Antibacterial and Antifungal Activities of Vasostatin-1, the N-terminal Fragment of Chromogranin A*

Vasostatin-1, the natural N-terminal 1–76 chromogranin A (CGA)-derived fragment in bovine sequence, has been purified from chromaffin secretory granules and identified by sequencing and matrix-assisted laser desorption time-of-flight mass spectrometry. This peptide, which displays antibacterial activity against Gram-pos-itive bacteria at micromolar concentrations, is also able to kill a large variety of filamentous fungi and yeast cells in the 1–10 m M range. We have found that the C-terminal moiety of vasostatin-1 is essential for the antifungal activity, and shorter active peptides have been synthesized. In addition, from the comparison with the activity displayed by related peptides (human recombinant and rat synthetic fragments), we could determine that antibacterial and antifungal activities have different structural requirements. To assess for such activities in vivo , CGA and CGA-derived fragments were identified in secretory material released from human polymorphonuclear (39), when completed, PMNs were centrifuged (800 3 g ) for 10 min. The superna- tant was recovered for further analysis. Western Blot Analysis— Samples from PMNs secretions were sepa- rated by SDS-PAGE gels containing 15% acrylamide (40). In order to detect immunologically reactive fragments, proteins were electrotrans- ferred to nitrocellulose sheets (41). They were first soaked in 3% bovine serum albumin in 25 m M sodium phosphate containing 0.9% NaCl at pH 7.5 (NaCl/P i ). Nitrocellulose sheets were quickly washed with NaCl/P i and incubated 2 h at room temperature with monoclonal anti-CGA5A8 corresponding to anti-CGA 47–68 antiserum (28) diluted in NaCl/P i (2 m g/ml). The second antibody was an anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad). The nitrocellulose sheets were stained for enzyme activity in 100 m M NaCl, 50 m M MgCl 2 , 100 m M Tris-HCl, pH 8.5, containing 0.4 m M nitro blue tetrazolium and 0.38 m M 5-bromo- 4-chloro-3-indolyl phosphate.

A rapid and effective response to challenge pathogens is essential for the survival of all living organisms. The need for efficient agents increases with the expanding number of immunodeficient patients (chemotherapy, grafts, human immunodeficiency virus type-1 syndrome, prolonged antibiotherapy, etc.) and with the emergence of bacterial and fungal pathogens resistant to current therapies. Among the several different mechanisms that have evolved to act efficiently, the production of a large variety of natural antimicrobial peptides from both animals and plants is attracting increasing attention. The importance of these molecules is clearly established in the immune defense of invertebrates (1,2), whereas in vertebrates they act as a first line of innate defense against pathogens and in the control of the natural flora (1,3). These antimicrobial peptides are located at sites exposed to microbial invasion such as the epithelia of amphibian (3), mammals (4 -6), and insects (2,7). They are present in the hemolymph of insects and stored in the secretory granules of immune cells of mammals and birds (8 -11). In addition, ␣-defensins are expressed by human intestinal Paneth cells that are secretory epithelial cells located at the bottom of crypts in the small intestine (12). Furthermore, ␤-defensins that are produced in various epithelia such as psoriatic skin (6) and trachea (13,14) are not stored in cytoplasmic granules, and their local concentration is directly related to their synthesis and secretion rates. Surveys of antimicrobial peptides have revealed links between the antimicrobial peptides found in vertebrates and those found in invertebrates (15)(16)(17)(18). Recently, new data have highlighted similarities between pathogen recognition, signaling pathways and effector mechanisms of innate immunity in Drosophila and mammals (19). Thus, it became apparent that innate immunity is an evolutionary ancient defense mechanism.
We have recently discovered new natural antimicrobial peptides derived from the processing of chromogranins (CGs) 1 and proenkephalin-A, which are secreted with catecholamines upon stimulation of chromaffin cells from adrenal gland (20 -24). These peptides may play a role in stress situations and act as one immediate protective barrier against infection.
