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SCHISTOSOMA MANSONI DERMASEPTIN-LIKE PEPTIDE: STRUCTURALAND FUNCTIONAL CHARACTERIZATIONAuthor(s): Gerry A. P. Quinn, Raymond Heymans, Franchesca Rondaj, Chris Shaw, and Marijke de Jong-BrinkSource: Journal of Parasitology, 91(6):1340-1351. 2005.Published By: American Society of ParasitologistsDOI: http://dx.doi.org/10.1645/GE-540R.1URL: http://www.bioone.org/doi/full/10.1645/GE-540R.1

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1340

J. Parasitol., 91(6), 2005, pp. 1340–1351q American Society of Parasitologists 2005

SCHISTOSOMA MANSONI DERMASEPTIN-LIKE PEPTIDE: STRUCTURAL ANDFUNCTIONAL CHARACTERIZATION

Gerry A. P. Quinn, Raymond Heymans*, Franchesca Rondaj*, Chris Shaw, and Marijke de Jong-Brink*†Department of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Northern Ireland BT52 1SA. e-mail:marijke.de.jong.brink@falw.vu.nl

ABSTRACT: Analysis of the Schistosoma mansoni peptidome for immunomodulatory molecules by solvent extraction and reverse-phase HPLC revealed a 27-amino-acid residue peptide from an extract of cercariae. Using matrix-assisted, laser desorption–ionization, time-of-flight mass spectrometry, the peptide yielded a protonated molecular ion [M 1 H]1 of m/z 2789. The un-equivocal sequence was deduced by automated Edman degradation as: DLWNSIKDMAAAAGRAALNAVTGMVNQ. The pep-tide exhibited an 80.76% identity with dermaseptin 3.1 from the leaf frog Agalychnis annae, and was therefore named Schistosomamansoni dermaseptin-like peptide (SmDLP). Immunocytochemical staining using a primary antidermaseptin B2 antibody locatedSmDLP in acetabular glands of cercariae, in and around schistosomula, and in adult worms and their eggs. Dot-blotting confirmedits presence in extracts (cercariae and worms) and excretion/secretion (E/S) products (transforming cercariae and eggs). This wascorroborated by use of a MALDI-ToF spectra database of E/S products from cercariae. Functional characterization of the peptideindicated that SmDLP had typical amphipathic antimicrobial peptide properties, i.e., the ability to lyse human erythrocytes causinga decrease in the levels of nitric oxide produced by monocytic cells. This last function strongly suggests that SmDLP plays avital role in the parasite’s immunoevasion strategy. The possibility that schistosomes acquired this gene from amphibians hasbeen discussed by constructing a phylogenetic tree.

Schistosoma mansoni is a dioecious trematode that has hada long association with humans and is uniquely adapted toevade human immunity (Contis and David, 1996). For its lifecycle, the parasite needs 2 hosts, i.e., humans as the definitivehost (Incani et al., 2001) and a freshwater snail Biomphalariaglabrata as the intermediate host. Cercariae that leave the snailhost are able to penetrate human skin, where they can remainfor 48–72 hr before migrating via the lungs to take up perma-nent residence in the blood vessels of the intestinal mesenteryas mature, paired (male and female) worms (He et al., 1992;McKerrow and Doenhoff, 1998; Bartlett et al., 2000; Dorsey etal., 2002; Curwen and Wilson, 2003; McKerrow, 2003). Themajority of the eggs that are produced by adult female wormscan penetrate the intestinal wall and leave the host with feces.In water, miricidia hatch from the eggs and continue the lifecycle in a molluscan intermediate host. Part of the eggs canbecome lodged in the presinusoidal capillaries of the liver andspleen, which can lead to hepatosplenomegaly, portal hyperten-sion, and eventually death (Bilharz, 1856; Graham, 2002).

Immunological reactivity against naive parasitic infectionfollows 2 different forms. The primary response, which is themost important line of defense, takes the form of innate im-munity. It involves the skin barrier, macrophages, mast cells,NK cells, chemokines, interferons (INFs), complement, patternrecognition receptors (PRRs), and cytokines (Fusco et al., 1993;Wang et al., 1999; Wolowczuk et al., 1999; Witko-Sarsat et al.,2000; Angeli et al., 2001; Ratzinger et al., 2002; Deng et al.,2003). The acquired immune response, the second line of de-fense, which is mainly facilitated by T-cells (cellular response;Ramalho-Pinto et al., 1976) and B cells (humoral response,which involves IgE; Smithers and Terry, 1965), takes at least2 days to counteract the parasite and is less effective and moreinvolved in secondary infections. One of the most ingeniousfacets of the infection process with a compatible schistosomeis the myriad of immunological evasion strategies that the par-

Received 8 April 2005; accepted 29 April 2005.* Faculty of Earth- and Life Sciences, Vrije Universiteit, De Boelelaan

1087 Amsterdam 1081 HV, The Netherlands.† To whom correspondence should be addressed.

asite utilizes as soon as it enters the skin. The excretory/secre-tory (E/S) products, which are released from transforming cer-cariae, play a key role in this evasion strategy (Pleass et al.,2000; Ramaswamy et al., 2000; Angeli et al., 2001; Chen etal., 2002; Coelho-Castelo et al., 2002; Rao et al., 2002; Shal-doum, 2002). Although significant efforts have been employedto characterize these products by means of polyacrylamide gelelectrophoresis (PAGE), the limitations imposed by this processmean that only proteins larger than 8 kDa, namely the proteo-me, have previously been analyzed (see Verjovski-Almeida etal., 2004; Wilson et al., 2004). A consideration of the role ofthe peptidome (which can be defined as peptides with a molec-ular mass of less than 8 kDa) in the interactions between cer-cariae and the definitive host stages has been almost neglectedin the field of schistosome research, with the exception of neu-ropeptide immunological studies conducted by Duvaux-Miretet al. (1992). With recent advances in mass spectrometry (MS),it is now possible to systematically identify the lower molecularmass components within the E/S products, namely the pepti-dome, and to investigate whether these molecules are involvedin the parasite’s immunoevasion strategy. With these tech-niques, we have identified a dermaseptinlike peptide in the pep-tidome of S. mansoni (SmDLP), which has (in addition to an-timicrobial properties) the ability to lower the amount of NOproduced by RAW 264.7 cells. This indicates that SmDLP mayplay a role in the parasite’s immune evasion strategy.

