Activity of buforin II alone and in combination with azithromycin and minocycline against Cryptosporidium parvum in cell culture

Andrea Giacometti*, Oscar Cirioni, Maria Simona Del Prete, Francesco Barchiesi, Anna Fineo and Giorgio Scalise

Institute of Infectious Diseases and Public Health, University of Ancona, Ancona, Italy


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The in vitro anti-cryptosporidial activity of buforin II alone and in combination with azithromycin and minocycline was investigated. Buforin II showed moderate activity, which increased with increasing concentration to 55.7% suppression of growth at 20 µM. Moreover, its activity was enhanced when it was combined with either azithromycin or minocycline with >90% parasite reduction at the highest concentration tested. Buforin II may be active in inhibiting Cryptosporidium parvum growth in vitro upon combination with either azithromycin or minocycline.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cryptosporidium parvum is a protozoan organism that produces severe and prolonged diarrhoea in immunodeficient individuals.1 So far no compound has received regulatory approval for treatment or prevention of this infection.2

In recent years a number of antimicrobial peptides have been isolated from a wide range of animal, plant and bacterial species.3 These compounds provide a host-defence system to combat infections: it was reported that they rapidly perturbed the membrane function of pathogenic microorganisms through the formation of ion channel pores spanning the biological membranes.3 Amphibians are rich in antimicrobial molecules: >20 different peptides have been isolated from the skins and stomachs of Xenopus laevis, Bombina spp., Phyllomedusa spp. and Rana catesbeiana.3,4

Buforin II is a peptide isolated from the stomach of the Asian toad Bufo bufo gargarizans. It has been used as a wound-healing agent in traditional Korean medicine. Previous studies showed that it displayed antimicrobial activities against a broad spectrum of Gram-positive and Gram-negative bacteria. Furthermore, it showed a high activity against Candida albicans, Saccharomyces cerevisiae and Cryptococcus neoformans.5 Recent reports provide evidence that polycationic peptides have a positive interaction upon combination with hydrophobic antibiotics, such as macrolides and tetracyclines.6 Among macrolides, azithromycin is currently in clinical trials as an anti-cryptosporidial agent, while there is little published information on the anti-cryptosporidial activity of tetracyclines, such as minocycline.7,8

The aim of the present study was to investigate the anti-cryptosporidial in vitro activity of buforin II alone, and combined with azithromycin and minocycline.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Drugs

Buforin II (Sigma–Aldrich, Milan, Italy) was solubilized in phosphate-buffered saline (PBS, pH 7.2), azithromycin (Pfizer/Roerig, Rome, Italy) was dissolved in 50% methanol/50% acetone and minocycline (Sigma–Aldrich) was dissolved in distilled water. Drug solutions were made fresh on the day of assay.

Organisms and parasite preparation

Three clinical isolates of C. parvum, without genotype determination, were tested separately. Oocysts were pre-treated in a bleach solution containing nine parts sterile deionized water and one part 5.25% sodium hypochlorite for 10 min, washed twice in sterile water, centrifuged again and resuspended in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker Inc., Walkersville, MD, USA). 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, resuspended in DMEM and counted in a haemocytometer before being added to cell cultures.

Cell cultures

A549 cells from human lung carcinoma (BioWhittaker) were cultured in 25 cm2 tissue culture flasks. Medium consisted of DMEM with 10% fetal calf serum (BioWhittaker), 1% L-glutamine (BioWhittaker), 20 mM N-2-hydroxyethylpiperazine-N-ethanesulphonic acid (Sigma– Aldrich), penicillin G (100 U/mL), streptomycin (100 mg/L) and amphotericin B (0.5 mg/L). Cells were lifted using a solution of 0.25% trypsin and 0.53 mM EDTA in PBS and quantified in a haemocytometer. Forty-eight hours before infection, 105 viable A549 cells were plated on to 35 mm diameter tissue culture plates. Viability was assessed by trypan blue exclusion.

Susceptibility test

Buforin II was tested at concentrations of 0.2, 2 and 20 µM. Azithromycin and minocycline were tested at concentrations of 0.5, 2 and 8 mg/L. The infection of the cell monolayer was initiated by adding 104 sporozoites in a volume of 0.2 mL medium. After incubation for 4 h at 37°C to allow attachment and penetration of sporozoites, the monolayers were washed with DMEM, and then 5 mL of penicillin-, streptomycin- and amphotericin B-free growth medium containing the antimicrobial agents to be tested was added. Antibiotic-free plates were used as controls in the study. Experiments were performed in triplicate. The monolayers were incubated for 48 h at 37°C in 5% CO2. Following four washes in PBS to remove free oocysts and non-adherent epithelial cells, the monolayers were observed under Nomarski interference contrast optics at 1000x. Parasite growth was assessed in 50 random oil fields. Only meronts and gamonts were enumerated to avoid counting non-viable, but adherent, sporozoites or merozoites.9

Cytotoxicity assay

The cytotoxicities of buforin II and its 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.

