1 Institute of Infectious Diseases and Public Health, University of Ancona; 2 Department of Biomedical Sciences and Technology, University of Udine; 3 National Laboratory CIB, Area Science Park, Padriciano 99, 34012 Trieste, Italy
Received 7 June 2002; returned 26 June 2002; revised 22 October 2002; accepted 13 January 2003
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Abstract |
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Keywords: cathelicidins, Cryptosporidium parvum, susceptibility
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Introduction |
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A variety of peptides with antimicrobial activity are found in nature.4 These molecules are active in vitro against a variety of microorganisms, and may be used as templates for the design of novel anti-infective drugs.5 The cathelicidin peptide family is a myeloid component of innate host defences in mammals.6,7 Cathelicidin peptides are heterogeneous in size and sequence and exhibit marked structural diversity, including -helical, proline-rich, tryptophan-rich and disulphide bridged peptides.6 Most of them share a net positive charge and an amphipathic character; these probably account for their ability to interact with and rapidly render permeable the membranes of target organisms.
The cathelicidin peptides have a diverse range of susceptible organisms. Helical peptides such as SMAP-29 and BMAP-28, and the two-disulphide bridged protegrin-1 (PG-1), for instance, rapidly inactivate Gram-negative and Gram-positive bacteria and fungal species in the micromolar or sub-micromolar range. Conversely, peptides with proline-rich sequences, such as Bac7 and its N-terminal derivative Bac7(1-35), have a more restricted spectrum of antibacterial activity, being effective mainly against Gram-negative species.6,8
Despite a wealth of published data on their antibacterial and antifungal activities, the effects of SMAP-29, BMAP-28, PG-1 and Bac7(1-35) on protozoan parasites have not been explored extensively. In the present study, these molecules were evaluated for their activity against the enteric protozoan parasite C. parvum. A cell culture system and double fluorogenic staining were used to investigate their anticryptosporidial activity.
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Materials and methods |
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Pooled C. parvum oocysts, isolated from stools of three patients with AIDS, were used throughout the study. Oocysts were pre-treated for 10 min in a bleach solution containing nine parts sterile deionized water and one part 5.25% sodium hypochlorite, washed twice in sterile water, centrifuged again, suspended in Dulbeccos modified Eagles medium (DMEM) (BioWhittaker Inc., Walkersville, MD, USA) and enumerated in a haemocytometer chamber. Oocyst viability was estimated using an excystation procedure and by vital dye staining.911 Excystation of sporozoites was achieved by incubating oocysts in phosphate-buffered saline (PBS, pH 7.2) containing 0.25% trypsin and 0.75% sodium taurocholate for 60 min at 37°C. Free sporozoites were isolated from excysted oocysts by passage through a polycarbonate filter (2.0 µm pore size), and counted in a haemocytometer. Sporozoite viability was confirmed by double-staining with fluorescein diacetate and propidium iodide.12 Oocysts and sporozoites were separately resuspended in PBS 0.1 mL. Following the addition of 40 µg/mL fluorescein diacetate (0.1 mL) and 20 µg/ml propidium iodide (0.15 mL) and incubation at room temperature for 5 min, the mixtures were further diluted 1:1 with PBS and analysed by flow cytometry.12 The parasite population in each sample was determined using forward and sideways scatter plots. Appropriate analysis regions for non-viable parasites were established by identifying and including 0.95% of organisms that incorporated only propidium iodide following heat-killing, and viable cells by identifying and including 0.95% of freshly harvested organisms that converted fluorescein diacetate to fluorescein when incubated with fluorescein diacetate only.