MATERIALS AND METHODS

Parasite cultivation

Cycles of S. mansoni (Puerto Rican strain) were maintained by pas-sage through snails (B. glabrata) and mice (Mus musculus; type: Swissalbino). All animals were kept in accordance with EEC Council Direc-tive 86/669/EEC on the approximation of laws, regulations, and admin-istrative provisions of the Member States regarding the protection ofanimals used for experimental and other scientific purposes. The inter-mediate host (B. glabrata) was induced to shed S. mansoni cercariaeon a weekly basis. From 6 wk after infection and onwards, cercariaewere obtained via induced shedding (Sluiters et al., 1980). Typically,50 thoroughly cleaned snails were placed in a perspex tank (10 cm 310 cm 3 15 cm) with 150 ml of copper-free water at room temperature;a strong directional light was shone on them for 2 hr, stimulating therelease of cercariae from the snails. After shedding, the snails were

QUINN ET AL.—S. MANSONI DERMASEPTIN-LIKE PEPTIDE 1341

placed back in their tanks and the resulting water with cercariae waspassed through filter paper. To ensure that the sample consisted solelyof cercariae without contamination from snail products, the filter paperswere then washed 3 times in copper-free water, centrifuged (2,000 g for20 min) and concentrated on a speed vac. The number of cercariae wasassessed microscopically by taking the average number of 3 samples of10 ml and adding iodine (to visualize and kill the cercariae). Typically,there were around 75,000 cercariae in 150 ml of water. This figure couldvary according to the maturity and the progression of infection in thesnails. For Trichobilharzia ocellata, ducks were used as definitive hostand Lymnaea stagnalis snails as the intermediate host. Three weeksafter being exposed to cercariae, schistosome eggs were isolated fromthe feces of the ducks for 10 successive days; after that period, theducks were killed. The miricidia hatching from these eggs were usedto infect snails with a shell height of 10–12 mm. About 6–8 wk afterinfection, the snails shed cercariae, which were collected once a weekusing a similar procedure as described above.

Extraction of proteins

From cercariae and adult worm pairs: All solutions were preparedusing Milli-Qt water. Solvents were HPLC grade and the rest of thereagents were analytical grade. Approximately 1 mg of freeze-dried S.mansoni cercariae or adult worm pairs were homogenized in an extrac-tion solvent of acetic acid/acid alcohol (1 M ethanol:0.7 M HCl [3:1])at 220 C. The homogenate was then stirred in the extraction solvent (4C for 24 hr) and finally centrifuged again (3,000 g for 1 hr). The su-pernatant from this solution was separated by centrifugation and air-dried.

The resulting extracted material from the worms was used for im-munodotting (see below), and that of the cercariae extract was resus-pended in 300 ml of 0.05% trifluoroacetic acid (TFA). Peptide separa-tion was performed by injecting 300 ml of the crude extract onto aphenomenex reverse-phase chromatographic column C-18 (2.1 mm 330 mm) HPLC system. Peptides were purified using a linear gradientfrom 0 to 80% acetonitrile (ACN) containing 0.05% TFA/water for 60min at 250 ml/min. Separation was continuously monitored at A214 nm.Fractions were hand collected in polypropylene tubes and stored at 4 C.

From E/S products: The cercariae obtained (as indicated above) wereincubated in sterile copper-free water with 5% gentamycin (20 min) andsubsequently mechanically transformed into schistosomula by passagethrough a syringe (0.3-mm microlance needle on a 50-ml syringe) 10times. Alternatively, a solution of cercariae in copper-free water wasadded to a Petri dish coated with linoleic acid (0.9 g/ml; Sigma, St.Louis, Missouri) and incubated at 37 C for 2 hr. Both methods resultedin the transformation of the cercariae into schistosomula, which wasconfirmed by microscopic examination. The resultant solution was cen-trifuged (2,000 g for 30 min at 4 C) and the supernatant with E/Sproducts collected. This was passed through a 0.2-mm disposable filter.Larger proteins (.8,000 Da) in the E/S products were separated byadding a mixture of acetone/HCl/H20 at a ratio of 40:1:6 and then pre-cipitated overnight between 0 and 4 C. The sediment from this precip-itation was then separated by centrifugation and the supernatant wassubjected to further purification using an activated (following manufac-turers instructions) Sep-Pak separation column (Supelco Lc-18, 3-mltubes, Bellefonte, Pennsylvania). The supernatant was dehydrated(speed vac) to a volume of 10–20 ml to remove the ACN. The extractwas then redissolved in 300 ml of TFA and injected into a phenomenexC18 column (2.1 mm 3 30 mm) rpHPLC system using a linear gradientfrom 25 to 60% acetonitrile (ACN) containing 0.073% TFA/water for30 min at 250 ml/min. Separation was continuously monitored at A214

nm and A254 nm.

Purification and characterization of SmDLP

Each fraction from the rpHPLC was subjected to mass spectrometricanalysis by a MALDI-ToF mass spectrometer (PerSeptive BiosystemsVoyager-DE Mass Spectrometer, Hertfordshire, U.K.). The purer frac-tions were then subjected to automated Edman degradation (Chen etal., 2004).

Bioinformatic analysis

The sequences obtained by Edman degradation were compared withprotein databases using the FASTA 3.3 program on the European Bioin-

formatics Institute (EBI) site (http://www.ebi.ac.uk/Tools/index.html).The secondary structures of the peptides were analyzed using the DeepView program (Deep View/Swiss-Pdb Viewer 3.7, SP5) on the EX-PASY site (http://us.expasy.org/spdbv/text/refs.htm).

Immunolocalization of SmDLP

SmDLP origins: To ascertain the origins of SmDLP, we used a poly-clonal antibody raised to a dermaseptin consensus region (raised in arabbit; Lacombe et al., 2000). This antibody recognized SmDLP. Twotechniques were used, i.e., immunodotting to detect the presence ofSmDLP in fractions of the parasite and their secreted products, andimmunocytochemistry to locate the origins of SmDLP in cercariae,adult worms, and eggs.

Immunodotting: Extracts of cercariae, and fractions thereof, were ob-tained as previously described. This was the same for extracts of cer-cariae E/S products and their corresponding fractions. Samples of theseextracts and of the fractions obtained by fractionating the extracts (ona Waters size fractionation column [HPGPC] with a maximum cut offof 40,000 Da) were used for immunodotting. Extracts of adult worms(WESm) were also prepared as described previously for cercarial ex-tracts. WESm, soluble egg antigen (SEA), and the E/S products of S.mansoni eggs (EST) and dilutions thereof were also immunodotted withantidermaseptin. Synthetic dermaseptin was prepared as stated previ-ously. Immunodotting was performed according to the method of Zenget al. (1999). Briefly, aliquots (1 ml) of the samples, and dilutions andfractions thereof, were pipetted onto a grid of precoated nitrocellulosepaper and left to air-dry for 30 min. Antidermaseptin was diluted inTBS gelatin buffer at 1:750. The paper was rinsed twice with TBS-gelbuffer (10 min). The second antibody (peroxidase-conjugated swine an-tibody against rabbit IgG, SWARPO; DAKO, Glostrup, Denmark), wasthen diluted in TBS gel buffer (1:100) and incubated with the nitrocel-lulose paper at room temperature (60 min) on a rotary shaker. Thenitrocellulose paper was rinsed 3 times in Tris (TBS) buffer (3 3 10min). The immunoprecipitate on the nitrocellulose paper was visualizedby adding 0.5 ml of 3,39-diaminobenzidine (DAB) solution (Sigma), 4.5ml of TBS buffer, and 50 ml of 10% nickel and 2 ml of H2O2. This wasleft for 20 min before rinsing with distilled water (2 3 10 min) andthen dried between clean sheets of filter paper.