Analysis of results

The activities of each agent alone and in combination were evaluated by counting parasites from plates with antimicrobial-supplemented medium compared with control plates. The percentage cytotoxicity was calculated from the optical density (OD) as follows: [(mean OD of uninfected cells – mean OD of infected cells)/mean OD of uninfected cells] x 100.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In control plates the average number of parasites in 50 random oil fields was 50.3 (range 32.9–56.8). Buforin II showed moderate activity, which increased with increasing concentration to 55.7% suppression of growth at 20 µM. Azithromycin and minocycline, at the highest concentrations tested, produced a decrease in parasite count of 32.1 and 24.6%, respectively.

The activity of buforin II was enhanced when combined with either azithromycin or minocycline, with >90% parasite reduction at the highest concentration tested. Buforin II 20 µM with azithromycin 8 mg/L showed the highest activity (94.4%), with an increase in the additive effect of 6%. The combination of buforin II 20 µM and minocycline 8 mg/L demonstrated less potency (90.5%), although it showed the highest increase in the additive effect (12%). The results are summarized in the TableGo.


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Table. Inhibitory effects of buforin II in combination with azithromycin and minocycline on C. parvum in A549 cells: parasite count and percentage reduction versus control plates without antimicrobials
 
The cytotoxic effect was practically absent at all concentrations of drugs tested alone (–7.3 to 10.2 percentage cytotoxicity) and at all buforin II combinations (–6.0 to 9.1 percentage cytotoxicity).

Until now, limited or no effect of proposed anti-cryptosporidial therapies has been observed and in a number of instances withdrawal of the drugs resulted in re-emergence of the infection.1,2

In our experiments buforin II showed some activity against C. parvum. It has been suggested that the mode of action of this compound may be different to that of other antimicrobial peptides. Though buforin II may interact with biological membranes like other peptides, the length of its amphipathic region is about 24 Å and for this reason it cannot span the whole biological membrane (>30 Å for the same region). Thus the ion channel model suggested for other peptides cannot be applied directly to buforin II. However, several buforin II molecules could associate with the membrane by electrostatic forces and form water-filled pores, which could perturb the cell osmotic balance.

The interaction between peptides and macrolides or tetracycline has not yet been studied extensively. The antimicrobial activity of macrolides and tetracyclines results from their ability to inhibit protein synthesis by binding to the transpeptidation site of the larger ribosomal subunit Buforin II forms an amphipathic {alpha}-helical structure, although it is limited to residues Pro-11 to Lys-21. Amphipathic compounds present properties of synergy with lipophilic and amphiphilic agents such as rifampicin, macrolides, fusidic acid and novobiocin.6 This phenomenon probably allows a positive interaction between amphipathic peptides and other lipophilic or amphiphilic antibiotics. Taken together, our results indicate that buforin II might have a role in treatment of C. parvum infections, especially when it is combined with other drugs. Further studies are needed to assess the efficacy and safety of these compounds fully.


    Notes
 
* Correspondence address: Clinica Malattie Infettive—c/o Azienda Ospedaliera Umberto I, Piazza Cappelli 1, I-60121 Ancona, Italy. Tel: +39-071-596-3467; Fax: +39-071-596-3468; E-mail: cmalinf{at}popcsi.unian.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Griffiths, J. K. (1998). Human cryptosporidiosis: epidemiology, transmission, clinical disease, treatment, and diagnosis. Advances in Parasitology 40, 37–85.[ISI][Medline]

2 . Anonymous (1997). 1997 USPHS/IDSA guidelines for the prevention of opportunistic infections in persons infected with human immunodeficiency virus. USPHS/IDSA Prevention of Opportunistic Infections Working Group. Morbidity and Mortality Weekly Reports 46, 1–46.

3 . Hancock, R. E. & Chapple, D. S. (1999). Peptide antibiotics. Antimicrobial Agents and Chemotherapy 43, 1317–23.[Free Full Text]

4 . Clark, D. P., Durell, S., Maloy, W. L. & Zasloff, M. (1994). Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin. Journal of Biological Chemistry 269, 10849–55.[Abstract/Free Full Text]

5 . Park, C. B., Kim, M. S. & Kim, S. C. (1996). A novel antimicrobial peptide from Bufo bufo gargarizans. Biochemical and Biophysical Research Communications 218, 408–13.[ISI][Medline]

6 . Vaara, M. & Porro, M. (1996). Group of peptides that act synergistically with hydrophobic antibiotics against gram-negative enteric bacteria. Antimicrobial Agents and Chemotherapy 40, 1801–5.[Abstract]

7 . Blanshard, C., Shanson, D. C. & Gazzard, B. G. (1997). Pilot studies of azithromycin, letrazuril and paromomycin in the treatment of cryptosporidiosis. International Journal of STD and AIDS 8, 124–9.[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. & Brillhart, D. B. (1994). Comparative development of Cryptosporidium parvum (Apicomplexa) in 11 continuous host cell lines. FEMS Microbiology Letters 118, 233–6.[ISI][Medline]

Received 20 April 2000; returned 2 August 2000; revised 31 August 2000; accepted 1 October 2000





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