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J. Biol. Chem., Vol. 277, Issue 25, 22353-22360, June 21, 2002

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Pore-forming Polypeptides of the Pathogenic Protozoon Naegleria fowleri^*

Rosa Herbst^§, Claudia Ott^¶, Thomas Jacobs, Thomas Marti, Francine Marciano-Cabral^**, and Matthias Leippe^§

From the  Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany, the ^§ Molecular Parasitology Group, Research Center for Infectious Diseases, Röntgenring 11, 97070 Würzburg, Germany, and the ^** Department of Microbiology and Immunology, Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia 23298

Received for publication, February 13, 2002, and in revised form, April 9, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The free-living amoeboflagellate and potential human pathogen Naegleria fowleri causes the often fatal disease primary amoebic meningoencephalitis. The molecular repertoire responsible for the cytolytic and tissue-destructive activity of this amoeboid protozoon is largely unknown. We isolated two pore-forming polypeptides from extracts of highly virulent trophozoites of N. fowleri by measuring their membrane-permeabilizing activity. N-terminal sequencing and subsequent molecular cloning yielded the complete primary structures and revealed that the two polypeptides are isoforms. Both polypeptides share similar structural properties with antimicrobial and cytolytic polypeptides of the protozoon Entamoeba histolytica (amoebapores) and of cytotoxic natural killer (NK) and T cells of human (granulysin) and pig (NK-lysin), all characterized by a structure of amphipathic -helices and an invariant framework of cysteine residues involved in disulfide bonds. In contrast to the aforementioned proteins, the Naegleria polypeptides both are processed from large precursor molecules containing additional isoforms of substantial sequence divergence. Moreover, biochemical characterization of the isolated polypeptides in combination with mass determination showed that they are N-glycosylated and variably processed at the C terminus. The biological activity of the purified polypeptides of Naegleria was examined toward human cells and bacteria, and it was found that these factors, named naegleriapores, are active against both types of target cells, which is in good agreement with their proposed biological role as a broad-spectrum effector molecule.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The free-living, bacteria-feeding amoeboflagellate Naegleria fowleri is distributed worldwide in soil and warm pools of water. The term amoeboflagellate is used to describe amoebae that can transform into flagellates. N. fowleri is a facultative parasite capable of carrying out its full life-cycle without the intervention of a parasitic stage (1). Portals of entry for human infection are the nasal passages and olfactory neuroepithelium during swimming and bathing in hot baths or hot springs. The invasive stage of N. fowleri is primarily the amoebic form. After traveling along the olfactory nerves to the brain they invade the olfactory bulbs and then spread to the more posterior regions of the brain (2, 3). Within the brain they provoke inflammation and cause extensive destruction of tissue (4). This rapidly fatal infection, known as "primary amoebic meningoencephalitis" usually results in death within 72 h after the onset of symptoms.

The molecular mechanism of invasion and pathogenesis of primary amoebic meningoencephalitis are still unclear. In vitro, intact amoebae (5) as well as cell-free extracts thereof (6) exert remarkable cytolytic and tissue-destructive activity. Among several factors to which pathogenicity has been attributed such as phospholipases (7), undefined hemolytic factors (6, 8-10), and a secreted cysteine proteinase (11), the most attractive candidate for mediating the enormous tissue-destructive capacity is a protein that has been described more than a decade ago (12) to form pores in target cell membranes. It was found that the pore-forming activity was membrane-bound, however, the responsible protein was never characterized at the molecular level. This study was directed at isolating the pore-forming protein of N. fowleri, subsequently identifying the gene that is coding for this factor and determining its primary structure. We purified two polypeptides that display potent pore-forming activity and that were found after their biochemical and molecular characterization to be glycoproteins each contained in a multipeptide precursor structure. These polypeptides were found to kill prokaryotic as well as eukaryotic target cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Amoebae-- A highly virulent strain of N. fowleri (ATCC 30894), LEEmp, was used in this investigation. Samples of brain tissue from infected mice containing amoebae were cultured at 37 °C in Oxoid medium supplemented with serum and hemin to obtain amoebae. Amoebae were cultured axenically for 4 days prior to use (13).