Immunocytochemistry: Mice were sedated with hypnorm and dor-micum (Janssen Pharmaceutica, Beersel, Belgium). Both narcotics werefirst diluted in water (1:1) and then a mixture (1:1) of these 2 solutionswas made. A dose of 15 ml/g body weight was injected intraperitoneallyin each mouse. The mice were infected with S. mansoni cercariae col-lected as described previously. The left and right ears were exposed to1,000 cercariae each and the shaved abdomen with 2,000 cercariae.Some abdominally infected mice were allowed to recover and left untilpatency. Parts of the intestinal wall and its mesentery containing adultworms and pieces of liver tissue with egg granulomas were excised,fixed, and prepared for staining with antidermaseptin as described fortissue sections infected with cercariae. Other mice were killed after aperiod of 30, 60, and 120 min exposure; both ears and the abdominalskin were excised, fixed in 4% paraformaldehyde (PFA) in 0.1 M phos-phate buffer (overnight) or in Bouin’s (5 ml formalaldehyde, 15 mlpicric acid, 1 ml acetic acid), and subsequently embedded in paraffin.

Free-swimming cercariae were collected and pelleted by centrifuga-tion. The pellet was quickly frozen in liquid nitrogen-cooled freon,freeze-dried at 280 C, fixed in PFA vapor for 1 hr at 60 C, and directlytransferred to paraffin. The tissue sections prepared (7 mm thickness)were adhered to double-coated (0.5% gelatin, 0.5% chrome-alum indistilled water) microscope slides. The sections were dewaxed and re-hydrated via a degrading alcohol series including methanol with 1%acetic acid and 0.1% hydrogen peroxide to inhibit endogenous peroxi-dases. These were then rinsed in TBS gelatin buffer, pH 7.4, (2 3 10min) and incubated with anti-SmDLP/TBS gelatin buffer (1:750 and/or1:1,000) overnight at 4 C. After rinsing again with TBS gelatin buffer(2 3 10 min), the sections were incubated with the secondary antibod-ies: 1:100 diluted peroxidase-conjugated swine antirabbit immunoglob-ulins (SWARPO; DAKO) for 60 min at room temperature. Subsequent-ly, the sections were rinsed in Tris buffer (2 3 15 min). The peroxidasereaction was visualized with 0.05% 3,39-diaminobenzidine (DAB; Sig-ma) in TBS containing 0.01% hydrogen peroxide (3–15 min). To ensurethe specificity of the antibody and the immunocytochemical staining,controls included in the procedure were (1) omission of the first anti-

1342 THE JOURNAL OF PARASITOLOGY, VOL. 91, NO. 6, DECEMBER 2005

body, (2) use of preimmune serum, and (3) staining with an antiserumnot recognizing SmDLP as anti-CD11c (clone N418) but recognizinginfiltrating dendritic cells and granulocytes.

Solid phase peptide synthesis

To further investigate the physiological effects of SmDLP, we madea synthetic variety of SmDLP. It was anticipated that the various phys-iological experiments seen here as well as those that are not publishednecessitated a large quantity of the peptide at a known concentration.An amidated all-L-amino acid form of SmDLP was prepared by step-wise solid-phase FastMoc synthesis (Chen et al., 2004).

Effects of SmDLP

Microorganisms: One of the first reported effects of dermaseptinswas their antimicrobial activity (Mor et al., 1991). Consequently, toascertain the microbiacidal profile of SmDLP, a liquid growth inhibitionassay was performed. This was achieved using gram-negative bacteria(Escherichia coli), gram-positive bacteria (Staphylococcus aureus andMicrococcus luteus), and yeast (Candida albicans) by a microdilutionmethod (Bulet et al., 1993).

Bacteria were subcultured on nutrient agar and the yeast were grownon yeast extract agar. One isolated colony of the test organism wassubcultured into nutrient broth and grown overnight at 37 C on a rotaryshaker at 150 rpm. The concentration of the organisms was measuredon a hemocytometer using Trypan Blue (Sigma-Aldrich, Dorset, U.K).Microbial suspensions were diluted to approximately 1 3 105 organismsper ml.

Synthetic SmDLP and the control antibiotics were dissolved in phos-phate-buffered saline (PBS) 1/10 w/v. A serial dilution was made ofSmDLP and control antibiotics; the highest concentration used for theassay was 33 mg/ml, then a 10-fold dilution (3.3 mg/ml) was tested,followed by a series of double dilutions down to 6.5 mg/ml. Antibac-terial activity was assessed using a microtiter plate filled with 50 ml ofbacteria in midlog phase and 25 ml of peptide/antibiotic. Positive anti-microbial controls were ampicillin for E. coli, tetracycline for S. aureusand M. luteus, and 70% alcohol for C. albicans. The negative controlwas nutrient broth. Tests were incubated for 24 hr at 37 C under agi-tation. The minimum inhibitory concentration (MIC) was defined as thelowest concentration at which no bacteria were optically visible after 6hr at 37 C. The minimum bactericidal concentration (MBC) was deter-mined as the lowest concentration of peptide at which no visible col-onies were seen after subculturing from the test wells onto nutrient agar/yeast extract agar and incubating overnight at 37 C.

Erythrocytes: Mammalian erythrocytes (red blood cells [RBCs]) wereused to ascertain the hemolytic potential of SmDLP. Fresh human bloodwith EDTA was rinsed 3 times with PBS pH 7.4 at a ratio of 1:3.3 andcentrifuged (1 min at 12,000 g). Approximately 1.25 3 108 RBCs werethen incubated for 2 hr at 37 C in various solutions. These includeddistilled water for a control of 100% hemolysis, phosphate-bufferedsaline (PBS) for a reference control, bovine serum albumin as a negativecontrol, and PBS containing different concentrations of SmDLP. Allsolutions were subsequently centrifuged (1 min at 12,000 g) and thesupernatants were transferred into a 96-well (500-ml wells) microtiterplate. The hemolytic activity (detectable as hemoglobin leakage) wasmeasured with a spectrophotometer at 405 nm.

A similar procedure was followed for nucleated duck (Anas platy-rhynchus) RBCs. Approximately 0.3 3 108 duck RBCs were used be-cause they are much larger than the human variety. Concentrations ofSmDLP varied from 1 3 1026 to 5 3 1023 M. Because of the insolubilityof SmDLP, in later experiments a solvent, dimethyl sulphoxide (DMSO)was used in a 5% solution to dissolve higher concentrations of SmDLP.Appropriate controls for the DMSO were observed.

RAW 264.7 cells: Because we assumed that SmDLP is released inthe skin and, therefore, will interact with the innate immune system,we chose RAW 264.7 monocytic cells (ATCC TIB-71) to investigatethe possible role of the peptide. The cells were cultured in RPMI-1640medium (Gibco/BRL Laboratories, Grand Island, New York) supple-mented with 10% heat-inactivated fetal calf serum (Gibco/BRL), 5 31025 M b-mercaptoethanol, 2 mM L-glutamine, 50 mg/ml streptomycin,and 1,000 U/ml penicillin-G. Subcultures were prepared 2 or 3 times/wk by scraping and transferring the cells into a new culture flask withnew RPMI-1640 medium. For optimal culture, the cells were incubated

at 37 C in a moist environment with 5% CO2. Monocytes (RAW 264.7)were cultured with SmDLP in the following manner. They were washedin RPMI-1640 medium, centrifuged (10 min at 1,200 g) and resuspend-ed in this medium. Aliquots of this suspension (5 3 105 cells/500 mlmedium enumerated with a Burker–Turk cell-counter) were added to a24-well plate and preincubated for 60 min. To test the option thatSmDLP might have a direct effect on the production of nitric oxide(NO) by monocytes, the RAW 264.7 cells were incubated with lipo-polysaccharide (LPS) (E. coli 0111:B4, Difco; Detroit, Michigan) forstimulation, using SmDLP (synthetic; dimethyl sulphoxide at a concen-tration of 5% for dissolving the stock solution), or with both LPS andSmDLP. To investigate the option that SmDLP might be lytic to RBCsand hence by means of oxyhemoglobin indirectly affect the amount ofNO produced by RAW 264.7 cells, the cells were incubated with un-lysed (in buffer) and lysed (in distilled water) RBCs, and with RBCspreincubated with SmDLP. In the case of erythrocyte lysis (by distilledwater or SmDLP), the preincubation fluid was used before and afterbeing centrifuged for removal of membrane remnants.

