In-vitro activity of polycationic peptides against Cryptosporidium parvum, Pneumocystis carinii and yeast clinical isolates

Andrea Giacometti, Oscar Cirioni*, Francesco Barchiesi, Francesca Caselli and Giorgio Scalise

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The in-vitro activity of magainin II, indolicidin and ranalexin against 14 clinical isolates of eukaryotic microorganisms was evaluated. Antifungal susceptibility testing was performed by broth microdilution, and activity against Pneumocystis carinii and Cryptosporidium parvum was determined by inoculation on to cell monolayers. For yeasts, peptide MICs and MFCs ranged from 6.25 to >50 mg/L. Ranalexin showed the highest activity against Candidaspp., while magainin II demonstrated greatest anticryptococcal activity. The peptides suppressed the growth of P. carinii by >=50% and >=90% at 5 and 50 µ M, respectively, with the exception of indolicidin. Ranalexin, the most effective compound against C. parvum,suppressed its growth by >=40% at 50 µM.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In recent years, a number of antimicrobial cationic peptides, which play an important defensive role against pathogenic organisms, have been isolated from various biological sources.1,2 In mammals, including man, they are the predominant protein species in the neutrophil and are also found on the surface of the tongue, trachea, lungs and upper intestine, and are thought to be a major antibacterial defence on mucosal surfaces.2 Even though their precise mechanism of action remains unknown, they are thought to act through the formation of ion channel pores spanning the membranes of bacteria, fungi, erythrocytes, artificial lipid bilayers and protozoa. In the present study, the antimicrobial activity of three polycationic peptides was evaluated against several eukarotic organisms causing severe infections in humans.


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

Magainin II, indolicidin and ranalexin were obtained from Sigma Aldrich S.r.l. (Milan, Italy). The peptides were dissolved in phosphate-buffered saline (pH 7.2), yielding 1000 mg/L stock solution. Stock solution of fluconazole (Pfizer, New York, NY, USA) was prepared using RPMI 1640 medium (Sigma Aldrich) buffered to pH 7.0 with 0.165 M morpholine propanesulphonic acid (MOPS) buffer (Gibco Laboratories, Milan, Italy) as solvent. Trimethoprim (Sigma Aldrich) was dissolved in 50% methanol:50% acetone at a concentration of 1 g/L. Sulphamethoxazole (Sigma Aldrich) was dissolved in dimethylsulphoxide (DMSO) at 1 g/L. Solutions of drugs were made fresh on the day of assay or stored at –80°C in the dark for short periods.

Yeast isolates

Eight clinical yeast isolates were evaluated. They included two isolates each of Candida albicans(CA1866, CA3028), Cryptococcus neoformansvar. neoformans(CN2881, CN2715), Candida krusei(CK2837, 2871), and one isolate each of Candida tropicalis(CT3086)and Candida glabrata(CG1413).

Pneumocystis carinii preparation and cell culture

These procedures have been described in detail previously.3 Briefly, culture was initiated by adding 103 Pneumocystis carinii organisms to an adherent layer of 50– 70% confluent A549 cells maintained in 96-well tissue culture plates (Becton Dickinson Italia, Milan, Italy). After incubation for 4 h at 37°C in 5% CO2 to allow attachment and penetration of sporozoites, the monolayers were washed with Dulbecco's modified Eagle's medium (DMEM) (Bio-Whittaker, Walkersville, MD, USA) to remove non-invasive sporozoites, residual cysts and non-adherent epithelial cells, and 200 µL of new growth medium with or without antimicrobial agents was added. Infected cell cultures were kept at 37°C in 5% CO2.

Cryptosporidium parvum preparation and cell cultures

Three strains of C. parvum were used throughout the study. The parasite was cultured as described previously.4 Briefly, 48 h before parasite inoculation, A549 cells were plated on to 35 mm diameter tissue culture plates at a concentration of 105 viable cells in a total volume of 5 mL. The infection of the cell monolayer was initiated by adding 105 pooled sporozoites in a volume of 0.2 mL 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.