In the present paper we report that bovine vasostatin-1, an abundant natural secreted peptide (24 -25), well conserved along evolution and corresponding to the N-terminal domain of bovine chromogranin A (CGA 1-76 ), displays both antibacterial and antifungal activities. By using recombinant and synthetic peptides corresponding to vasostatin-related fragments from various species, structural features necessary for the expression of antibacterial and antifungal activities have been pointed out. We have shown that the C-terminal moiety of vasostatin-1 displays a potent antifungal activity, and we have prepared several synthetic antifungal vasostatin-1-derived fragments. Furthermore, in order to study the biological function of CGA-derived peptides in innate immunity, we have investigated their presence in material secreted from human polymorphonuclear neutrophils.

MATERIALS AND METHODS
Purification of Natural Bovine Vasostatin-1 (CGA  ), Production of Human Recombinant VS-1 (Ser-Thr-Ala CGA , and Rat CGA  Synthetic Peptides-Secretory granules were isolated from bovine ad-renal medulla (26), and soluble proteins were separated from membranes after lysis and centrifugation (27). CGA 1-76 was purified by HPLC on a Macherey Nagel Nucleosil 300 -5C18 column (4 ϫ 250 mm; particle size 5 m and pore size 100 nm) with the Applied Biosystems HPLC system 140 B. Absorbance was monitored at 214 nm, and the solvent system consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B). Material was eluted at a flow rate of 0.7 ml/min using successively a gradient of 0 -30% B in A over 15 min followed by a gradient of 30 -50% over 40 min and achieved by a gradient of 50 -100% over 10 min. Each fraction was manually collected and concentrated by evaporation. Human recombinant Ser-Thr-Ala-CGA 1-78 peptide (VS-1) was cloned, purified, and characterized as described previously (28). Rat CGA 7-57 peptide was synthesized on an ABI 431 A (Applied Biosystems) peptide synthesizer using the standard procedures of Fmoc (9-fluorenylmethoxycarbonyl) chemistry (29). Formation of the disulfide bridge between Cys 17 and Cys 38 was obtained in the presence of N-ethyldiisopropylamine at pH 8.5, leaving the mixture to stand under open atmosphere for 24 h until complete reaction, according to Ellman test (30). After reduction of the disulfide bridge in the presence of 8 M guanidine HCl in 1 M Tris-HCl, pH 8.5, containing 4 mM EDTA, S-pyridylation of cysteine residues was obtained by treatment with 4-vinylpyridine (31). The reduced and alkylated peptide was then isolated by HPLC on a Macherey Nagel Nucleosil 300 -5C18 column. In order to oxidize natural bovine vasostatin-1 and recombinant human VS-1, the oxidizing agent was prepared by addition of formic acid to 30% hydrogen peroxide (v/v, 9:1) and stirring at 20°C for 45 min. This mixture was then chilled at 0°C, and the oxidative agent (100 l) was added to the protein. The reaction was performed at 0°C for 30 min, stopped by dilution with water (500 l). Samples were then concentrated by evaporation but not to dryness. This washing step was repeated three times to eliminate completely performic acid.
Purification of VS-1-derived Peptides-Recombinant VS-1 was digested for 18 h at 30°C by endoproteinase Glu-C at a protein to enzyme weight ratio of 50:1 in 50 mM Tris-HCl, pH 8.3. Released fragments were separated by HPLC using a Macherey Nagel Lichrospher 100 -5-RP-18 column (3 ϫ 250 mm; particle size 5 m and pore size 100 nm). Absorbance was monitored at 214 nm, and the solvent system consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B). Material was eluted at a flow rate of 0.7 ml/min using successively a gradient 0 -10% solvent B in 5 min, followed by a gradient 10 -40% over 55 min.
Sequence Analysis-The sequence of purified peptides was determined in our laboratory by automatic Edman degradation onto an Applied Biosystems 473A microsequencer. Samples purified by HPLC were loaded on Polybrene-treated and precycled glass fiber filters (25). Phenylthiohydantoin (PTH)-derivatives were identified by chromatography on a PTH C18 column (2.1 ϫ 200 mm).
Mass Spectra Analysis-Determination of molecular mass was carried out on a Brucker BIFLEX TM matrix-assisted laser desorption timeof-flight mass spectrometer (MALDI-TOF) according to the procedure previously reported (23). A micromolar analyte solution (0.5 l) was applied to the matrix and dried under moderate vacuum. After fast spreading and evaporation of the solvent, we obtained a thin layer of matrix crystals (32,33). This preparation was washed by applying 1 l of a 0.5% trifluoroacetic acid in water and then flushed after a few seconds.