Protein Purification-- Freshly harvested and washed amoebae were extracted overnight with 5 volumes of 10% acetic acid. The extract was centrifuged at 150,000 × g at 4 °C for 1 h, and the resulting supernatant was passed through a C₁₈ 12-cc (2 g) Sep-Pak cartridge (Waters). The cartridge was washed with 0.1% trifluoroacetic acid (TFA),¹ and adsorbed material was stepwise eluted with 30-100% acetonitrile, 0.1% TFA (10 ml for each 10%-step). Proteins eluted with 50% acetonitrile, 0.1% TFA were lyophilized, resuspended in 50 mM sodium acetate, pH 4.5, and loaded onto a Mono S HR5/5 cation exchange column (Amersham Biosciences, Inc.) equilibrated with the same buffer. Adsorbed material was eluted by washing the column with 50 mM sodium acetate, pH 4.5 (5 ml), and by the use of a 0-500 mM NaCl gradient (25 ml). Pore-forming material representing naegleriapore A was eluted with 260 mM NaCl. After lyophilization of the active fractions, proteins were redissolved in 0.1% TFA and subsequently subjected to reversed-phase HPLC using a C3 PRP-3 column (Hamilton) connected to a 130A separation system (Applied Biosystems) and equilibrated with 0.1% TFA. The column was washed with 0.1% TFA (3 min), and peptides were eluted with a linear gradient of 0-45% acetonitrile, 0.1% TFA for 45 min and at a flow rate of 0.2 ml/min. Naegleriapore A was eluted with 48% acetonitrile from the column. Proteins eluted with 60-70% acetonitrile, 0.1% TFA from the initial Sep-Pak cartridge were directly applied to reversed-phase HPLC as described above. Naegleriapore B was eluted with 52% acetonitrile from the HPLC column.

Protein Analysis and N-terminal Sequencing-- Tricine-SDS-PAGE was performed according to Schägger and von Jagow (14). For N-terminal sequencing, naegleriapores purified by reverse-phase HPLC were analyzed using a gas-phase protein sequencer (model 437 A, Applied Biosystems). The method applied did not allow the detection of cysteine residues. The concentration of purified peptides was determined by measuring absorbance at 214 nm; the extinction coefficients were calculated using the respective sequence information (15).

Molecular Cloning-- Specific DNA fragments for each isoform were amplified from a cDNA library of N. fowleri using degenerate sense and antisense oligonucleotides deduced from the respective N-terminal amino acid sequence of naegleriapore A (sense: 5'-GATGC(A/T)GAATGTGAAATTG-3'; antisense: 5'-C(T/C)TTTTG(A/C)ATTTC(A/T)GCTTG-3') and naegleriapore B (sense: 5'-GG(A/T)TGTGAAATTTGTGAATGG-3'; antisense: 5'-CAAAC(A/C)AT(A/T)GCTGGTGGTTC-3'). The ZAP cDNA library from N. fowleri was constructed according to the protocol of the manufacturer (Stratagene), and its screening with the radiolabeled probes was performed as described previously (16). Hybridizing phages were isolated, the plasmids were released according to the instructions of the manufacturer (Stratagene), and the nucleotide sequences were determined from both strands. For primer extension experiments, the oligonucleotides 5'-CAGTGTTATTCTTTGGGCAC-3' for naegleriapore A and 5'-CAATTTGTGGCTTGGTC-3' for naegleriapore B were used. The nucleotide sequence of the extension product was determined according to the anchored PCR technique (17). Genomic fragments were amplified from genomic DNA of N. fowleri using oligonucleotide primers according to the 5'- and 3'-ends of the coding cDNA sequences and sequenced on both strands.

Southern and Northern Blot Analysis-- Southern and Northern blotting were performed according to published procedures (18); hybridization and washing conditions were described previously (19).

Deglycosylation and Glycan Detection of Naegleriapores-- Peptides (1 µg) were deglycosylated with 10 units of N-glycosidase F (Boehringer, Mannheim) at 37 °C overnight according to the manufacturer's instruction. On Tricine-SDS-PAGE, the mobility of deglycosylated peptides was higher than that of native peptides. After blotting onto the nitrocellulose membrane, the deglycosylated and native peptides were analyzed using a glycan detection assay according to the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim).

