Short-Term Exposure to Membrane-Active Antibiotics Inhibits Cryptosporidium parvum Infection in Cell Culture

Abstract

A cell culture system and double fluorogenic staining were used to study the susceptibility of Cryptosporidium parvum to membrane-active antibiotics. Buforin II and magainin II exerted a cytotoxic effect on sporozoites but did not consistently affect oocyst viability. Lasalocid and nigericin demonstrated less activity against sporozoites but reduced the infectivity of oocysts.

Several compounds have been proposed as anticryptosporidial agents (3, 4, 9, 14). The membrane-active polyether ionophores are lipid-soluble molecules that transport polar cations across the cell membranes. Lasalocid, a polyether carboxylic acid ionophore, was isolated from Streptomyces lasaliensis. It disrupts membrane potential and stimulates ATPase activity in mitochondria (5, 10, 11). Nigericin, isolated from Streptomyces hygroscopicus, is another polyether ionophore which exerts similar activity (5). Today only lasalocid is therapeutically useful: it is an effective anticoccidial drug for poultry and farm animals. The vertebrate polycationic peptides demonstrate a broad spectrum of antimicrobial activity. Their mechanisms of action are under investigation: perturbation of the membrane function, formation of transient channels, and attachment to cytosolic targets are those recently proposed (6, 7, 13). In the present study, short-term exposures to membrane effectors were performed to investigate the anticryptosporidial activity of these compounds.

Pooled Cryptosporidium parvum oocysts were suspended in Dulbecco's modified Eagle's medium (BioWhittaker Inc., Walkersville, Md.) and enumerated in a hemocytometer chamber. Oocyst viability was estimated by using an excystation procedure and vital dye staining (3).

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 were counted in a hemocytometer. Sporozoite viability was confirmed by double staining with fluorescein diacetate and propidium iodide (2, 3, 12).

Oocysts and sporozoites were separately resuspended in 0.1 ml of PBS. Following the addition of 0.1 ml of 40-μg/ml fluorescein diacetate and 0.15 ml of 20-μg/ml propidium iodide and incubation at room temperature for 5 min, the incubation mixtures were further diluted 1:1 with PBS and analyzed by flow cytometry (1).

All drugs were provided by Sigma-Aldrich (Milan, Italy). Buforin II and magainin II were examined at concentrations of 10 and 100 μg/ml. Lasalocid and nigericin were examined at concentrations of 0.1 and 1 μg/ml. Oocysts (5 × 10³ organisms/ml) and sporozoite suspensions (2 × 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 in two half-series: the first series was examined by flow cytometry after double staining; the second was serially diluted in 10 mmol of 20 mM HEPES buffer (pH 7.2) to minimize the carryover effect and was plated onto a cell monolayer. Experiments were performed in triplicate.

A-549 cells (BioWhittaker) were maintained in Dulbecco's modified Eagle's medium 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 by trypan blue exclusion. The 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 postinfection in 100 random fields. The results from flow cytometry were reported as percentages of viable organisms, while from the cell culture they were evaluated by comparing parasite counts from plates infected with drug-exposed organisms with counts from control plates infected with nonexposed organisms. Each value was reported as the geometric mean of three experiments.

Flow cytometry demonstrated differences in the percentages of the viable populations (Tables 1 and 2). In the sporozoite series, the percentage of the viable population rapidly fell under 10% after 20- and 60-min exposures to the peptides at concentrations of 100 and 10 μg/ml, respectively. In contrast, the viable sporozoite population remained above 70 and 50% after 180 min of exposure to the ionophores at concentrations of 0.1 and 1 μg/ml, respectively. In the oocyst series, the percentage of the viable organisms remained above 65% after 180 min of exposure to the highest peptide concentrations, while it fell under 50% after 180 min of exposure to the ionophores at a concentration of 1 μg/ml.

TABLE 1.