Antibacterial Activity-Bacteria were precultured aerobically at 37°C in a Mueller-Hinton Broth medium, pH 7.3. The following bacteria strains were tested: Micrococcus luteus (A270), Bacillus megaterium (MA), Bacillus cereus (ATCC11778), Bacillus subtilis (QB935), Streptococcus pyogenes, Mycobacterium fortuitum (K23430), Staphylococcus aureus, Listeria monocytogenes (S47014), Escherichia coli (D22 and D31), Enterobacter cloacae, Salmonella typhimurium, Klebsiella pneumonia, and Pseudomonas aeruginosa (ATCC82118). Bacteria were suspended in the Mueller-Hinton Broth medium, and antibacterial activity was tested by measuring the inhibition of bacterial growth (34). Aqueous peptidic solutions (10 l) were incubated in microtiter plates with 100 l of a midlogarithmic phase culture of bacteria with a starting absorbance of 0.001 at 620 nm (35). Microbial growth was assessed by the increase of absorbance after 16 h incubation at 30°C. The A 620 nm value of control cultures growing in the absence of peptide was taken as 100%.
Antifungal Activity-Filamentous fungi were grown on medium including a five-cereal medium, and the spores were harvested as described previously (36). The following filamentous fungi strains were used: Alternaria brassicola (MUCL 20297), Trichophyton mentagro-phytes, Nectria hematococca (160.2.2), Fusarium culmorum (MUCL 30162), Fusarium oxyporum (MUCL 909), Neurospora crassa (CBS 327-54), and Aspergillus fumigatus. Yeast cells were precultured on a Sabouraud medium, and two yeast strains were tested, Saccharomyces cerevisiae (TGY481 pJM600) and Candida albicans. In order to test the antifungal activity of CGA-derived peptides, spores (final concentration, 10 4 spores/ml) were suspended in a growth medium containing Potato Dextrose Broth (Difco, in half-strength) and yeast cells in Sabouraud medium with starting absorbance at 620 nm of 0.001. These media were supplemented with tetracycline (10 g/ml) and cefotaxime (0.1 g/ml). Fungal growth was assessed after appropriate incubation period, specific to each fungi (24 or 48 h at 30°C). Aliquots of peptide extract (20 l) were incubated in microtiter plates with 80 l of fungal spores or yeast cultures. Growth of fungi was monitored microscopically and also evaluated by measuring the culture absorbance at 595 nm using a microplate reader. Yeast cells growth was assessed by the increase of absorbance at 620 nm.
Isolation of Peptides and Proteins Released from Polymorphonuclear Neutrophils (PMNs)-Human PMNs were prepared to 98% homogeneity, as described previously (37), from buffy coat of healthy donors of either sex, kindly provided by the Center de Transfusion Sanguine de Strasbourg (France). PMNs were suspended in a buffer solution containing 140 mM NaCl, 5 mM KCl, 1.1 mM CaCl 2 , 0.1 mM EGTA, and 10 mM Hepes, pH 7.3, at 5 ϫ 10 6 cells per ml. Exocytosis of the content of the specific and primary granules of PMNs was initiated at room temperature by application of 2.3 nM LukS-PV and 0.6 nM LukF-PV, the two components of leukocidin from S. aureus (38). The secretion was monitored by flow cytometry as described previously (39), and when completed, PMNs were centrifuged (800 ϫ g) for 10 min. The supernatant was recovered for further analysis.
Western Blot Analysis-Samples from PMNs secretions were separated by SDS-PAGE gels containing 15% acrylamide (40). In order to detect immunologically reactive fragments, proteins were electrotransferred to nitrocellulose sheets (41). They were first soaked in 3% bovine serum albumin in 25 mM sodium phosphate containing 0.9% NaCl at pH 7.5 (NaCl/P i ). Nitrocellulose sheets were quickly washed with NaCl/P i and incubated 2 h at room temperature with monoclonal anti-CGA5A8 corresponding to anti-CGA 47-68 antiserum (28) diluted in NaCl/P i (2 g/ml). The second antibody was an anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad). The nitrocellulose sheets were stained for enzyme activity in 100 mM NaCl, 50 mM MgCl 2 , 100 mM Tris-HCl, pH 8.5, containing 0.4 mM nitro blue tetrazolium and 0.38 mM 5-bromo-4-chloro-3-indolyl phosphate.