Mass Spectrometry-- After deglycosylation samples were dialyzed against 5% acetic acid, vacuum-dried, solubilized in 0.1% TFA, and mixed with the same volume of saturated trans-sinapinic acid in acetonitrile, 0.1% TFA as UV-absorbing matrix. About 10 µl of the samples was spotted onto a stainless steel probe tip and dried at room temperature. Measurements were performed using a Bruker Relex MALDI-TOF. Ions were formed by laser desorption at 337 nm using an N₂ laser. Spectra were recorded with an acceleration voltage of 35 kV in the linear mode. The mass spectrometer was calibrated with bovine serum albumin (BSA) (Sigma) and carbonic anhydrase from bovine erythrocytes (Sigma Chemical Co.) as external standards.

Antibody Generation and Western Blotting-- Antibodies against naegleriapore A and B were raised in chickens by injecting purified proteins subcutaneously. Antibodies were purified from egg yolk according to the water dilution method (20). Immunoblotting and development of the blots were carried out as described (21).

Indirect Immunofluorescence-- Amoebae were fixed with 3% paraformaldehyde in 0.2 M sodium cacodylate, pH 7.2, and rendered permeable by 0.05% saponin (Sigma) in 4.5 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 147 mM NaCl, 2.5 mM KCl, pH 7.4, 320 mosmol kg¹ (PBS-A). After blocking with 2% fetal calf serum in PBS-A, the cells were incubated at 20 °C for 30 min with anti-naegleriapore A or anti-naegleriapore B. As a control, amoebae were also incubated with the respective preimmune antibodies. After several washes with PBS-A, the amoebae were incubated with dichlorotriazinylaminofluorescein (DTAF)-conjugated anti-chicken IgY from rabbit (Dianova) at 20 °C for 30 min in the dark and washed carefully with PBS-A. Nuclei were stained with propidium iodide (5 µg/ml), and samples were analyzed by confocal microscopy with the Leica TCS NT confocal laser scanning system in combination with a Leica DMR microscope.

Other Peptides-- Amoebapore A was purified from Entamoeba histolytica HM-1:IMSS as described previously (22). Magainin I and cecropin A and B were purchased from Sigma.

Pore-forming Activity Assay-- Pore-forming activity of samples was determined by measuring fluorometrically the dissipation of a valinomycin-induced membrane potential in liposomes (23). Fluorescence was measured by a fluorescence spectrophotometer (model LS 50B, PerkinElmer Life Sciences) using excitation and emission wavelengths of 620 and 670 nm, respectively. Pore-forming activity was measured as the initial change in fluorescence intensity over time after adding the sample. One unit of activity was defined as a fluorescence increase to 5% of the pre-valinomycin intensity in 1 min at 25 °C.

Antibacterial Assay-- The bacterial strains used were Bacillus subtilis (strain 60015) (22) and Escherichia coli K-12 D31 (24). Bacteria were grown in Luria-Bertani (LB) medium and subsequently inoculated in LB medium for growth to mid-logarithmic phase. After centrifugation, bacteria were washed twice with and resuspended in 20 mM MES, pH 5.5, containing 25 mM NaCl. A 96-well microtiterplate (Greiner) was precoated with 0.1% BSA for 15 min prior its use in the assay. Peptides in 0.01% TFA were serially diluted 2-fold in 20 mM MES, pH 5.5, 25 mM NaCl. 1 × 10⁵ bacteria (25 µl) were incubated with the diluted peptides (25 µl), and 2 µM of the fluorescent dye Sytox green (50 µl, in 20 mM MES, pH 5.5, 25 mM NaCl) (Molecular Probes) at 37 °C for 1 h. Perturbation of the bacterial cytoplasmic membrane allows the dye to cross this membrane and to intercalate with the DNA. When excited at 495 nm the binding of the dye to DNA resulted in an increase of emitted fluorescence at 538 nm, which was measured in a fluoroscan II microtiterplate reader (Labsystems). Antibacterial activity of the peptides was expressed as the percentage of permeabilized bacteria. For maximum permeabilization of the bacteria (100% value), cells were incubated with 70% ethanol for 5 min.