Peptide synthesis
Fmoc-L-Pro-PEG-PS and PAL-PEG-PS resins, coupling reagents for peptide synthesis, and Fmoc-amino acids were purchased from Applied Biosystems (Foster City, CA, USA). Peptide synthesis-grade N,N-dimethylformamide, N-methyl-2-pyrrolidone, dichloromethane and HPLC-grade acetonitrile were from Biosolve (Valkenswaard, The Netherlands). Trifluoroacetic acid and N-methylmorpholine were from Acros Chimica (Beerse, Belgium). BMAP-28 (27 residues), SMAP-29 (28 residues), PG-1 (18 residues) and Bac7(1-35) (35 residues) were synthesized by the solid phase method, using a Milligen 9050 synthesizer and Fmoc chemistry. PG-1, BMAP-28 and SMAP-29 were synthesized as C-terminally amidated peptides using PAL-PEG-PS resin (0.16 meq/g). Fmoc-L-Pro-PEG-PS (0.17 meq/g) was used for the synthesis of Bac7(1-35). Fmoc-protected amino acids were added in a six-fold molar excess with respect to resin substitution for most coupling steps, and coupling reactions (30 min) were carried out with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate. As several couplings were predicted to be difficult, the synthesis of BMAP-28 and SMAP-29 was carried out at 48°C by heating the jacketed column and the solvent solutions. An eight-fold molar excess of Fmoc-protected amino acid and the highly efficient acylating reagent O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate were used for difficult couplings. Following Fmoc deprotection and before the addition of the subsequent residue, the resin was saturated with a solution of dichloromethane/N,N-dimethylformamide/N-methyl-2-pyrrolidone (1:1:1) containing 1% Triton X-100 and 2 M ethylencarbonate. Amino acid side-chains were protected as follows: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulphonyl (Arg), t-butoxycarbonyl (Lys, Trp), trityl (His, Cys) and t-butyl (Ser, Tyr, Thr). Cleavage from the resin and deprotection of synthesized peptides were carried out with a solution of 90% trifluoroacetic acid, 3% water, 1% triisopropylsilane and 2% each of phenol, 1,2-ethanedithiol and thioanisole. After repeated precipitation with methylbuthylether, peptides were purified using RP-HPLC on a C18 Delta-Pak column (Waters, Bedford, MA, USA), using an appropriate 060% acetonitrile gradient in 0.1% trifluoroacetic acid. Crude and reduced PG-1 was air oxidized for 36 h at room temperature, dissolved in 0.1 M TrisHCl buffer, pH 8.5 at a concentration of 0.1 mg/mL, and purified with RP-HPLC. The molecular masses of the synthetic peptides were determined by electrospray mass spectrometry. An API I instrument (Perkin Elmer SCIEX) was used to assess the quality control of each synthesis.
In vitro experiments
The compounds were dissolved in distilled H2O at 20 times the required maximal concentration. Successive dilutions of the peptides were prepared in 0.01% acetic acid containing 0.2% bovine serum albumin in polypropylene tubes. The dilutions were examined at concentrations of 10 and 100 µg/ml. Oocyst (5 x 103 organisms/mL) and sporozoite suspensions (2 x 104 organisms/mL) were exposed to each compound for 0, 5, 10, 15, 20, 30, 40, 50, 60, 120 and 180 min at 37°C. Duplicate samples (0.1 mL) were withdrawn and separated into two half-series: the first series was examined by flow cytometry after double-staining, the second was serially diluted in 10 mM HEPES buffer (pH 7.2) to minimize the carryover effect, and plated onto a cell monolayer. Experiments were performed in triplicate. A-549 cells (BioWhittaker) were maintained in DMEM with 10% fetal calf serum (BioWhittaker), 1% L-glutamine (BioWhittaker), 20 mM HEPES, penicillin G (100 U/mL), streptomycin (100 µg/mL) and amphotericin B (0.5 µg/mL). Viability was assessed using Trypan Blue exclusion. Infection of the cell monolayer was initiated by adding 0.1 mL of drug-exposed organism suspensions. Infected cell cultures were kept at 37°C in 5% CO2 throughout the study. Parasite growth was assessed at 48 h post-infection in 100 random fields.
The results from flow cytometry were reported as a percentage of viable organisms. The results from cell culture were evaluated by counting parasites from plates infected with drug-exposed organisms, compared with control plates infected with non-exposed organisms. Each value was reported as the geometric mean of three experiments.