To measure the activation of the RAW 264.7 monocytic cells, thereleased amount of nitric oxide (NO) was determined by means of aGriess assay. Briefly, after centrifugation of the microtiter plate, thesupernatant from each test well, i.e., 50 ml of each sample, was pipettedinto a 96-well microtiter plate together with 50 ml of Griess reagent(1% sulphanilamide/0.1% naphthylethylene diamine dihydrochloride/2.5% H3PO4) at room temperature for 10 min. The concentration of NOwas spectrophotometrically determined using a standard curve withknown NO concentrations on a microplate reader at a wavelength of540 nm. Readings from each sample cultured were performed in trip-licate, and the average value was used.

Data analysis and statistics

In experiments on hemolytic activity of SmDLP on human RBCs(detectable as hemoglobin leakage), the measurements were used toconstruct a graph of increasing hemolysis (as detected by a change inabsorbance at 405 nm) against the log 10 concentration of SmDLP. Theoptical density (OD) from RBCs with water, which represents 100%hemolysis, (water average OD 5 2.92; standard deviation [SD] 50.0311) and the OD from RBCs in PBS, which represents 0% hemolysis(average 5 0.2883; SD5 0.008273) were added to the graph. Valuesare means 6 the standard error of the mean (SEM) of 3 readings. Adose-response curve was then fitted to the data. The same approach wascarried out for the experiment using duck RBCs, except the valuespresented were means 6 the SEM obtained in 3 independent experi-ments. In the experiments in which the levels of NO produced by mono-cytes were measured, the concentration of NO was spectrophotometri-cally determined using a standard curve with known NO concentrationson a microplate reader at a wavelength of 540 nm. Readings from eachsample cultured were performed in triplicate and the average value wasused. The figures indicate the numerical average of 3 readings. For thenext experiment on monocytes, the same statistical methods were usedexcept that the values are means 6 SEM for 3 readings in the experi-ment. The Student’s t-test (unpaired) was performed on this final result.

Molecular phylogeny of SmDLP

We examined the evolutionary relationships between SmDLP andother homologous sequences taken from a Blast search in NCBI (thedatabase abbreviated code for organism and protein [NCBI] is giventogether with species and type of peptide) by constructing a phylo-genetic tree from alignments of protein sequences. The following se-quences were used: Q5P4L8-AZOSE, Azoarcus sp., hypothetical (bac-terium); Q7Q0E1-ANOGA, Anopheles gambiae str, enzyme (insect);Q5PBL9-ANAMA, Anaplasma marginale, heme protein (bacterium);DDSL-PHYDS P83639, dermadistinctin L, Phyllomedusa distincta(monkey frog); Q9HPP5-HALN1, Halobacterium sp, hypotheticalprotein (bacterium); Q800S0-AGACL, dermaseptin-like DRP-AC-3,Agalychnis callidryas (red-eyed leaf frog); Q5T400-Human, OT-THUMP00000018721, DERB-PHYBI, dermatoxin precursor, Phyllo-medusa bicolor, (two-colored leaf frog); SmDLP (S. mansoni); VPN-BPMU, DNA circulation protein, bacteriophage Mu, (64-kDa virionprotein); DRG2-PHYBI, dermaseptin DRG2, Phyllomedusa bicolor(two-colored leaf frog); HIS5-RHILO, imidazole glycerol phosphatesynthase subunit hisH, Rhizobium loti (Mesorhizobium loti); TI17-

QUINN ET AL.—S. MANSONI DERMASEPTIN-LIKE PEPTIDE 1343

FIGURE 1. (A) Reverse-phase HPLC chromatogram of a Schistosoma mansoni cercarial freeze-dried extract. The retention time of SmDLP isapproximately 47 min. (B) MALDI-ToF/MS spectrograph of fraction 47 with a predominant ion being observed at 2789 m/z [M 1 H]1.

DROME probable mitochondrial import inner membrane translocasesubunit Tim17 4, Drosophila melanogaster (fruit fly); DMS3-PACDA,dermaseptin PD-3–3 precursor, Pachymedusa dacnicolor (giant mex-ican leaf frog); DRS3-AGAAN, dermaseptin AA-3–1 precursor, Aga-lychnis annae (yellow-eye leaf frog); and DMS2-PHYBI, adenore-gulin precursor, Phyllomedusa bicolor (two-colored leaf frog).

The guide tree, which resembles a phylogenetic tree, is built usingthe neighbor-joining method (NJ). This works on a matrix of distancesbetween all pairs of sequences. The distances are related to the degreeof divergence between the sequences considered. The calculated dis-tance values appear in parentheses.

RESULTS

Isolation, purification, and charicterisation of SmDLP

To identify potential peptides involved in parasite–host in-teractions, cercariae extracts were prepared and subjected toreverse-phase HPLC. Analysis of the MALDI-ToF MS ionspectra from these fractions led us to focus on a pure and rel-atively abundant molecule with a mass ranging from 2796 m/z[M 1 H]1 to 2799 m/z [M 1 H]1, which was mainly locatedin fraction 47 (Fig. 1A). This elution position suggested a rel-atively hydrophobic molecule. Further fine-tuning of the MAL-DI-ToF MS (using standard calibrants) yielded a peptide ofatomic weight of 2789 m/z [M 1 H]1 (Fig. 1B). Examinationof our parasite database, which contained all the MALDI-ToFmass spectra from fractionations of both cercariae and their E/S products, repeatedly revealed an ion of 2787–2797 m/z [M1 H]1 (data not shown). This was consistently observed in 3independent experiments in 2 different laboratories confirmingthe common occurrence of this peptide. The complete sequencededuced by automated Edman degradation of fraction 47 fromcercariae is shown (Figs. 2A,B) and is consistent with the massdetermined by MALDI-ToF MS. This sequence was then usedin the Fasta 3.3 protein homology search program, where thepeptide exhibited an 80.76% identity with dermaseptin 3.1 fromA. annae (blue-sided leaf frog, Wechselberger, 1998) and wasaccordingly named Schistosoma mansoni dermaseptinlike pep-tide (SmDLP). The amino acid sequence of this protein can beaccessed through Swiss-Prot and TrEMBL knowledge base un-der the accession number P83914.

The secondary structure of SmDLP was assessed using thePDB Deep View Program, which is an interactive 3-dimension-al program. This enabled a putative prediction on the structure–function relationship. Figures 3A and 3B clearly display the

amphipathic character of SmDLP, which conforms to the lateststructural observations on the dermaseptin class of moleculesusing X-ray crystallography (Kustanovich et al., 2002; Lequinet al., 2003). The large conserved tryptophan group (see Fig.2) also seems to impart a topological orientation to the molecule(Fig. 3).