Cytotoxicity Assay-- Jurkat T cells were maintained at a density of 1-6 × 10⁵ per ml in suspension cultures with RPMI 1640 medium (Invitrogen) supplemented with 10% inactivated fetal bovine serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin sulfate (0.1 mg/ml). Cells were washed with and suspended in 20 mM MES, pH 5.5, 150 mM NaCl before used in the assay.

A 96-well microtiterplate was precoated with 0.1% BSA before use. Peptides in 0.01% TFA were 2-fold serial diluted in 20 mM MES, pH 5.5, 150 mM NaCl. The diluted peptides (50 µl) were then incubated with 5 × 10⁴ Jurkat cells in 20 mM MES, pH 5.5, 150 mM NaCl, including 20% Alamar Blue (50 µl) (Alamar Bioscience, Sacramento, CA) at 37 °C under 5% CO₂ for 1 h. For maximum lysis (100% value) Jurkat cells were incubated with Alamar Blue in 1% Triton X-100 in 20 mM MES, pH 5.5, 150 mM NaCl and for minimum lysis (0% value) in 20 mM MES, pH 5.5, 150 mM NaCl. Viable cells metabolized the dye Alamar Blue resulting in an increase of fluorescence by excitation/emission at 538/590 nm. Cytotoxic activity was calculated as follows: % cytotoxicity = 100 × (0% value  experimental value/0% value  100% value).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Purification-- We purified two polypeptides with pore-forming activity from acid extracts of N. fowleri by a combination of hydrophobic interaction and cation exchange chromatography. Final purification was achieved by reverse-phase HPLC. The purification yielded apparently homogeneous material with molecular masses of 10 and 13 kDa as judged by SDS-PAGE (Fig. 1). The purified polypeptides named naegleriapore A and B, respectively, were subjected to N-terminal sequencing, resulting in a single amino acid sequence up to residue 34 (DAEXEIXKFVIQQVEAFIESXHSQAEIQKELNKL) for naegleriapore A and up to residue 77 (SVIGXEIXEWLVATAEGFVXKTKPQIEQELLQICAKLGPYEQIXDQLVLMELPDIIDQIIAKEPPAIVXSQVKIXXG) for naegleriapore B confirming the purity of the peptides.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Different steps of purification of pore-forming peptides of N. fowleri. A, proteins eluted with 50% acetonitrile, 0.1% TFA from a Sep-Pak cartridge were subjected to Mono S cation exchange chromatography. The dotted line indicates the NaCl gradient; fractions containing proteins exerting pore-forming activity are marked with a bar. The inset shows the elution profile of this protein supplied to reversed-phase HPLC. The dotted line here represents the acetonitrile gradient, and the arrow marks the peak representing naegleriapore A (I). B, proteins eluted with 60-70% acetonitrile from the Sep-Pak cartridge were directly supplied to reversed-phase HPLC. The dotted line represents the acetonitrile gradient, and the arrow marks the pore-forming peptide naegleriapore B (II). C, naegleriapore A (I) and B (II) (1 µg each) were separated on a Tricine/SDS-gel followed by silver staining. Molecular masses are indicated at the right.

Molecular Characterization-- Using degenerate primers, specific DNA fragments of 90 and 197 bp were amplified representing naegleriapore A and B, respectively. Screening of a cDNA library of N. fowleri and subsequent primer extension experiments resulted in the complete cDNA sequences. The cDNAs for naegleriapores A and B contain a short 5'-untranslated region of 37 and 36 bp, an open-reading frame of 921 and 1452 bp, followed by a short 3'-untranslated region of 31 and 24 bp, respectively. Northern blot analyses confirmed a single transcript for naegleriapore A and B of 1.0 and 1.5 kb, respectively, which corresponded to the estimated size of the complete precursor mRNA of each isoform, and Southern blot analyses indicated that each naegleriapore precursor is encoded by a single copy gene (Fig. 2). The size of the cDNAs indicated that the pore-forming polypeptides derive from substantially larger precursor molecules. Deduction of amino acid sequences of 308- and 486-amino acid residues revealed that both precursor proteins start with a sequence, which has the characteristics of a typical signal peptide. The putative signal peptide is followed by several elements, which all are characterized by a virtually invariant motif of six cysteine residues within their primary structures and which are connected by short intervening sequences without cysteine residues. This cysteine residue motif is known from other membrane-active polypeptides defined as the protein family of saposin-like proteins (SAPLIPs) (25). The SAPLIP motif is found three times in the precursor of naegleriapore A and five times in that of naegleriapore B. The isolated pore-forming peptides naegleriapore A and naegleriapore B represent the second and the first SAPLIP element within their corresponding precursor, respectively (Fig. 3). Given that each SAPLIP element starts with four residues preceding the first cysteine residue and ends with the sixth cysteine residue of the motif, a sequence identity of 19 to 31% was observed between the SAPLIP domains.