RESULTS
In addition to catecholamines, material present in bovine chromaffin intragranular matrix contains a large number of peptides derived from chromogranins and proenkephalin-A. After separation by HPLC, antibacterial activity against M. luteus (Gram-positive bacteria) was detected in several fractions, and we focused on a major active fraction eluted at 47% B (Fig. 1A). Automatic Edman degradation revealed a unique sequence (LPVNSPMNKGDT) corresponding to the N-terminal end of bovine CGA (42). To determine further the full primary structure of this N-terminal CGA-derived fragment, mass spectrometry analysis was obtained using the MALDI-TOF technique (Fig. 1B). The experimental molecular mass values of 8583.9, 4292.9, and 2863.6 correspond to the ratio m/z (z ϭ 1, 2 and 3) and are in agreement with the theoretical molecular mass of the peptide CGA 1-76 (8584.9 Da), named vasostatin-1, including the disulfide bridge Cys 17 -Cys 38 (43). The alignment of the primary structure of this natural fragment with the corresponding fragments of several species (43)(44)(45)(46)(47)(48) shows the high conservation of this sequence along the evolution (Fig. 2).

Structural and Biological Characterization of the Antimicrobial Activity of Vasostatin-1-derived Fragments
Natural Bovine Vasostatin-1 (CGA  )-When tested against several Gram-positive and -negative bacteria, natural bovine vasostatin-1 displays antibacterial activity against M. luteus and B. megaterium after 16 h incubation at 30°C, inhibiting completely bacterial growth at 2 and 0.2 M, respectively (Table I). In contrast, no activity is detectable at the concentration of 10 M against other Gram-positive bacteria such as B. cereus, B. subtilis, S. pyogenes, M. fortuitum, S. aureus, L. monocytogenes, and several Gram-negative bacteria such as E. coli D31 and D22, E. cloacae, S. typhimurium, K. pneumoniae, and P. aeruginosa (data not shown).
To complete the spectrum of activity of vasostatin-1, we also tested its ability to affect the growth of filamentous fungi and yeast cells (Table I). Interestingly, this peptide in the concentration range of 1-10 M is highly active against a variety of filamentous fungi including N. crassa, A. fumigatus, A. brassicola, N. hematococca, F. culmorum, and F. oxyporum, but it is inactive against T. mentagrophytes. In addition, it is active against the yeast forms of S. cerevisiae and C. albicans at the concentration of 10 M (Table I). Furthermore, after removal of medium containing vasostatin-1 and substitution with fresh vasostatin-1-free growth medium, fungi are unable to restart its growth, strongly suggesting that vasostatin-1 possesses fungicidal activity. In parallel experiments, we have found that vasostatin-1 is not able to induce the lysis of bovine erythrocytes (data not shown). In addition, the antimicrobial properties of vasostatin-1 against M. luteus, B. megaterium, and N. crassa disappear after treatment with a mixture of proteolytic enzymes such as trypsin, chymotrypsin, and the protease V8 of S. aureus, indicating that the activity is due to peptidic material.
The importance of the disulfide bridge for the antibacterial and antifungal activities of natural vasostatin-1 was then examined. After reduction, alkylation using 4-vinylpyridine and desalting by HPLC, the peptide was modified on the two cysteine residues (Cys 17 and Cys 38 ), which were S-pyridylethylated as verified by MALDI-TOF analysis (data not shown). The modified fragment (pS-vasostatin-1) is still active against the growth of M. luteus and B. megaterium but at the concentration of 10 and 5 M, respectively (Table II). In addition, modification of disulfide bridge does not alter antifungal activity since the pS-vasostatin-1 inhibits the growth of N. crassa at a concentration of 3 M similarly to the natural form (Table II). These data indicate that the intactness of the disulfide bridge is not crucial for antifungal property but seems to be important for full antibacterial activity.