In accordance with other research groups (Kustanovich et al.,2002), we analyzed the hydrophobicity of the C-terminal endof SmDLP and other dermaseptins, from the glycine/basic ami-no acid residue located in the middle of the peptides to the endof the C-terminal using values given by Kyte and Doolittle(1982), (see Table I). We observed that the dermaseptins witha positive hydrophobicity in this region were hemolytic and thatthe dermaseptins with negative hydrophobicity in the C-termi-nal portion were not hemolytic (see Table I). This hemolyticeffect seemed to coincide with the absence of a GEQ/NEQtripeptide motif at the C-terminus of dermaseptins, which canbe removed by posttranslational processing (Table I). This tableis based on the data available so far for erythrocyte lysis bydermaseptins. However not all dermaseptins have been testedfor this activity.

Immunolocalization

Immunodotting: The original fraction of SmDLP (cercarialextract) from which the sequence was obtained proved to reactpositively to the dermaseptin antibody (see Fig. 4A). Althoughthe polyclonal antibody used in these experiments was raisedto a synthetically prepared consensus sequence in dermaseptins(Lacombe et al., 2000), it recognized the original fraction fromwhich SmDLP was sequenced (by Edman degradation), as wellas synthetic SmDLP. It was observed that antidermaseptin wasimmunologically reactive to whole E/S products of cercariaeand subsequently to fractionated E/S products. Bovine serumalbumin and omission of the primary antibody were used ascontrols in these experiments. Furthermore, extracts of adultworms (WESm) as well as secreted egg antigens (SEA) and E/S products of S. mansoni eggs (EST) were investigated by im-munodotting and appeared to be positive to this dermaseptinantibody (Fig. 4B). Interestingly, SEA, EST, and syntheticSmDLP proved to be antidermaseptin positive even at low con-centrations. WESm (of which the protein concentration is un-

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FIGURE 2. Diagram of homologies between preprodermaseptins: Dermaseptin AA3.1, which demonstrated the greatest similarity to SmDLPand to Dermaseptin B2, which was also immunopositive with the antiserum raised to the consensus sequence to which the antidermaseptinantiserum was raised. The signal peptide, the acidic propiece, and the antimicrobial progenitor sequence are illustrated. Gaps have been introducedto maximize sequence similarities. Identical (grey background) and similar (black background) amino acid residues are highlighted. The firsthomology (A) uses the dermaseptin consensus region as an anchor sequence and illustrates why the dermaseptin consensus antibody might havereacted with SmDLP. The second homology (B) does not have an anchor sequence and aligns the consensus peptide sequence to which theantibody was raised to the preproregion of the peptides. Alignments were performed using Vector NTI protein homology software.

FIGURE 3. (A) Side view of the Swiss model Deep View representation of the secondary structure of SmDLP. (B) N-terminal view of theSwiss model Deep View representation of the secondary structure of SmDLP. The arrangement of the hydrophilic (black) and the hydrophobic(nearly white) residues reveal the amphipathic nature of the SmDLP. Neutral residues are grey.

known) appeared less immunopositive as compared to SEA andEST.

Immunocytochemistry

A clear difference was observed between cercariae and schis-tosomula. Pretransformation cercariae displayed very weak im-munopositivity to antidermaseptin in the postacetabular glands.In histological sections of mouse ear and abdominal skin thathad been exposed to cercariae, the schistosomula displayed anintense staining as compared to the negative control (Fig. 5A;see 5F as an example for the negative controls). Sections ofadult worm pairs also displayed clearly localized immunopos-itivity. In male worms, immunopositivity could be found close-ly associated with the tegument at the edges of the body wallof these flattened organisms encompassing a female (Fig. 5B).This area was often marked by closely associated erythrocytesor their membrane remnants (ghosts). The male gonad (testes;Fig. 5C) also showed immunopositivity. The same holds for thegonad in female worms, i.e., the oocytes were positive, whereas

the vitelline cells produced in the vitelline glands were clearlynegative with the antiserum applied (Fig. 5D). Eggs in livertissue and those passing through the intestinal wall were alsoclearly immunopositive (Fig. 5E; 5F for the negative control).This immunopositivity gradually decreased when the eggs be-came encapsulated by a well-developed, dense granuloma.

These data confirm the presence of SmDLP (as indicated bythe results of the immunodotting) in extracts of transformingcercariae/schistosomula, adult worms, eggs (SEA), and theproducts that are released, i.e., E/S products and EST.

Solid-phase synthesis of SmDLP

To investigate the physiological effects of SmDLP, the pep-tide was synthesized by the solid-phase method in the form ofSmDLP-amide. This was analyzed for purity by MALDI-ToFMS (Fig. 6).

Bioactivity of SmDLPRole of SmDLP: To test for potential bioactivity of SmDLP

that might play a role in parasite–host interaction, we focused

QUINN ET AL.—S. MANSONI DERMASEPTIN-LIKE PEPTIDE 1345

TABLE I. A hydrophobic comparison of amino acids of the C-terminal ends of selected dermaseptin peptides. Hydrophobicity values of the C-terminal ends of dermaseptin peptides were calculated from data by Kyte and Doolittle (1982). Some of these peptides have the C-terminaltripeptide motifs GEQ or NEQ outlined in bold. The data on hemolytic capacity of the whole peptide were taken from literature mentioned.*Abbreviation: Derm 5 Dermaseptin.

C-terminal ends of peptideC-terminal ends (calculated)

Hydrophobicity ofwhole peptide Hemolytic activity

SmDLP (16–27) AALNAVTGMVNQDerm*AA3.1(16–30)AALNAVTGMVNQGEQDerm B4(17–27) AVLNTVTDMVNQDerm S2 (19–33) AVLNAVTNMANQNEQDerm S4 (13–24) AVLNTVTDMVNQGEQ

17.920.514.725.714.727.4

Yes (Experimental data)No (Wechselberger, 1998)Yes (Charpentier et al., 1998)No (Wechselberger, 1998)Yes (Charpentier et al., 1998)

FIGURE 4. Immunodotting with antidermaseptin. (A) E/S products of transforming cercariae of Schistosoma mansoni and Trichobilharziaocellata, which have been subjected to size fractionation. 1–20, size fractionation of S. mansoni E/S products; 21–38, size fractionation of T.ocellata E/S products; 39, 40, methanol washout of purification of S. mansoni E/S products and T. ocellata E/S products; 41, S. mansoni E/Sproducts before purification; 42, T. ocellata E/S products before purification; 43, BSA control; 44, blank, no products, just first and secondantibodies (AB); 45, S. mansoni E/S products purification washout; 46, T. ocellata E/S products purification washout; 47, S. mansoni E/S productswithout first AB; 48, T. ocellata E/S products without first AB; 49, BSA without first AB; 50, just second AB. (B) Ten-fold serial dilutions ofsoluble egg antigens (SEA), worm extract (WESm), and the E/S products of eggs (EST) of S. mansoni. In the dilutions of SEA the concentrationof proteins ranged from 5,000 ng/1 ml to 0.1 ng/1 ml (numbers 1–10). 12–21, serial dilutions of WESm; 23–32, serial dilutions of EST (proteinconcentrations unknown). Numbers 11 and 22, 1023 M synthetic SmDLP (positive control).

on 2 primary activities, i.e., erythrocyte hemolytic potential(Fig. 7) and nitric oxide suppression (Fig. 8). In previous re-search (Coulson et al., 1998), it was observed that oxyhemo-globin, which is released by lysed RBCs, (partly) scavengesnitric oxide (NO) produced by LPS-stimulated monocytes/mac-rophages. Therefore, we examined the effect of syntheticSmDLP on the levels of NO produced by (LPS-stimulated)monocytes in 2 ways. First, we added SmDLP directly to themedium in which the monocytic cells were cultured, and sec-ond, we added SmDLP indirectly through erythrocyte lysis(oxyhemoglobin) caused by SmDLP.