View larger version (76K): [in this window] [in a new window]   Fig. 2.   Northern and Southern analysis of naegleriapore A and B. A, total RNA (10 µg) of N. fowleri was separated on a formaldehyde-agarose gel and hybridized with the cDNA of naegleriapore A and naegleriapore B, respectively. RNA size markers are indicated at the left. B, genomic DNA (10 µg) prepared from N. fowleri was digested with the restriction enzymes indicated and hybridized with the cDNA of naegleriapore A and B, respectively. DNA size markers are indicated in the middle.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Alignment of amino acid sequences of the SAPLIP elements of naegleriapore precursors with amoebapores, NK-lysin, and granulysin. Disulfide bonds are outlined as connecting horizontal lines between the six conserved cysteine residues (shaded in gray). The arrow indicates the site of post-translational processing at the C terminus of granulysin. N-terminal sequences determined experimentally were boxed. Shown also is a schematic presentation of the precursor molecules of naegleriapores. SAPLIP elements (gray boxes) within the precursor molecules are interrupted by spacer regions (dark gray boxes). Those elements representing the purified peptides naegleriapore A and B are the light boxes in the respective precursor molecule. Signal peptides of the precursors are shown in black, and the N-glycosylation sites are marked Y.

Sequencing of the genes coding for naegleriapore A and B, respectively, amplified from genomic DNA of N. fowleri revealed that the sequences are identical to the respective cDNA except that the genomic sequences are interrupted by introns. The gene coding for the naegleriapore A precursor contains three introns of 97, 69, and 47 bp in size, whereas the gene coding for the precursor of naegleriapore B contain a single intron of 316 bp. Three introns share the same 5'-donor splicing site (5'-GTAAGT-3') with the exception of the first intron of the naegleriapore A precursor, which is slightly modified to 5'-GTACGT-3'. At the 3'-acceptor site a consensus sequence was not identified.

Biochemical Characterization-- Putative N-glycosylation sites within the amino acid sequences of the mature pore-forming peptides and the finding that the molecular mass of purified naegleriapores observed upon SDS-PAGE exceeded the mass of one SAPLIP element prompted us to analyze whether the native peptides are glycosylated. Both peptides were indeed reactive in a glycan detection assay (Fig. 4A). After digestion with N-glycosidase F both polypeptides shifted to an apparent molecular mass of ~7 kDa and became negative in the glycan detection assay upon SDS-PAGE.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Deglycosylation and MALDI-TOF analysis of naegleriapore A and B. A, deglycosylation of naegleriapore A and B. Peptides were incubated without () or with (+) N-glycosidase F and subjected to Tricine-SDS-PAGE. After electrophoresis, the proteins were silver-stained or blotted onto nitrocellulose and subjected to a glycan detection assay. B, MALDI-TOF spectra of deglycosylated naegleriapore isoforms. The experimental molecular masses obtained for each peptide by MALDI-TOF are shown at the respective peak. C, amino acid sequences of mature naegleriapore A and B. The arrows indicate the theoretical molecular masses calculated for differently processed C termini. For the calculation it was taken into account that the cysteine residues are involved in disulfide bonds and that the methionine residues are oxidized during the experimental procedure. Asterisks mark the N-glycosylation motifs within the amino acid sequences of the polypeptides.