Antifungal susceptibility testing

Antifungal activities of peptides and fluconazole were determined using a broth microdilution procedure following the guidelines proposed by the NCCLS.5 Final test concentrations were as follows: 100 to 0.19 mg/L for fluconazole and 50 to 0.19 mg/L for the peptides. To determine MFCs, 50 µL samples were withdrawn from the well containing the MIC or greater, and inoculated in duplicate on to Sabouraud dextrose agar plates. The MFC was defined as the lowest concentration of the drug at which no growth was detected after 48–72 h of incubation.

Anti-pneumocystis susceptibility testing

The peptides were tested at the following concentrations: 0.5, 5 and 50 µM. Trimethoprim–sulphamethoxazole (TMP– SMX), used as a reference drug combination, was tested at concentrations of 0.8 and 3.2 mg/L, and 4 and 16 mg/L, respectively. Antibiotic-free plates were used as controls in the study. Experiments were performed in triplicate. After 72 h, samples of supernatant were withdrawn from each triplicate well and evaluated for P. carinii trophozoite and cyst counts by Giemsa and methenamine silver staining.

Anti-cryptosporidial susceptibility testing

The peptides were examined at concentrations of 0.5, 5 and 50 µM. Antibiotic-free plates were used as controls. Experiments were performed in triplicate. Parasite growth was assessed at 48 h post-infection in 50 random oil-immersion fields. 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. Only meronts and gamonts were enumerated to avoid counting non-viable, but adherent, sporozoites or merozoites.6

Analysis of results

As mentioned above, the antifungal activity of the peptides was compared with that of fluconazole using a broth microdilution procedure.

The anti-P. carinii activity was evaluated via parasite counts from plates with antimicrobial-supplemented medium compared with control plates lacking drugs. The number of P. carinii organisms was calculated by counting 50 oil-immersion fields of each of three slides. The 50% and 90% inhibitory concentrations (IC50 and IC90, respectively) were defined as the concentrations required to produce 50% and 90% reductions in the counts of mean cysts or nucleii, compared with controls, after 72 h incubation in the presence of drugs.

The anti-cryptosporidial activities of each agent were evaluated by counting parasites from plates with antimicrobial-supplemented medium compared with control plates lacking drugs.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antifungal susceptibility testing

Table I shows the antifungal activity of the peptides compared with that of fluconazole. One strain of C. albicansand both strains of C. kruseiwere highly resistant to fluconazole. Peptide MICs and MFCs both ranged from 6.25 to >50 mg/L. Ranalexin showed good in-vitro activity against both isolates of C. albicans,with a fungicidal activity against the fluconazole-resistant strain 3028 corresponding to the MIC (12.5 mg/L). Although magainin II exerted a potent anti-cryptococcal activity against both isolates tested (MICs 6.25–12.5 mg/L), only against strain 2715 was it shown to be truly fungicidal. Both ranalexin and indolicidin were fungicidal against the strains of C. kruseiat concentrations <=25 mg/L (Table I).


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Table I. Antifungal activity of magainin II, ranalexin, indolicidin and fluconazole against eight clinical yeast isolates
 
Anti-pneumocystis susceptibility testing

The average number of parasites grown in the absence of antibiotic was 42.8 (range 21–63) when calculated by counting 50 oil-immersion fields. All membrane active peptides demonstrated similar activity against P. carinii. Magainin II at concentrations of 5 and 50 µM caused a decrease in both cyst (51.2% and 98.5%, respectively) and trophozoite (59.5% and 99.2%, respectively) counts. At the same concentrations, ranalexin also reduced cyst (53.6% and 98.7%) and trophozoite (57.7% and 99.0%) counts. Finally, at concentrations of 5 and 50 µM, indolicidin, although slightly less effective, produced a decrease in cyst counts of 45.6% and 95.1%, and in trophozoite counts of 48.9% and 97.6%, respectively. The IC50 and IC90 values showed that the peptides were as effective as TMP–SMX, but only at the highest concentration.

