In vitro anti-cryptosporidial activity of cationic peptides alone and in combination with inhibitors of ion transport systems

Andrea Giacometti*, Oscar Cirioni, Francesco Barchiesi, Fausto Ancarani and Giorgio Scalise

Institute of Infectious Diseases and Public Health, University of Ancona, Piazza Cappelli 1, 60121 Ancona, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The anti-cryptosporidial activity of four cationic peptides alone and in combination with five ion transport system (ITS) inhibitors was investigated for six clinical isolates of Cryptosporidium parvum recovered from stools of AIDS patients. The susceptibility tests were performed by inoculating the protozoa on to cell monolayers and determining the parasite count after 48 h incubation at 37°C. The culture medium was supplemented with serial dilutions of cecropin P1, magainin II, indolicidin and ranalexin alone or in combination with amiloride and its analogues. No agent was able to inhibit parasite growth completely. The peptides had some inhibitory effect on parasite growth: cecropin P1, magainin II, indolicidin and ranalexin at a concentration of 50 µM produced 30.6, 33.2, 38.5 and 42.1% reductions, respectively, in schizont count. Conversely, the ITS inhibitors were scarcely effective. Positive interaction was demonstrated when the peptides were tested in combination with ITS inhibitors.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cryptosporidium parvum is a major cause of diarrhoeal disease in a wide range of mammals. Several antimicrobial agents have been used in vitro in animals or in humans without success.1 The discovery of vertebrate polycationic peptides isolated from the ventral skin of toads and frogs2 and subsequently the identification of other peptides in the giant silk moth3 and in pig intestine4 have opened a new area of research into antimicrobial agents. The amino acid sequences of several cationic peptides and the structural requirements for their biological activity have been determined.2 These compounds could perturb membrane functions responsible for osmotic balance in susceptible target organisms. It has been suggested that the mode of action of these molecules on the membranes of bacteria, fungi, protozoa and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes without requiring a specific target receptor.5 Association of several such peptides would form a water-filled pore which would serve as an ion-conducting, anion-selective channel.

The ion transport system (ITS) is the target of amiloride, a substituted pyrazinoyl guanidine therapeutically useful as a potassium-sparing diuretic, and its analogue bearing substituents either on the 5-amino nitrogen or on a terminal guanidine nitrogen atom.6 Amiloride analogues such as 5-(N,N-dimethyl)amiloride (DMA), 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and, particularly, 5-(N-methyl-N-isobutyl)amiloride (MIBA) are selective inhibitors of Na+/H+ antiport. These agents are able to affect the mechanisms that regulate the intracellular pH and therefore cause intracellular acidification. Moreover, benzamil, a Na-benzyl derivative of amiloride, is a selective and potent blocker of Na+/H+ and Na+/Ca2+ channels.7 The ability of these drugs to suppress the Na+/H+ antiport in tumour cells and their efficiency in causing cell killing have yet to be evaluated. Little information is available about their toxicity, pharmacokinetics and mechanisms of interaction with other molecules, either in vitro or in vivo.

Cationic peptides and amiloride analogues act primarily on the structure or function of biological membranes. Therefore one can speculate about a possible synergic interaction between these two different groups of molecules. In the present study we have investigated the in vitro activity of four cationic peptides, tested alone and in combination with amiloride and its analogues, on the growth of C. parvum in the A549 cell line.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Organisms

Oocysts of C. parvum were isolated from stools of six different patients with AIDS. Stool specimens were stored at 4°C in 2.5% (w/v) potassium dichromate (Sigma-Aldrich, Milan, Italy) for up to 4 months until processing.

Drugs

Cecropin P1, magainin II, indolicidin and ranalexin (all from Sigma-Aldrich) were solubilized in phosphate-buffered saline (PBS, pH 7.2) (BioWhittaker, Walkersville, USA), yielding 1 mM stock solutions. Amiloride, DMA, EIPA, MIBA and benzamil (all from Sigma-Aldrich) were dissolved in 2% dimethylsulphoxide (DMSO) and then brought to the final concentration of 1 mM in distilled water.

Parasite preparation

These procedures have been described in detail previously.8 Briefly, stools were homogenized in physiological saline and filtered through a metal sieve to remove coarse debris. Fatty material was removed by ether sedimentation: stools were separated into 9 mL aliquots and ether (1 mL) was mixed with each aliquot, which was then centrifuged (200g for 20 min). The supernatant with the fatty plug was discarded. Oocysts were successively purified and concentrated by flotation in Sheather's sugar solution (500 g sucrose and 6.5 g phenol in 320 mL of distilled water). The upper layer was removed and collected. Contaminating bacteria were eliminated by three washes in sterile distilled water followed by two washes in 0.05% sodium hypochlorite and finally by incubation in PBS containing penicillin G (2 MU/L), streptomycin (2000 mg/L) and amphotericin B (10 mg/L) for 4 h at 37°C. Excystation of sporozoites was achieved by incubating oocysts in PBS containing 0.25% trypsin and 0.75% sodium taurocholate for 60 min at 37°C. Free sporozoites were pelleted by centrifugation (200g for 20 min) and resuspended in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker). Finally, the six isolates were pooled, counted in a haemocytometer and aliquoted for culture.