After performic oxidation of vasostatin-1, all peptide molecules were modified, as shown from MALDI-TOF analysis that revealed the presence of five oxidations corresponding to the modifications of cystine (cysteic acids Cys 17 (Table II). These data indicate that performic oxidation induces the complete loss of the antibacterial activity and strongly decreases the antifungal activity. The conformational changes induced by the oxidative treatment inhibit the antimicrobial activity of vasostatin-1.
Human Recombinant Ser-Thr-Ala-CGA 1-78 (VS-1)-Recombinant VS-1 corresponds to the sequence of human CGA 1-78 bearing the tripeptide STA at the N-terminal end. This peptide was expressed in E. coli, purified (28), and tested after sequencing and mass spectrometry analysis. The experimental mass values of 9069.7, 4536.1, and 3024.1 correspond to the ratio m/z (z ϭ 1, 2 and 3) and fully agree with the theoretical mass of 9069.5 Da corresponding to the recombinant fragment including the disulfide bridge (data not shown). This fragment is active against the growth of M. luteus and B. megaterium at a concentration of 30 M (Table I). In addition, it inhibits the growth of several fungi such as N. crassa, A. brassicola, N. hematococca, and F. culmorum at concentration range of 1-10 M (Table I). However, VS-1 is inactive against the growth of yeast cells at a concentration up to 50 M (Table I).
After reduction and alkylation, VS-1 was modified by Spyridylethylation of the two cysteine residues (Cys 20 and Cys 41 ) as indicated by MALDI-TOF analysis (data not shown). This modified fragment (pS-VS-1) is inactive against the growth of M. luteus at a concentration up to 30 M, but it is active against B. megaterium at a concentration of 20 M (Table  II). pS-VS-1 inhibits the growth of N. crassa at a concentration of 5 M, similarly to unmodified VS-1 (Table II). These data indicate that the alkylation of VS-1 leads to the specific loss of its antibacterial activity against M. luteus but does not alter the antibacterial activity against B. megaterium nor its antifungal activity.
The oxidized form of recombinant VS-1 is inactive against the growth of M. luteus and B. megaterium at a concentration up to 30 M, but it kills N. crassa at a concentration of 20 M (Table II).
Synthetic Rat CGA 7-57 -The synthetic rat CGA 7-57 peptide was purified by HPLC and analyzed by sequencing and MALDI-TOF mass spectrometry. The experimental molecular mass values of 5654.4 and 2827.1 correspond to the ratio m/z (z ϭ 1 and 2) and well agree with the expected mass of 5655.7 Da for the rat CGA 7-57 fragment with two cysteine residues  (Table I); it is inactive against the growth of yeast cells at a concentration of 50 M.
The importance of the disulfide bridge was examined after preparation of the synthetic peptide including the disulfide loop Cys 17 -Cys 38 . The formation of the disulfide bridge was controlled by the reaction with Ellman's reagent and MALDI-TOF mass spectrometry (data not shown). After chemical modification, the synthetic peptide was purified by HPLC. The antibacterial test revealed that the synthetic rat CGA 7-57 with disulfide bridge is inactive against M. luteus and B. megaterium at a concentration of 30 M (Table I). In contrast, this peptide kills N. crassa, A. brassicola, N. hematococca, and F. culmorum at a concentration of 10 -20 M (Table I). These data indicate that synthetic peptides corresponding to rat CGA 7-57 with or without the disulfide bridge possess similar antifungal activity.
In conclusion, natural vasostatin-1 is the most active peptide, since the fragments rat CGA 7-57 seem too short to display the antibacterial activity. Furthermore, in natural vasostatin-1 the presence of the disulfide bridge is crucial for this antibacterial activity, but in contrast it seems not to be important for the antifungal activity. We then attempted to characterize the shorter antifungal peptide after proteolytic digestion of rhVS-1 and purification of the generated fragments.