Anti-cryptosporidial susceptibility testing

In control plates without drugs, the average number of parasites in 50 random oil fields was 33.8 (range 21–48). A high preponderance of meronts over microgamonts was observed. No compound had a good inhibitory effect against C. parvum, and both were equally effective. Ranalexin, the most active compound, suppressed parasite growth by >40% at 50 µM. The activity of these agents is summarized in Table II.


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Table II. Anti-cryptosporidial activity of magainin II, ranalexin, indolicidin: percentage reduction in the number of parasites
 
In the last few years, the widespread use of new antifungals, especially fluconazole, has selected for Candidaspp. that are primarily resistant to this triazole such as C. krusei, or whose resistance is easily inducible such as C. glabrata.7 In addition, in the AIDS population there is an increasing incidence of fungal infections, particularly those caused by C. albicansand C. neoformans, which are resistant to standard dosing regimens of this drug.7,8 The difficulties involved in the therapy of fungal infections indicate an urgent need for new therapeutic strategies. In this study we investigated the in-vitro activity of three peptides against the four most common species of Candida, and against two isolates of C. neoformans. Although we used only a small number of isolates, our preliminary results underline two important features regarding the antifungal activity of these compounds. First, our data seems to indicate a specific activity related to the peptide/yeast species considered. For example, magainin II showed potent antifungal actvity against all yeast isolates tested with the exception of C. albicans. On the other hand, ranalexin showed the best antifungal activity against C. albicansand C. krusei. Secondly, our data show the potential fungicidal properties of peptides. Interestingly, magainin II was fungicidal against C. neoformans2715 at the same MIC (6.25 mg/L). Similarly, both ranalexin and indolicidin were fungicidal against C. krusei2837 at the MIC.

In addition, our study provides data which indicate that polycationic peptides are as effective against P. carinii as TMP–SMX, but only at the highest concentration tested.

Finally, these peptides demonstrated poor activity against C. parvum. Taken together, our results show that polycationic peptides are effective in inhibiting several eukaryotic organisms. This fact makes these compounds potentially valuable as an adjuvant for antimicrobial chemotherapy. Further investigations are needed to characterize the mechanism of action of these agents.


    Notes
 
* Corresponding address. Clinica Malattie Infettive -c/o Azienda Ospedaliera Umberto I, Piazza Cappelli, 1, I-60121 Ancona, Italy. Tel: +39-71-596-3467; Fax: +39-71-596-3468; E-mail: cmalin{at}popcsi.unian.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Cannon, M. (1987). Antimicrobial peptides. A family of wound healers. Nature 328, 478.[ISI][Medline]

2 . Hancock, R. E. W. (1997). Antibacterial peptides and the outer membranes of Gram-negative bacilli. Journal of Medical Microbiology 46, 1–3.[ISI][Medline]

3 . Cirioni, O., Giacometti, A. & Scalise, G. (1997). In-vitro activity of atovaquone, sulphamethoxazole and dapsone alone and combined with inhibitors of dihydrofolate reductase and macrolides against Pneumocystis carinii. Journal of Antimicrobial Chemotherapy 39, 45–51.

4 . 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]

5 . National Committe for Clinical Laboratory Standards. (1995). Reference Method for Broth Dilution Antifungal Susceptibility Testing for Yeasts: Approved Standard M27-A. NCCLS, Villanova, PA.

6 . 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, 45–50.[ISI][Medline]

7 . Rex, J. H., Rinaldi, M. G. & Pfaller, M. A. (1995). Resistance of Candidaspecies to fluconazole. Antimicrobial Agents and Chemoherapy 38, 1–8.

8 . Zuger, A., Louie, E., Holzman, R. S., Simberkoff, M. S. & Rahal, J. J.(1986). Cryptococcal disease in patients with acquired immunodeficiency syndrome: diagnostic features and outcome of treatment. Annals of Internal Medicine 104, 234–40.[ISI][Medline]

Received 21 October 1998; returned 1 March 1999; revised 29 March 1999; accepted 27 April 1999