MALDI-TOF analysis of the deglycosylated peptides to identify the C terminus of naegleriapore A and B gave molecular masses of 8.783 and 8.871 kDa for naegleriapore A and 8.906, 8.977, 9.057, and 9.128 kDa for naegleriapore B (Fig. 4B). The multiple masses for each peptide can be explained by variable processing at the C terminus. The experimentally obtained molecular masses are in a good agreement with the calculated masses for both peptides provided that six half-cystines are present and that the methionine residues of the peptides were oxidized during the purification process as observed with other SAPLIPs (26) (Fig. 4C).

Cellular Localization of Naegleriapore A and B-- Polyclonal antibodies against the pore-forming peptides were raised in chickens and tested for their specificity by immunoblotting with purified naegleriapore A and B. Each antibody recognizes only its antigen and did not cross-react with the other naegleriapore isoform (Fig. 5). These peptide-specific antibodies were then used to localize the naegleriapore isoforms inside the amoebae. As judged by indirect immunofluorescence, both peptides had a distinctly focal distribution in the cytoplasm of the cells assuming an intracellular storage in granular vesicles (Fig. 6).

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Specificity of anti-naegleriapores A and B. Purified naegleriapore A (NP-A) and naegleriapore B (NP-B) were blotted each (1 µg) onto nitrocellulose and subsequently incubated with polyclonal antibodies against naegleriapore A (anti-NP-A) and naegleriapore B (anti-NP-B), respectively.

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Cellular localization of naegleriapore A and B using indirect immunofluorescence microscopy. Paraformaldehyde-fixed trophozoites of N. fowleri were permeabilized with saponin and incubated with the antibodies raised against naegleriapore A and B in chickens. The insets show trophozoites incubated with the respective preimmune antibodies. After incubation with DTAF-conjugated anti-chicken IgY and staining of the nuclei with propidium iodide, the cells were analyzed by confocal laser microscopy. The bar represents 5 µm.

Biological Activity-- As outlined above, naegleriapores A and B were isolated from extracts of N. fowleri by purifying the entities responsible for the pore-forming activity as determined by measuring the dissipation of a valinomycin-induced diffusion potential in liposomes. The specific pore-forming activities of naegleriapore A and B are 3.1 ± 1.7 units pmol¹ and 3.7 ± 1.3 units pmol¹, respectively. Because both peptides have been found to be glycosylated, we wanted to determine whether glycosylation is essential for activity. After treatment of purified naegleriapores with N-glycosidase F and subsequent rechromatography on reversed-phase HPLC, it became evident that naegleriapore A possesses one sugar moiety, whereas naegleriapore B has two. This is in good agreement with the potential glycosylation sites within the primary structures. Comparison of the pore-forming activity of incompletely and completely deglycosylated peptides revealed no substantial negative effect upon glycan removal: Although a slight decrease in pore-forming activity was found upon deglycosylation of naegleriapore B, the pore-forming activity of naegleriapore A increases when it was deglycosylated (Fig. 7).

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Effect of deglycosylation on pore-forming activity of naegleriapores. Peptides were incubated without () or with (+) N-glycosidase F and subsequently purified using reversed-phase HPLC. For naegleriapore B, a peptide containing one sugar moiety was separated from the completely deglycosylated peptide. In the upper panel, 1 µg of each peptide was run on a Tricine-SDS-PAGE followed by silver staining. Molecular masses of marker proteins are shown at the right. Specific pore-forming activity of equal amounts of peptides was estimated by measuring the dissipation of a valinomycin-induced diffusion potential. The lower panel shows the activities of the incompletely and completely deglycosylated peptides relative to the respective glycosylated form. Values are expressed as median and range of three measurements.