Identification of Antifungal rhVS-1-derived Peptides
After digestion of recombinant VS-1 by endoproteinase Glu-C, short fragments were separated by HPLC (Fig. 3A). The analysis by sequencing and MALDI-TOF mass spectrometry of the different peaks indicate the presence of 5 major cleavage sites located, in the vasostatin-1 sequence at positions Glu 13 -Val 14 , Glu 40 -Thr 41 , Glu 46 -Arg 47 , Glu 60 -Leu 61 , and Glu 71 -Arg 72 . Then, the antifungal properties of the peptides included in the different peaks were tested. An active peptide corresponding to CGA 47-60 with an experimental molecular mass of 1732.9 Da (Fig. 3B; expected molecular mass, 1733.1 Da) is active against N. crassa showing a minimal inhibitory concentration giving 100% inhibition of 7 M. Incubation of fungi with synthetic peptide corresponding to CGA 47-60 at increasing concentrations from 3 to 10 M (Table III) induces a decrease of fungal proliferation and the inhibition in the growth of filaments. For instance, as illustrated in Fig. 4, incubation of A. brassicola with increasing concentrations of synthetic peptide CGA [47][48][49][50][51][52][53][54][55][56][57][58][59][60] (3 M-10 M) shows that a complete stop was reached at 10 M.
In order to investigate the structural features crucial for the antifungal activity of this highly conservative C-terminal moiety of VS-1 (Fig. 2), we decided to examine the role of the tripeptide Arg 43 -Gly 44 -Asp 45 and the length of the peptidic chain, testing the activities of the synthetic peptides CGA 41-60 , CGA  , and CGA 47-70 , respectively (Table III)

Characterization of CGA-derived Fragments in Secretions of PMNs
The secretions released from human PMNs were submitted to Western blot immunodetection (Fig. 5) with monoclonal anti-CGA 5A8, directed against the epitope CGA 47-68 (28). We immunodetected several N-terminal CGA-derived fragments corresponding to the processing of CGA in secretions of PMNs. The difference between the apparent and theoretical molecular mass of CGA and CGA-derived fragments possibly results from post-translational modifications (O-glycosylation, phosphorylation) and the abundance of acidic amino acids (25%) causing a reduced migration in SDS-PAGE gels (25,54). By taking into account the primary structure of human CGA (42), the apparent molecular mass of the different released fragments, the reactivity observed with the monoclonal antisera, and the cleavage points previously reported (25), the immunodetected bands are likely to correspond, respectively, to the following: the native protein (band 1), CGA 1-394 (band 2), CGA 1-272 (band 3), CGA 1-209 (band 4), CGA 1-115 (band 5), and CGA 1-78 (band 6). The presence of diffuse bands results from the presence of post-translational modifications (O-glycosylation and phospho-rylation) (49) and from an endogenous cleavage point (Val 3 -Asn 4 ) located at the N-terminal end of the protein as it has been previously reported for bovine CGA (25). These data reveal the presence of CGA-derived fragments in secretions of PMNs with a pattern similar to that described for secretions of stimulated chromaffin cells (50). In conclusion, CGA-derived fragments are secreted by PMNs and thus may locally be recovered in specific infectious fluids where they exert their antimicrobial activity.

DISCUSSION
Chromogranins/secretogranins (CGs) constitute a family of acidic secretory glycophosphoproteins widely expressed in a large number of endocrine and neuroendocrine cells and in neurons (50 -53). At the subcellular level, chromogranins are exclusively found in the soluble core of hormone and neurotransmitter storage vesicles and are released during exocytosis. CGs are considered as precursor proteins that are processed into peptides within the secretory granules (25,50,54,55). Chromogranin A (CGA), the major member (40% of total soluble bovine chromaffin granule proteins) of this family, has been extensively studied. CGA is a ubiquitous 48-kDa secretory protein stored and released from most endocrine and neuroendocrine cells and neurons. Different biological activities, in relation to the autocrine or paracrine role in hormone secretion, have been attributed to peptides located along the sequence of CGA (for review see Ref. 56). Among these peptides, the collective term of vasostatins refers to bovine N-terminal fragments CGA 1-76 (vasostatin-1) and CGA 1-113 (vasostatin-2) to specify their vascular inhibitory effects as has been originally reported (57). Our previous study on the natural processing of CGA has indicated that vasostatins are predominantly generated in the matrix of chromaffin granules and co-released with catecholamines