Cell cultures

Cells (BioWhittaker) were maintained in 25 cm2 tissue culture flasks. The medium consisted of DMEM with 10% fetal calf serum (BioWhittaker), 1% l-glutamine (BioWhittaker), 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES) (Sigma-Aldrich), penicillin G (100 mU/L), streptomycin (100 mg/L) and amphotericin B (0.5 mg/L). Cells were lifted from the surface of flasks using a solution of 0.25% trypsin and 0.53 mM EDTA in PBS and quantified with a haemocytometer. Forty-eight hours before parasite inoculation, A549 cells were plated into 35 mm diameter tissue culture plates at a concentration of 105 viable cells in a total volume of 5 mL. Viability was assessed by trypan blue exclusion. Infection of the cell monolayer was initiated by adding 105 pooled sporozoites in 50 µL of medium. After incubation for 4 h at 37°C in 5% CO2 to allow attachment and penetration of sporozoites, the monolayers were washed with DMEM to remove non-invasive sporozoites, residual oocysts and non-adherent epithelial cells, and 5 mL of new growth medium with or without antimicrobial agents was added. Infected cell cultures were kept at 37°C in 5% CO2 throughout the study.

In vitro studies

Cecropin P1, magainin II, indolicidin and ranalexin were examined singly at concentrations of 0.5, 5 and 50 µM. In experiments to test drug interactions, the three above-mentioned concentrations of each cationic peptide were tested in combination with each ITS inhibitor at concentrations of 5 and 50 µM. Antibiotic-free plates were used as controls. Experiments were performed in triplicate. C. parvum pooled sporozoites were added at a concentration of 105 sporozoites per plate. The monolayers were incubated for 72 h at 37°C in 5% CO2. Following four washes in PBS to remove free oocysts and non-adherent epithelial cells, 5 mL of new growth medium was added and the monolayers were observed under Nomarski interference contrast optics at x1000 magnification.9 Parasite growth was assessed at 48 h after infection in 50 random fields. Only meronts and gamonts were enumerated, to avoid counting non-viable, but adherent, sporozoites or merozoites.

Cytotoxicity assay

The cytotoxicities of drugs and their combinations were determined by the CellTiter 96 AQ cell proliferation assay (Promega Corp., Lyon, France). Controls for each cytotoxicity assay included (i) uninfected cells incubated in DMEM, (ii) infected cells incubated in DMEM and (iii) cells exposed to a freeze–thaw lysate containing 104 oocyst equivalents in DMEM. The cytotoxicity levels were indicated as no (0–5%), mild (6–25%), moderate (26–50%) and severe (51–100%) cytotoxicity.

Analysis of results

The anti-cryptosporidial activity of each compound and combination was evaluated by comparing the parasite count from plates with antimicrobial-supplemented medium with that from control plates without antimicrobials. The number of parasites was calculated as the mean of the number of organisms observed in three monolayers exposed to the same concentration of drug, by microscopic examination of 50 random fields from each monolayer. Each drug concentration was defined as inhibitory if it caused a significant decrease in parasite count when compared with control plates. The significance of differences was evaluated by Student's t test. To assess the efficacy of drug combinations, the significance of differences between results obtained by testing the peptides in combination with amiloride and its analogues were compared with those of control plates by Student's t test. A P value of <=0.05 was considered significant.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cationic peptides, because of their small size and antimicrobial potency, may have therapeutic potential in the treatment of infections.10 In our study an in vitro culture system for C. parvum was used to measure the anticryptosporidial activity of four biologically active peptides alone and in combination with agents known as selective calcium channel blockers or selective inhibitors of Na+/H+ antiport.

In control plates without drugs the average number of parasites in 50 random fields was 36.8 (range 29.7–48.1). A high preponderance of meronts over microgamonts was observed. Macrogamonts were not seen at 48 h after infection. A significant inhibitory effect on parasite growth was noted for each peptide at the concentrations of 5 and 50 µM (TableGo). All peptides were similarly effective, although ranalexin exhibited the highest activity with Meront and gamont growth suppressed by >40% at 50 µM. Cecropin P1, magainin II, indolicidin and ranalexin at concentrations of 5 µM produced a decrease in parasite counts of 11.8, 13.7, 12.2 and 14.7%, respectively. The same peptides at concentrations of 50 µM produced a decrease in parasite counts of 30.6, 33.2, 38.5 and 42.1%, respectively. No peptide was able to inhibit parasite growth completely. Amiloride, DMA, EIPA, MIBA and benzamil had no significant inhibitory effects on C. parvum (data not shown).