To strengthen the notion that pore-forming peptides are involved in the pathogenicity of the parasite, the cytotoxic activity of naegleriapore A and B were tested toward human target cells. Both peptides exert cytotoxic activity against Jurkat cells, but naegleriapore B was twice as active as naegleriapore A (Fig. 8A).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Activity of naegleriapores against different target cells. A, cytotoxic activity of naegleriapores. The incubation of naegleriapore A (NP-A) and naegleriapore B (NP-B) in various concentrations with Jurkat cells resulted in metabolically inactive target cells, which was measured fluorometrically using the dye Alamar Blue. The decrease of fluorescence compared with viable, metabolically active cells is expressed in percent cytotoxicity. B, antibacterial activity of naegleriapores against Gram-positive B. subtilis compared with other antibacterial peptides. Naegleriapores (NP-A, NP-B), amoebapore A (AP-A), magainin I, and the cecropins were incubated with Gram-positive bacteria in various concentrations for 1 h at 37 °C. Membrane damage of bacteria was measured fluorometrically using the dye Sytox green. The binding of the dye to the DNA in the damaged target cell resulted in an increase of fluorescence. Antibacterial activity of the peptides was compared with completely lysed bacteria and expressed as percent permeabilized bacteria.

Moreover, naegleriapore A displays antibacterial activity toward the Gram-positive bacterium B. subtilis comparable to other antibacterial peptides such as magainin I from Xenopus laevis (27) and cecropin A and B from Hyalophora cecropia (28) (Fig. 8B). In concentrations up to 10 µM, naegleriapore A was inactive against the Gram-negative representative, E. coli D31 (data not shown). Naegleriapore B did not exert any antibacterial activity in concentration up to 10 µM against the bacteria tested here.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Pore-forming activities have been found in extracts of several parasites, e.g. Trypanosoma cruzi (29), Leishmania sp. (30-32), E. histolytica (23), and N. fowleri (12). Although the proteins mediating this activity have been presented as potential pathogenicity factors of the respective parasite, only the pore-forming peptides of E. histolytica were isolated as a single active entity from crude extracts and characterized in detail at the molecular level (22, 23, 33). This is the first report on the isolation and further molecular and functional characterization of pore-forming peptides of the parasitic protozoon N. fowleri, namely naegleriapores. The primary structure of naegleriapores clearly shows that the peptides belong to the distinct protein family of SAPLIPs, the members of which exert a favorable tertiary structure characterized by amphipathic helices and by six similarly located cysteine residues forming three disulfide bonds (25, 34). Members of this protein family that also share antibacterial and cytolytic activities were found in such diverse organisms as protozoa, i.e. amoebapores, the aforementioned pore-forming peptides of E. histolytica (35-37), and Mammalia, i.e. porcine NK-lysin (38) and human granulysin (39). The SAPLIP motif is also found in nonlytic polypeptides, e.g. saposins and surfactant-associated protein B (25). In addition to the structural similarities, all members of the SAPLIP family have in common their interaction with lipids, and it is assumed that the appearance of the SAPLIP domain is an early evolutionary event from which multiple forms with different specific functions have diverged (40).

The fold of a saposin-like protein has been elucidated for porcine NK-lysin by NMR spectroscopy (34) and yielded a bundle of five -helices connected by three disulfide bonds to a very stabile tertiary structure. Recently, this structure has been found conserved in the plant-specific domain of plant aspartic proteinase as evidenced by crystal structure analysis (41). The latter has been named a swaposin, because in sequence alignments it became apparent that the N- and C-terminal parts of the saposin-like domain are swapped.

Naegleriapores are each organized within a large precursor, and the processing of the mature peptides appears to be more complicated than it is in the case of amoebapores, the primary translation products of which each consist of one SAPLIP domain and a signal domain only. Each naegleriapore precursor contains several elements of additional naegleriapore isoforms, all revealing the structural SAPLIP motif. Besides this structural similarity to members of the SAPLIP family, the overall sequence identities of all naegleriapore isoforms to amoebapores of E. histolytica, porcine NK-lysin, and human granulysin are between 15 and 30% only. It is tempting to suggest that naegleriapores originating from free-living amoebae represent the archetype of saposin-like cytolytic and antimicrobial peptides that gave rise to the mammalian effector molecules NK-lysin and granulysin. Notably, the organization of several peptides within one precursor molecule was not only found with SAPLIPs, e.g. saposins A-D and surfactant-associated protein B (42-44); with respect to antibacterial peptides, magainins from frog skin and the apidaecins from honey bees are organized in multipreproproteins as well (45-47). The organization in and the processing of multiple peptides from one large precursor molecule may be an efficient way to synthesize different effector molecules at once and thereby to amplify the antibacterial response.