Erythrocyte lysis: Experiments indicated that a concentrationof 6.92 3 1024 M produced a 50% lysis of human RBCs, andan optimal lysis of 80% was obtained with 1 3 1023.1 MSmDLP, both determined as relative to 100% lysis in distilledwater (Fig. 7A). Higher concentrations of SmDLP could not betested because of its insolubility. Because the E/S products ofthe schistosome parasite T. ocellata, which is not compatiblewith the human host and has a duck as definitive host, alsoshowed an immunopositive reaction in immunodotting (Fig.4B) we also tested the effect of SmDLP on nucleated duckRBCs. SmDLP appeared to lyse duck RBCs. Although a similar

dose-response curve was observed as compared to humanRBCs (optimal lysis at a concentration of 5 3 103 M), the dataobtained with duck RBCs showed a larger standard error of themean (Fig. 7B).

To test the effect of SmDLP on levels of NO produced bymonocytic cells (RAW 264.7), we used LPS as a NO stimulatorand considered suppressive effects below the level of LPS stim-ulation. The direct effect of SmDLP on the NO concentrationwas examined and compared with appropriate controls, includ-ing RBCs. Monocytic cells (stimulated with LPS) were incu-bated with either SmDLP or human RBCs for 18 hr at 37 C.The effects on NO production by RAW 264.7 cells can be seenin Figure 8A. These results indicate that NO production is stim-ulated at optimal levels by the addition of LPS, but to a lowerextent as compared to the presence of RBCs. Surprisingly,SmDLP also had a direct inhibiting effect on the amount of NOproduced by LPS-stimulated RAW 264.7 cells (reducing theamount of NO produced by half). Similarly, addition of RBCsalone was also observed to lower the amount of NO producedupon LPS stimulation to almost 0. In combination, SmDLP andRBCs (incubated with LPS-stimulated RAW 264.7 cells) werealso observed to reduce the levels of NO in the supernatant.

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FIGURE 5. Micrographs of histological sections immunocytochemically stained with antidermaseptin and counterstained with hematoxylin andeosin. Bar 5 20 mm. (A) Mouse ear skin after being exposed for 2 hr to cercariae of Schistosoma mansoni, showing the strongly immunopositiveschistosomula. h, hair follicle; sc, stratum corneum of the epidermis. (B) Immunopositive tegument (arrow) at the edge of the flattened body wallof an adult male in close association with erythrocytes (e) or their membrane remnants (ghosts). (C) Immunopositive gonad (testes) in male worm.(D) Immunopositive gonad (oocytes in ovary) in a female worm. The vitelline cells (v) and the vitelline glands are negative. (E) Liver of an 8-wk-infected mouse showing an immunopositive egg. (F) Liver section stained for CD11c (clone N418; control, not counterstained). The egg isimmunonegative, whereas the infiltrating cells (dendritic cells and granulocytes) at the periphery of the granuloma are immunopositive with thisantiserum.

Although the data of only 1 experiment are presented in Figure8A, the same tendency was found in experiments performedunder slightly different conditions and, therefore, a direct com-parison was not possible. The effects of simultaneously addedRBCs and LPS on the release of NO from monocytes confirmsearlier data (Carr and Morrison, 1984) that LPS could not stim-ulate RAW 264.7 cells in the presence of RBCs because LPSbinds to erythrocyte membrane.

For that reason, RBCs were preincubated with SmDLP (or

distilled water as a control) and after removing the erythrocytemembranes by centrifugation the supernatant was added to themonocytes. Combinations of intact RBCs, SmDLP, and LPSserved as controls (Fig. 8B). The results obtained show that thehigh NO levels were measured in the supernatant of the LPS-stimulated RAW 264.7 cells. In contrast, when RBCs were pre-incubated with SmDLP and the resulting supernatant was addedtogether with LPS to RAW 264.7 cells, NO levels were almostzero (P , 0.01); the same was found in the control reaction (P

QUINN ET AL.—S. MANSONI DERMASEPTIN-LIKE PEPTIDE 1347

FIGURE 6. MALDI-ToF/MS of synthetic SmDLP showing a clearpeak for SmDLP at 2789 m/z [M 1 H]1; other peaks represent thecrystallization matrix (CHCA). If there were any other impurities insSmDLP above a relative abundance of 10, then the sample would havebeen repurified.

FIGURE 8. (A) Effect of a combination of sSmDLP (1 3 1023 M)and RBCs (5 3 107) on NO levels produced by lipopolysaccharide(LPS)-stimulated RAW 264.7 cells. The numerical average of 3 read-ings is presented. (B) Effects on NO production by LPS-stimulatedRAW 264.7 cells by a combination of the supernatant of intact RBCs(I RBC) and of RBCs that have been lysed by adding 1 3 1023 MSmDLP (LRBC). Values are means 6 the standard error of the mean(SEM) for 3 readings in the experiment.

FIGURE 7. (A) Synthetic SmDLP-induced hemolysis of human RBCs. The graph illustrates increasing hemolysis as detected by a change inabsorbance (at 405 nm) against the log10 concentration of SmDLP. An optimal hemolysis of 80% of the RBCs was obtained at a concentrationof 1 3 1023.1 M SmDLP with an EC 50 value of 6.92 3 1024 M, using a scale in which RBCs in water represent 100% hemolysis and those inPBS represent 0% hemolysis. Values are means 6 the standard error of the mean (SEM) of 3 readings. PBS average OD 5 0.28835, standarddeviation (SD) 5 0.0083; water average OD 5 2.92, SD 5 0.0311. (B) Effects of synthetic SmDLP on duck RBCs. SmDLP is observed to belytic to the RBC at 5 3 1023 M, using a scale in which RBCs in water represents 100% hemolysis and those in PBS represent 0% hemolysis.Values presented are means 6 SEM for 3 independent experiments.

, 0.01). The difference in using lysed RBCs as opposed tousing intact RBCs together with SmDLP and LPS was alsosignificant (P , 0.01). These results showed that SmDLP hadboth a direct and an indirect effect on the amount of NO pro-duced by the monocytic cell line.

