of NPY and [Lys13]NPY(13–36) on C. albicansa
| Peptide | Concn of peptide (μM) | Incubation time (h) | Viable cells (%)b |
|---|---|---|---|
| NPY | 50 | 0 | 100 |
| 0.5 | 10 | ||
| 2 | 8 | ||
| 4 | 5 | ||
| 100 | 0 | 100 | |
| 0.5 | 11 | ||
| 2 | 10 | ||
| 4 | 3 | ||
| [Lys13]NPY(13–36) | 5 | 0 | 100 |
| 0.5 | 7 | ||
| 2 | 7 | ||
| 4 | 10 | ||
| 10 | 0 | 100 | |
| 0.5 | 1 | ||
| 2 | 1 | ||
| 4 | 2 |
Different concentrations of peptides were added to fungal suspensions (optical density at 600 nm = 0.5), and at designated times suitably diluted aliquots were withdrawn and plated on Sabouraud dextrose agar plates. The plates were incubated for 24 h at 30°C for colony counting.
Expressed as the ratio of viable cells in the peptide-treated culture to the number of viable cells in the control culture. The values are means of duplicate experiments.
Thus, we found that the fungicidal activity of porcine NPY for C. albicans was enhanced significantly by N-terminal truncation. The enhancement of the activity in the truncated analogs can be explained, at least partly, by the net charge. The truncated analogs have lost charged amino acids in the N-terminal sequence (Lys4, Asp6, Glu10, and Asp11), and the net charge of the analogs was more positive than that of the intact NPY. Such an increase in the net positive charge of the analogs is expected to cause stronger electrostatic interactions with the target membrane. The increase in negative charges by replacement of the N termini with acidic amino acids or acylation of the α-amino groups is considered to weaken this interaction. In the case of cecropin P1 from pig intestine, a free amino terminus was identified to be essential for its high antimicrobial activity (9). Because the truncated analog of NPY with lysine at the N terminus retained high antimicrobial activity after acylation, the ɛ-amino group of the N-terminal lysine seems to be able to substitute for the α-amino group.
Acknowledgments
We thank Kazuo Omi (Shionogi Research Laboratories, Shionogi and Co., Ltd.) for helpful comments concerning the kinetic kill assay in which dilution plating techniques were used.
Mayumi Shimizu was an Industrial Technology Researcher in the New Energy and Industrial Technology Development Organization.
REFERENCES
- 1.Blondelle S E, Houghten R A. Design of model amphipathic peptides having potent antimicrobial activities. Biochemistry. 1992;31:12688–12694. doi: 10.1021/bi00165a020. [DOI] [PubMed] [Google Scholar]
- 2.Langner C A, Lodge J K, Travis S J, Caldwell J E, Lu T, Li Q, Bryant M L, Devadas B, Gokel G W, Kobayashi G S, Gordon J I. 4-Oxatetradecanoic acid is fungicidal for Cryptococcus neoformans and inhibits replication of human immunodeficiency virus I. J Biol Chem. 1992;267:17159–17169. [PubMed] [Google Scholar]
- 3.Larhammar D, Söderberg C, Blomqvist A G. Evolution of the neuropeptide Y family of peptides. In: Colmers W F, Wahlestedt C, editors. The biology of neuropeptide Y and related peptides. Totowa, N.J: Humana Press; 1993. pp. 1–30. [Google Scholar]
- 4.Mor A, Hani K, Nicolas P. The vertebrate peptide antibiotics dermaseptins have overlapping structural features but target specific microorganisms. J Biol Chem. 1994;269:31635–31641. [PubMed] [Google Scholar]
- 5.Murase S, Yumoto N, Petukhov M G, Yoshikawa S. Acylation of the α-amino group in neuropeptide Y(12–36) increases binding affinity for the Y2 receptor. J Biochem. 1996;119:37–41. doi: 10.1093/oxfordjournals.jbchem.a021213. [DOI] [PubMed] [Google Scholar]
- 6.Schwartz T W, Fuhlendorff J, Kjems L L, Kristensen M S, Vervelde M, O’Hare M, Krstenansky J L, Bjørnholm B. Signal epitopes in the three-dimensional structure of neuropeptide Y. Ann NY Acad Sci. 1990;611:35–47. doi: 10.1111/j.1749-6632.1990.tb48920.x. [DOI] [PubMed] [Google Scholar]
- 7.Uegaki K, Murase S, Nemoto N, Kobayashi Y, Yoshikawa S, Yumoto N. Effects of covalent dimerization on the structure and function of the carboxy-terminal fragment of neuropeptide Y. Biochem Biophys Res Commun. 1997;241:737–743. doi: 10.1006/bbrc.1997.7661. [DOI] [PubMed] [Google Scholar]
- 8.Vouldoukis I, Shai Y, Nicolas P, Mor A. Broad spectrum antibiotic activity of skin-PYY. FEBS Lett. 1996;380:237–240. doi: 10.1016/0014-5793(96)00050-6. [DOI] [PubMed] [Google Scholar]
- 9.Vunnam S, Juvvadi P, Merrifield R B. Synthesis and antibacterial action of cecropin and proline-arginine-rich peptides from pig intestine. J Pept Res. 1997;49:59–66. doi: 10.1111/j.1399-3011.1997.tb01121.x. [DOI] [PubMed] [Google Scholar]
- 10.Wahlestedt C, Reis D J. Neuropeptide Y-related peptides and their receptors—are the receptors potential therapeutic drug targets? Annu Rev Pharmacol Toxicol. 1993;32:309–352. doi: 10.1146/annurev.pa.33.040193.001521. [DOI] [PubMed] [Google Scholar]