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Results |
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Flow cytometry demonstrated four populations of organisms: (1) green-fluorescent organisms (viable cells), (2) red-fluorescent organisms (non-viable organisms), (3) organisms that showed both red and green fluorescence (injured or dying cells), (4) particles that showed no fluorescence (cellular debris or cystic forms that are poorly permeable to fluorescein diacetate). In the sporozoite series, the percentage of viable population rapidly decreased to non-detectable levels after 15 and 60 min exposure to the peptides, at concentrations of 100 and 10 µg/mL, respectively. SMAP-29 exerted the highest activity, determining complete suppression of sporozoite viability after 10 min exposure (Figure 1). In the oocyst series, 6580% of the organisms remained viable after 180 min exposure to the highest peptide concentrations, BMAP-28, PG-1 and Bac7(1-35) producing a reduction of 21.0%, 25.8% and 27.2% at 100 µg/mL, respectively. SMAP-29 suppressed the growth of meronts and gamonts by 34.7%.
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The average number of parasites in plates infected with non-exposed sporozoites was 73.4 (range 55.489.6), while in plates infected with non-exposed oocysts it was 45.4 (range 36.059.2). In the sporozoite series, complete inhibition of parasite growth was observed after 20 and 60 min exposure to the peptides at concentrations of 100 and 10 µg/mL, respectively (Figure 2). In the oocyst series, no compound tested alone produced complete inhibition of parasite growth. BMAP-28, PG-1 and Bac7(1-35) were similarly active, effecting a reduction of 15.7%, 19.5% and 22.3% after 180 min exposure at 100 µg/mL, respectively. SMAP-29 showed the highest activity: after 180 min exposure it suppressed the growth of meronts and gamonts by >40%.
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Discussion |
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Antimicrobial peptides are an important component of the innate immunity of all living species. Their spectrum of activity is broad, and can include bacteria, fungi, enveloped viruses and protozoa. By virtue of these characteristics, these molecules may represent a useful therapeutic approach to infectious diseases.13 In this study, four mammalian antimicrobial peptides belonging to the cathelicidin peptide family were tested for anticryptosporidial activity. These molecules exhibit different structures: SMAP-29 and BMAP-28 are -helical in hydrophobic environments, PG-1 is a loop peptide owing to the presence of two disulphide bonds, Bac7(1-35) is an active fragment of the natural Bac7 and, based on the structure of other proline-rich peptides, may adopt a polyproline type II extended helix (Table 1).8,1417 We show here that all these peptides are toxic to excysted sporozoites. This result might reasonably be expected for SMAP-29, BMAP-28 and PG-1, since their spectra are broad and include a variety of prokaryotic and eukaryotic cells.6 Destabilization of the cytoplasmic membrane of sporozoites is probably the primary event, since previous studies have shown that the antibacterial effects of these peptides are mediated by rapidly and extensively rendering the target cell membranes permeable.6,14,15,18 In addition, similar to other peptides, they could have caused functional changes in the apical complex or surface molecules involved in attachment, invasion and intracellular development. Thus, in this manner, they could have led to alterations in the apical complex glycoprotein that contains a sporozoite ligand for epithelial cells.
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Flow cytometry confirmed that all these peptides were highly active against sporozoites. In contrast, they exhibited poor activity against non-excysted organisms that, inside the oocyst, are surrounded by a single-unit membrane and by the thick, two-layered, environmentally resistant oocyst wall. This wall may not permit the interactions required for membrane permeation by the helical peptides and PG-1, and may conceal important cellular targets of Bac7(1-35).
Importantly, the results obtained using the cell culture system and the double fluorogenic staining method are in good agreement, and concur to demonstrate that the peptides are especially effective against sporozoites. It is well known that the hosts are infected by ingesting oocysts, which travel through the gut lumen to the small intestine. Here they rupture, releasing sporozoites, and in this invasion process, Cryptosporidium focally disrupts the microvilli that cover the host cell and slides into the host cell, enveloping itself in the host cell membrane.1 Thus, the different potency demonstrated against sporozoites and oocysts suggests that the effects of these peptides could be exerted at different stages of the disease. In particular, the peptides, scarcely effective against intracellular organisms and oocysts, should act primarily during parasite replication. This occurs when Cryptosporidium ruptures out of the host cell, infects other host cells and completes the asexual stage of the life cycle. This interesting property would seem to recommend these peptides as leads for novel anticryptosporidial compounds.
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Acknowledgements |
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Footnotes |
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