Antimicrobial activity: Many of the dermaseptin peptideshave antimicrobial activity and well-documented effects on pro-karyotic and/or eukaryotic membranes (Mor et al., 1991). Todetermine whether SmDLP had also effects on prokaryotic and/or eukaryotic membranes we tested for microbiacidal activityagainst 3 prokaryotes M. luteus, S. aureus, and E. coli, and 1eukaryote, Candida albicans, all in exponential growth phase.The result of these experiments can be seen in Table II. SmDLPshowed a limited spectrum of antimicrobial activity against M.luteus and C. albicans. In contrast, the peptide had no visibleeffect on E. coli and S. aureus. Comparisons with standard an-

tibiotics revealed that SmDLP was 2 to 5 times less efficientthan conventional antibiotics having a minimum inhibitory con-centration (MIC) of 74.6nM against M. luteus and C. albicans.Within 1 dilution of SmDLP, a sharp response in the MIC wasobserved against M. luteus and C. albicans, presumably indi-cating a critical peptide threshold concentration. A proportion-ately higher concentration of SmDLP was necessary to observethe minimum bactericidal concentration (MBC). These dataconfirm the antimicrobial activities of SmDLP as predicted ear-lier by analyzing the hydrophobicity of this peptide.

DISCUSSION

The most significant finding presented here was the discoveryof a small peptide in the peptidome of S. mansoni cercariae andE/S products released during their transformation, specificallyS. mansoni DLP. The origin and dispersal of SmDLP in theprimary skin penetration stages was established using immu-nocytochemistry. The antiserum used in these studies was

1348 THE JOURNAL OF PARASITOLOGY, VOL. 91, NO. 6, DECEMBER 2005

TABLE II. In vitro effects synthetic SmDLP and control antibiotics onthe inhibition of microbial growth of gram-positive bacteria Staphylo-coccus aureus, Micrococcus luteus), gram-negative bacteria (Escherich-ia coli) and yeast (Candida albicans). Values presented are means ob-tained in 3 independent experiments. Abbreviation: N, no discernableeffects of the peptide.

M. luteus S. aureus E. coli C. albicans

MIC*Tetracycline 29 nM 59 nMAmpicillin 70 nMSmDLP 74.6 nM N N 74.6 nMAlcohol 17.5%

MBC†Tetracycline 234 nM 468 nMAmpicillin 140 nMSmDLP 1.18pM N N 74.6 nM

* The minimum inhibitory concentration (MIC) was defined as the lowest con-centration at which no bacteria were optically visible after 6 hr at 37 C.

† The minimum bactericidal concentration (MBC) determined as the lowest con-centration of peptide at which no visible colonies were seen after subculturingfrom the test wells onto nutrient agar/yeast extract agar and incubating overnightat 37 C.

raised to a designed consensus preproregion of the dermaseptins(Lacombe et al., 2000). This was necessitated because of dif-ficulties in raising an antibody to the mature region of derma-septins, SmDLP included, and also because of the lack of in-formation on the DNA sequence of the immature proSmDLP.Based on the sequence homologies to frog dermaseptins, weassumed that SmDLP would have a (pre-) propeptide form andwould eventually be cleaved to form the mature peptide. Ap-parently, the antidermaseptin antibody recognizes the maturepeptide SmDLP, because both the original extract and, moreconclusively, the synthetic variety were immunopositive.

The results indicated that antidermaseptin also recognizedSmDLP in histological sections of cercariae and schistosomula,and in adult worms and their eggs, showing the origin and lo-calization of SmDLP in S. mansoni. This confirmed the dataobtained by immunodotting. The fact that the postacetabularglands of cercariae showed very weak immunopositivity,whereas the schistosomula in the host’s skin were highly posi-tive, can be rationalized if we assume that the antiserum onlyrecognizes the mature form of SmDLP, which may be pro-cessed/released during skin penetration. We suppose thatSmDLP is released into the skin because it was clearly foundin the E/S products secreted by transforming cercariae.

Comparative bioinformatic analyses of primary structure mo-tifs were used to predict putative bioactivities of SmDLP, i.e.,the hemolytic and antimicrobial effects demonstrated here.Analysis of this sequence inferred that SmDLP would attach tomost membranes with an overall negative electrochemicalmembrane potential. The C-terminal region of SmDLP matchesa super-conserved region of dermaseptins, which is responsiblefor their binding capacity (Kustanovich et al., 2002; Lequin etal., 2003). It was predicted from an examination of the func-tional motifs in previous research on dermaseptins that theunique nature of the peptide’s N-terminal region and the overallcharge of 0 would cause SmDLP to be a weak antibiotic. Thisis concluded because the net positive charge in dermaseptins is

responsible for their lytic potential in membranes (Kustanovichet al., 2002).

These predictions were confirmed by the liquid inhibitionassay, which indicated that SmDLP was similar to other der-maseptins in its spectrum of activity but was a less potent an-timicrobial peptide (Caulfield and Cianci, 1985; Ghosh et al.,1997; Batista et al., 1999).

The hemolytic activity of the peptide was likewise confirmedin vitro using human RBCs. Similar effects were detected withnucleated duck erythrocytes, although the data obtained showeda larger standard error. This confirms our observation that afraction in the E/S products of T. ocellata was immunopositiveto antidermaseptin (data not shown). It may indicate that otherschistosomes also release a dermaseptin-like molecule, whichin the case of T. ocellata may be better adapted to the mem-brane of nucleated duck erythrocytes.

To analyze the hemolytic capacity of SmDLP in relationshipto its amino acid sequence, we compared the sequence motifsof other dermaseptins, which had already been investigated fortheir hemolytic capacity. A new observation, which was alsoput forward in this analysis, is that the hemolytic capacity ofsome dermaseptins can be related to common primary sequencemotifs. It is quite striking that dermaseptins that have a GEQ/NEQ tripeptide motif on their C-terminal end do not have anyreported hemolytic activity, and that those peptides that do nothave this motif, such as SmDLP and dermaseptin S4 (Charpen-tier et al., 1998), have a hemolytic effect. Binding studies per-formed on other dermaseptins suggest that SmDLP may bindto RBCs because of its C-terminal hydrophobicity, causing adepolarization effect, which in turn promotes RBC lysis. A crit-ical mass of peptide may also affect the orientation of nativeproteins in the RBCs, causing the formation of pores, compa-rable to electroporation. This is hypothesized from observationsby other researchers that, first, depolarization of the RBC mem-brane causes an almost instantaneous (,1 msec) formation ofpores (Kinosita and Tsong, 1977). Second, various proteins inthe erythrocyte membrane can adopt different conformations inthe membrane as a response to the changing electrochemicalpotential. This is thought to assist in the formation and expan-sion of the pores in the membrane (Kinosita and Tsong, 1977).It is known that mature RBCs have a membrane potential thatdiffers from that of other somatic cells and that, in commonwith prokaryotic organisms, they are susceptible to lysis byamphipathic a-helical peptides (Dathe and Wieprecht, 1999).This means that the lytic capacity of SmDLP may be confinedto a narrow spectrum of membranes. This is supported by ourobservation that SmDLP did not affect the viability of RAW264.7 cells when it was added to the medium in which theywere cultured.

An important question is why this peptide released by S.mansoni has hemolytic activity. Our immunocytochemical datashowed that the SmDLP is intimately associated with schisto-somula in the skin. This can be explained by the affinity of theouter surface of the schistosome for amphipathic molecules(Wilson, 1987). It corresponds with the observation from pre-vious researchers that schistosomula are usually covered by alayer of lysed RBCs as soon as they reach blood vessels (Golanet al., 1986; Shai, 1999). The same holds true for adult wormsresiding in the mesenterial blood vessels of the host’s intestine.They even ingest and live on RBCs (Zussman et al., 1970).