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Table. Anti-cryptosporidial activity of cationic peptides alone and in combination with ITS inhibitors against C. parvum (per cent reduction in the number of parasites compared with control plates)
 
The activity of the peptides remained virtually unchanged when they were tested in combination with ITS inhibitors at a concentration of 5 µM. In contrast, their activity was significantly improved when they were combined with ITS inhibitors at a concentration of 50 µM. Ranalexin 50 µM in combination with EIPA 50 µM or benzamil 50 µM showed the highest activity, with a c. 60% decrease in parasite counts (TableGo). As a working hypothesis, the proposed permeabilization of the biological membranes owing to the formation of ion-sized channels may enhance the activity of agents able to inhibit Na+/H+ and Na+/Ca2+ exchanges. Alternatively, the increased permeabilization may lead to severe perturbation of the intracellular ionic balance.

Cationic peptides and ITS inhibitors may be able to perturb biological membrane function, combining the effect of two or more different mechanisms. The membrane-active peptides are interesting compounds, but further research is needed to characterize in more detail the mode of action, the in vivo toxicity, the pharmacokinetics and the mechanisms of interaction with other molecules. The fact that cationic peptides are found in mammals and in insects hints that antimicrobial peptides may be a universal means for defence against infections. Our data suggest that by varying the amino acid sequence it may be possible to develop synthetic peptides with high antimicrobial activity combined with low toxicity for the host: with thousands of theoretical variants of small amino acid peptides, there is ample scope for designing improved synthetic peptides.


    Notes
 
* Corresponding author. Tel: +39-071-596-3467; Fax: +39-071-596-3468; E-mail: cmalinf{at}popcsi.unian.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . McDonald, V., Stables, R., Warhurst, D. C., Barer, M. R., Blewett, D. A., Chapman, H.D. et al. (1990). In-vitro cultivation of Cryptosporidium parvum and screening for anticryptosporidial drugs. Antimicrobial Agents and Chemotherapy 34, 1498–500.[ISI][Medline]

2 . Canon, M. (1987). Antimicrobial peptides. A family of wound healers. Nature 328, 478.[ISI][Medline]

3 . Boman, H. G. & Hultmark, D. (1987). Cell-free immunity in insects. Annual Review of Microbiology 41, 103-26.[ISI][Medline]

4 . Kleyman, T. R. & Cragoe, E. J., Jr (1988). Amiloride and its analogs as tools in the study of ion transport. Journal of Membrane Biology 105, 1–21.[ISI][Medline]

5 . Wade, D., Boman, A., Wahlin, B., Drain, C. M., Andreu, D., Boman, H. G. et al. (1990). All-d amino acid-containing channel-forming antibiotic peptides. Proceedings of the National Academy of Sciences, USA 87, 4761–5.[Abstract]

6 . Lee, J. Y., Boman, A., Chuanxin, S., Andersson, M., Mutt, H., Jörnall, H. et al. (1989). Antimicrobial peptides from pig intestine: isolation of a mammalian cecropin. Proceedings of the National Academy of Sciences of the USA 86, 9159–62.[Abstract]

7 . Garritsen, A., Ijzerman, A. P., Beukers, M. W., Cragoe, E. J., Jr & Soudijn, W. (1990). Interaction of amiloride and its analogues with adenosine A1 receptors in calf brain. Biochemical Pharmacology 40, 827–34.[ISI][Medline]

8 . Giacometti, A., Cirioni, O. & Scalise, G. (1996). In-vitro activity of macrolides alone and in combination with artemisin, atovaquone, dapsone, minocycline or pyrimethamine against Cryptosporidium parvum. Journal of Antimicrobial Chemotherapy 38, 399–408.[Abstract]

9 . Upton, S. J., Tilley, M., Nesterenko, M. V. & Brillhart, D. B. (1994). A simple and reliable method of producing in vitro infections of Cryptosporidium parvum (Apicomplexa). FEMS Microbiology Letters 118, 45–9.[ISI][Medline]

10 . Arrowood, M. J., Jaynes, J. M. & Healey, M. C. (1991). In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum. Antimicrobial Agents and Chemotherapy 35, 224–7.[ISI][Medline]

Received 28 January 1999; returned 6 August 1999; revised 1 September 1999; accepted 27 November 1999





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