Employing alternative strategies, we detected some but not all of the SAPLIPs, which theoretically should be synthesized in parallel with the naegleriapores by the amoeba.² Although they are apparently not true pore-forming peptides, these other SAPLIPs may well be additional elements of the antimicrobial arsenal of Naegleria.

Notably, naegleriapores are glycoproteins as opposed to being amoebapores, NK-lysin and granulysin. However, other SAPLIPs such as saposins and surfactant-associated protein B are glycosylated as well (25), and interestingly, the glycosylation sites are situated at the same position within their primary structure as found with naegleriapores. The deglycosylation of naegleriapores A and B has no significant negative effect on their pore-forming activity. The observed increase in activity of naegleriapore A in the liposome depolarization assay may be due to an elevated hydrophobicity of the peptide upon glycan removal. Likewise, it has been shown that the presence of the sugar moieties of saposin B (48) and saposin D (49) are not essential for activity, lipid binding, or protease resistance in vitro.

The finding that both peptides are cytotoxic to human cells strengthen the assumption that these peptides are involved in tissue destruction induced by the parasite. Recently, the crucial role of pore-forming peptides in the pathogenicity of parasitic organisms has been demonstrated in E. histolytica by transfection with amoebapore A-antisense RNA. The transformed amoebae, in which amoebapore A synthesis was substantially impaired, exerted reduced cytolytic activity and did not produce liver abscesses in the animal model (50).

Naegleriapore A displays similar activity against Gram-positive bacteria as the well-known antimicrobial peptides magainin I and cecropins but is less potent than amoebapore A. Naegleriapore A did not affect the representative Gram-negative bacteria E. coli, which may be shielded by their outer membrane. Likewise, it has been shown for amoebapores that only wall-less Gram-negative bacteria were effectively lysed by these peptides (22). Naegleriapore B does not exert any antibacterial activity here. Because only a limited number of bacteria was tested, this may indicate a divergent spectrum of target bacteria. Nonetheless, the fact that Naegleria is a facultative parasite only and feeds on bacteria suggests that the primary function of the membrane-permeabilizing polypeptides is killing of engulfed bacteria. The naegleriapores appeared to be contained in granules in which they are most likely compartmentalized together with other proteins, e.g. lysozymes, phospholipases, and proteases, that may act synergistically with the membranolytic peptides.

The pathogenicity of parasites is a complex process and is proposed to involve more than one essential factor. However, in addition to the high phagocytotic activity of the amoebae, the naegleriapores may play an important role during the lethal contact of naegleriae and host cells. The discharge of cytolytic factors upon contact with host tissues may facilitate invasion into the brain. Moreover, rapid killing of effector cells of the immune system by amoebic pore-forming peptides may explain why the human defense system is, at least in several cases, unable to prevent the invasive process. Accordingly, for the fatal conversion of a free-living, bacteria-hunting amoeba into a parasite that assaults the human body, the cytolytic armament and in particular the pore-forming peptides may play a pivotal role.

	FOOTNOTES

^* This work was supported by the Deutsche Forschungsgemeinschaft by Grant LE 1075/2-3 and by a Heisenberg fellowship (to M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF154046, AF154047, AF196308, and AF196309.

^¶ Present address: Institut für Allgemeine Botanik, University of Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany.

Present address: Megamedics, Hafenstrasse 32, 22880 Wedel, Germany.

To whom correspondence should be addressed. Tel.: 49-931-31-2151; Fax: 49-931-31-2578; E-mail: matthias.leippe@mail.uni-wuerzburg.de.

Published, JBC Papers in Press, April 10, 2002, DOI 10.1074/jbc.M201475200

² R. Herbst and M. Leippe, unpublished results.

	ABBREVIATIONS

The abbreviations used are: TFA, trifluoroacetic acid; HPLC, high performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DTAF, dichlorotriazinylaminofluorescein; MES, 4-morpholineethanesulfonic acid; SAPLIP, saposin-like protein; NK, natural killer.

	REFERENCES

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