Flow cytometry: effects of buforin II, magainin II, lasalocid, and nigericin on sporozoite viability

Time (min)	%a viable organisms in control solution or in antibiotics at 2 different concns
	Control	Buforin II		Magainin II		Lasalocid		Nigericin
		10 μg/ml	100 μg/ml	10 μg/ml	100 μg/ml	0.1 μg/ml	1 μg/ml	0.1 μg/ml	1 μg/ml
0	87.9	88.5	89.0	84.7	86.4	90.2	87.8	87.3	85.4
5	88.0	84.7	79.6	82.3	77.9	89.6	88.2	87.6	86.0
10	87.5	78.3	55.2	77.6	58.5	88.5	86.9	88.0	85.5
15	87.9	66.1	22.7	67.0	25.7	86.9	83.1	86.1	81.1
20	88.2	54.0	8.9	55.1	9.7	87.1	77.7	84.3	74.7
30	87.4	40.8	7.0	42.5	8.8	82.3	74.2	80.3	71.2
40	88.0	26.4	5.6	31.0	8.0	80.7	71.3	80.4	70.0
50	86.9	14.7	5.0	15.2	6.9	81.1	69.3	79.1	67.3
60	87.3	9.4	4.7	9.7	6.1	80.0	65.6	79.0	64.5
120	86.7	2.1	0.1	3.0	1.1	76.2	58.0	74.2	56.1
180	86.1	0.2	0.1	0.2	0.1	73.3	52.8	71.6	50.9

^aGeometric mean of three examinations. 

TABLE 2.

Flow cytometry: effects of buforin II, magainin II, lasalocid, and nigericin on oocyst viability

Time (min)	%a viable organisms in control solution or in antibiotics at 2 different concns
	Control	Buforin II		Magainin II		Lasalocid		Nigericin
		10 μg/ml	100 μg/ml	10 μg/ml	100 μg/ml	0.1 μg/ml	1 μg/ml	0.1 μg/ml	1 μg/ml
0	75.8	78.7	78.0	78.7	78.0	76.3	75.8	79.0	78.4
5	74.8	79.7	78.1	77.5	76.9	76.4	76.1	78.5	78.2
10	75.5	78.4	76.6	77.0	76.5	75.8	76.9	78.7	78.0
15	75.6	77.6	77.0	76.4	75.2	76.0	75.2	77.9	77.1
20	76.0	78.0	76.3	77.3	75.9	75.7	74.3	78.0	74.5
30	76.2	77.1	75.5	76.8	75.4	75.1	74.0	77.3	72.8
40	75.1	76.9	75.6	75.7	74.3	75.2	70.7	77.2	69.9
50	76.2	75.8	71.6	75.1	72.7	75.3	67.1	77.1	65.2
60	75.9	76.3	70.3	75.9	71.8	74.5	63.4	76.8	61.5
120	73.8	73.2	67.2	74.4	71.2	74.9	54.0	73.2	53.5
180	72.7	70.8	66.5	71.7	69.2	71.2	48.7	70.3	46.8

^aGeometric mean of three examinations. 

The average number of parasites in plates infected with nonexposed sporozoites was 76.9 (range, 63.3 to 87.6), while in plates infected with nonexposed oocysts it was 43.3 (range, 31.9 to 53.6). In the sporozoite series, complete inhibition of parasite growth was observed after 20 and 60 min of exposure to the peptides at concentrations of 100 and 10 μg/ml, respectively, and after 120 min of exposure to the ionophores at a concentration of 1 μg/ml. In the oocyst series, no compound produced complete inhibition of parasite growth. Buforin II and magainin II were similarly active, effecting respective reductions of 18.8 and 15.1% after 180 min of exposure at 100 μg/ml. The ionophores showed the highest activity against oocysts: after 180 min of exposure, they suppressed the growth of meronts and gamonts by more than 20 and 30% at concentrations of 0.1 and 1 μg/ml, respectively.

Most studies of the mode of action of cationic peptides have concerned their activity against gram-negative bacteria (6, 7). One could speculate that the excysted sporozoites are susceptible because their cytoplasmic membrane is somewhat structurally similar to the bacterial cytoplasmic membrane. However, inside oocysts they are surrounded by an additive single-unit membrane and by the thick, two-layered, environmentally resistant oocyst wall, which may explain the slight activity exerted by the peptides against nonexcysted organisms. In addition, the peptides could have caused functional changes in apical complex or surface molecules involved in attachment, invasion, and intracellular development: they could have determined alteration of the apical complex glycoprotein that contains a sporozoite ligand for epithelial cells (8). Nevertheless, flow cytometry confirmed that buforin II and magainin II were lethal for sporozoites. On the other hand, lasalocid and nigericin exerted the highest activity against oocysts.

Developing an in vitro drug screening system is an important step toward the initial identification of candidate anticryptosporidial compounds. In this study we used two different laboratory methods, a cell culture system and double fluorogenic staining, to evaluate the anticryptosporidial activity of short-term exposure to ionophores and cationic peptides. Further investigations are needed before firm conclusions about reliability can be drawn.