in the extracellular medium upon chromaffin cell stimulation (25). A study on the characterization of CGA-derived fragments, generated by the isolated retrogradely perfused bovine adrenal gland under basal conditions and during stimulation with acetylcholine, indicates that vasostatins are secreted from bovine adrenal medulla (58). In addition, vasostatins have been immunodetected in the large dense core vesicles traveling down sympathetic axons and are released from the nerve terminal in response to stimulation (59). The vasoinhibitory effect of vasostatins, which does not involve interference with endothelin-1 (a potent vasoconstrictor peptide) binding to its vascular receptor (60), has recently been associated with recombinant and synthetic human vasostatinderived fragments (61). Synthetic peptides corresponding to the sequence CGA 1-76 mimic natural vasostatin-1 in its inhibition of contractions induced by endothelin-1 (62). Furthermore, vasostatin-1 exhibits other biological activities such as the inhibition of parathyroid hormone secretion (63), the regulation of cell adhesion (64), and the neurotoxicity in neuronal/ microglial cell co-cultures (65). The predominant presence of vasostatin-1 in bovine chromaffin granules, but also in numerous neuroendocrine tissues, and its release from sympathetic nerves suggest an important function for this fragment. A role in the regulation of vascular smooth muscle has been reported (57), although the specific receptor has not been identified yet. It has also been demonstrated that the disulfide-bonded loop included in vasostatin-1 plays a crucial role for oligomerization of CGA (66). This N-terminal natural bovine CGA-derived fragment corresponds to a very conserved domain showing high yield of identity with human (97.3% (67)), pig (97.3% (43)), rat (85.5% (44)), mouse (86.8% (45)), ostrich CGA (80.2% (46)), equine CGA (92.1%), 3 and frog CGA (75.7% (48)), respectively. In all species examined so far the disulfide bridge Cys 17 -Cys 38 is conserved, and the sequence SILRHQNLLKELQ (CGA 50 -62 ) is strictly unchanged. Furthermore, it has been shown that vasostatin-1 interacts with the secretory vesicle membrane at pH 5.5 and dissociates from it at pH 7.5 (68). The biological role of these interactions is still not understood, but they may reflect some function of this domain in intragranular lattice organization.
Data reported in the present paper establish that bovine vasostatin-1, human recombinant VS-1, and rat synthetic CGA 7-57 possess antimicrobial activities at micromolar concentration. Bovine vasostatin-1 results from the cleavage of CGA at the dibasic site Lys 77 -Lys 78 and the subsequent action of carboxypeptidase B (25). This peptide which is composed of 76 residues with a disulfide bridge (Cys 17 -Cys 38 ) forming a loop (Fig. 2) possesses structural features specific for antimicrobial peptides as follows: (i) a global positive charge (ϩ3), (ii) an equilibrated number of polar (21:76) and hydrophobic residues (23:76), and (iii) the presence of a helical region corresponding to sequence CGA 40 -65 , as previously indicated (69).
In addition to its antibacterial activity, vasostatin-1 was found to display unexpected antifungal activity, provoking the death of filamentous fungi and yeast cells at micromolar concentrations. After removal of vasostatin-1 no re-growth of fungi or yeast cells was observed after a 24-h incubation demonstrating the fungicidal property of the peptide. In order to understand the molecular mechanisms implicated in the antibacterial and antifungal properties of vasostatin-1, we have attempted to relate these activities with some structural parameters.
Antimicrobial Activity in Relation with the Length of the CGA-derived Fragments-The loss of the antibacterial activity of synthetic rat CGA 7-57 , with and without the disulfide bridge, (Table I) indicates that the regions comprising 6 and 19 residues on the N-or C-terminal ends, respectively, are essential for the activity (Fig. 2). The 19 residues on C-terminal end (residues 58 -76) are important for the secondary structure of CGA 1-76 , probably with regard to the formation of helical structure. A previous study has shown the importance of the Nterminal sequence CGA [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] in the inhibition of parathyroid hormone secretion evoked by CGA   (62), whereas residues 1-5 on the N-terminal end are not necessary for such an activity (70). Concerning recombinant VS-1 peptide, the addition of STA and KK residues at the N-and C-terminal ends, respectively (Fig. 2), may be responsible of the antibacterial activity decrease.