QUINN ET AL.—S. MANSONI DERMASEPTIN-LIKE PEPTIDE 1349

FIGURE 9. Phylogenetic tree construction from alignments of proteinsequences (taken from a Blast search in NCBI) to SmDLP. The lettersrepresent the identification codes for the sequences as found in theNCBI and the numbers in brackets represent the relative homologies ofthe peptides/proteins.

Surprisingly, the tegument at the edges of the body wall offlattened male worms also showed immunopositivity with an-tidermaseptin. The fact that this area was often seen in closerelationship with RBCs (mainly their membrane remnants, orghosts) suggests that they are lysed here by SmDLP. This lysismay serve as protection of the female, which is enveloped bythe male, against NO. The same might be true for SmDLP,which has been immunologically demonstrated in both eggs andSEA. Because the eggs in the liver became gradually immu-nonegative for SmDLP during granuloma formation, this sug-gests that SmDLP is released from the eggs. This is supportedby the observation that SmDLP could also be found in the EST.The release of SmDLP supposedly protects the embryos devel-oping within the eggshell against NO produced by numerouscell types such as macrophages in the granulomas (Pearce andSher, 1987).

Other forms of dermaseptin that have been observed to behemolytic to RBCs are dermaseptin S4 (Krugliak et al., 2001,Navon-Venezia et al., 2002, Kustanovich et al., 2002), DS 01,and dermadistinctins (Batista et al., 1999). Surprisingly, der-maseptin S4 derivatives, which have an antimicrobial profilesimilar to that of SmDLP, have also been shown to preferen-tially lyse RBCs infected with Plasmodium falciparum. This isthought to be because of the alteration of the erythrocyte mem-brane’s protein/lipid composition, but has also been ascribed tothe new permeability pathways (NPP) that appear as the resultof infection by the parasite (Krugliak et al., 2001).

As mentioned above, hemolysis in the close vicinity of theparasite could be quite advantageous for the parasite because itis known from previous research that NO plays an importantrole in parasite elimination through macrophages, natural killer(NK) cells, platelets, endothelial cells, and eosinophils (Josephet al., 1983; Pearce and Sher, 1987; Oswald et al., 1994; As-cenzi et al., 2002). To study this possibility, various combina-tions of RBCs, LPS, and SmDLP were used. SmDLP appearedto have both a direct and an indirect effect on the levels of NOproduced by RAW 264.7 cells. The indirect effect was basedon the capacity of SmDLP to lyse RBCs. However, the NOreducing effect of oxyhemoglobin on monocytes was only ob-served when erythrocyte membranes and membrane fragmentswere removed by centrifugation. This is apparently caused bythe binding of LPS to erythrocyte membranes (Coulson et al.,1998). Because the relatively small SmDLP peptide is not re-moved by centrifugation and will remain in the fluid, it was notclear whether the low NO levels (in the presence of the super-natant of RBCs preincubated with SmDLP) resulted only froman indirect effect of the peptide.

Other researchers state that the absence of NO cannot beattributed to the scavenging effects of RBCs alone (James etal., 1998). This seems in line with our observation that SmDLPalso affects the RAW 264.7 cells in a direct manner, i.e., bylowering the amount of NO produced by RAW cells in theabsence of RBCs. The mechanism of this effect has not beenelucidated in this instance, but it has been demonstrated byother researchers that similar a-helical peptides, such as citropinand lesurin, can attach to or penetrate the monocyte membraneand down-regulate the inducible nitric oxide synthase (iNOS)system (Doyle et al., 2002, 2003). Peptides containing a C-terminal motif similar to that of SmDLP, such as the mastopor-an–galanin hybrid transportan, have been shown to traverse so-

matic cell membranes and associate with nuclear material (Poo-ga et al., 1998). This corresponds with the observations andconclusion of Ramaswamy et al. (1997). They found that whennaive mice were exposed to live cercariae, the iNOS systemappeared to be down-regulated during penetration. Because thiswas not the case when irradiated cercariae were used, the au-thors concluded that the E/S products of live S. mansoni cer-cariae must contain a factor that down-regulates the NO syn-thesis machinery. SmDLP might be this factor. Therefore, wepostulate from the evidence presented here that S. mansonicould negate the effects of NO in the host skin by releasingSmDLP, which causes the lysis of RBCs in the immediate vi-cinity of the parasite and hence lowers NO levels indirectly ordirectly, thereby affecting the iNOS system of monocytes.

In earlier experiments, several attempts were made to dis-cover the DNA sequence that coded for SmDLP by using de-generate primers designed to the N-terminal end of the peptide,with and without a hypothesized propeptide and the C-terminalsegment of the peptide. Because of the degenerate nature of theprimers, we were unable to locate this gene and, in this case,the possible number of combinations of primers was thoughttoo prohibitive for further analysis. This problem of obtainingthe DNA sequence from small peptides has also been encoun-tered by Andersson et al. (2003), who attempted to obtain thesequence of the Cecropin P1 encoding gene. Thirteen yearspassed before a homologous sequence was encountered in anEST clone (GenBank).

Until very recently, the presence of dermaseptins was exclu-sively confined to Neotropical leaf frogs. However, sequenceshomologous to dermaseptins have now also been identified inseveral other organisms such as bacteria, bacteriophages, in-sects, and nonparasitic worms (see Fig. 9). To answer the ques-tion of how schistosomes obtained the DLP-encoding gene, wehave assembled a phylogenetic diagram illustrating sequencehomologies based on a Blast search using the sequence ofSmDLP. The data illustrate that SmDLP has a very close rela-tionship to dermaseptins from frogs and appears to be verysimilar to a cluster of frog dermaseptins. This suggests thatschistosomes could have acquired the sequence of the gene di-rectly from an ancestoral frog host, indicating that dermaseptinis not only of survival value to frogs in terms of antimicrobial

1350 THE JOURNAL OF PARASITOLOGY, VOL. 91, NO. 6, DECEMBER 2005

activity, but also apparently to schistosomes, in the latter casenot only as an antimicrobial factor, but possibly more impor-tantly as a potential cell penetration peptide. It may be that thetransfer or capture of host DNA could be facilitated by hori-zontal gene transmission via an ancestral virus. Host gene cap-ture by parasites is not a new phenomenon. Evidence for thisis emerging from gene sequencing of retrotransposons in S.mansoni (Copeland et al., 2003), which is evidence of intimateretroviral activity. These viruses have the ability to enter thehost genome as endogenous proviruses (Imase et al., 2003) andto transfer parts of the genome to schistosomes.

ACKNOWLEDGMENTS

The authors would like to thank A. J. Bjourson for his support andadvice during our research, Marion Bergamin-Sassen and Carry Moor-er-van Delft for technical help, Daniel Berrar and Anton Pieneman fortheir help in preparing the figures of this manuscript, Claire Lacombe(Laboratoire de Bioactivation des Peptides, Institute Jacques Monod,CNRS- Universites Paris 6-Paris 7, France) for the antidermaseptin an-tiserum, Maria Yazdanbaksh (RU Leiden, The Netherlands) for provid-ing the SEA, and Peter Ashton (York, U.K.) for the EST of S. mansoni.

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