Antimicrobial Activity in Relation with the Disulfide Bridge-The opening of the disulfide bridge and the S-pyridylethylation of CGA-derived peptides induce a slight decrease in the antibacterial activity of vasostatin-1 and the loss of the inhibition against M. luteus for VS-1. In contrast, the activity of VS-1 against B. megaterium is fully preserved. In addition, we found that the opening of the disulfide bridge and the S-pyridylethylation of vasostatin-1 and VS-1 induce no alteration in the antifungal activity. A similar result is obtained with the two synthetic rat CGA 7-57 peptides indicating that the disulfide bridge is not crucial for the antifungal activity. Thus, it appears that the antibacterial and antifungal activities require different structural parameters.
Antimicrobial Activity in Relation with Oxidation-Performic oxidation of vasostatin-1 and of VS-1 induces the complete loss of the antibacterial activity against M. luteus and B. megaterium and partially modifies the antifungal activity at the concentration range of 20 -30 M. These data suggest the importance of methionine residues Met 7 , Met 15 , and Met 32 and cysteine residues Cys 17 and Cys 38 for the antimicrobial activity of vasostatin-1.
Antimicrobial Activity in Relation with Residue Modifications-Comparison of bovine vasostatin-1 and human VS-1 sequences shows two changes on Lys 36 /Gln 36 and Thr 73 /Ala 73 (Fig. 2). As Thr 73 /Ala 73 change corresponds to a conservative modification, the decrease of antibacterial activity is likely to result from the modification of Lys 36 which may induce electrostatic interactions with Glu 37 and Glu 40 to stabilize the active conformation.
In addition, comparison of vasostatin-1 sequence with that of rat synthetic CGA 7-57 indicates two changes on hydrophilic residues Glu 13 /Lys 13 and Lys 36 /Pro 36 (Fig. 2) that may be responsible for conformational changes. The chemical synthesis of peptides including prolyl residues in the sequence is well known to provide several molecules corresponding to different prolyl isomers and, among these structures, only one corresponds to the natural conformation (23). Thus, the presence of four prolyl residues in rat CGA 7-57 (Pro 29 , Pro 31 , Pro 33 , and Pro 36 ) may explain the attenuated antifungal activity (Fig. 2).
Biological Function-Vasostatin-1 is a 76-residue peptide displaying antibacterial and antifungal activities at micromolar concentrations in biological assays, as standardized here. The secretory granules present in adrenal chromaffin cells and other neural and neuroendocrine tissues represent a compartment storing several antibacterial peptides (secretolytin, chromacin, and enkelytin) for which a role in innate immunity during stress situations has been suggested (24). The new antifungal activity of vasostatin-1 described here looks remarkable because it supplements the antibacterial activity in innate immunity. Taken together these two antibacterial and antifungal activities would provide a highly beneficial survival strategy. To further characterize the biological function of vasostatin-1, secretion of PMNs was examined by Western blot. Indeed, the local inflammatory response may initiate the synthesis and the secretion of these peptides by immune cells at the concentration necessary for the antimicrobial activity, as we have recently shown for enkelytin (23). Our data indicate for the first time that stimulated immune cells (PMNs) release CGA and CGA-derived peptides, including vasostatin-1, resulting from the natural processing of the precursor (Fig. 5). The potency of antimicrobial activities associated with peptides may be modulated by different agents present in biological fluids (47), and the activity of antibacterial and antifungal peptides in biological fluids is still a matter of debate. In infectious fluids and in blood, there is a large variety of antimicrobial peptides that can act on same target with cooperative mode (23).
VS-1 expresses antifungal activity indicating that this recombinant fragment can be used for future structural investigation including NMR analysis and the understanding of the mode of action. Comparison of the human sequence with the corresponding bovine CGA fragment (Fig. 2) indicates 97.3% homology. Because of the high conservation of the N-terminal domain of CGA, the antibacterial and antifungal activities appear to have occurred early in evolution. Peptides with antifungal activity have been described in plants, insects, and invertebrates, and they fully protect these organisms against pathogenic invasion. In mammals vasostatin-1, which is the first natural antifungal CGA-derived peptide predominantly isolated so far from adrenal chromaffin cells, may represent a weapon in innate immunity during stress situations, a role that is presently under investigation in our laboratory.