In vitro efficacies of nitazoxanide and other thiazolides against Neospora caninum tachyzoites reveal antiparasitic activity independent of the nitro group

Abstract

The thiazolide nitazoxanide [2-acetolyloxy-N-(5-nitro-2-thiazolyl)benzamide] (NTZ) exhibits a broad spectrum of activities against a wide variety of intestinal and tissue-dwelling helminths, protozoa, and enteric bacteria infecting animals and humans. The drug has been postulated to act via reduction of its nitro group by nitroreductases, including pyruvate ferredoxin oxidoreductase. In this study, we investigated the efficacies of nitazoxanide and a number of other thiazolides against Neospora caninum tachyzoites in vitro. We employed real-time-PCR-based monitoring of tachyzoite adhesion, invasion, and intracellular proliferation, as well as electron microscopic visualization of the effects imposed by nitazoxanide. In addition, we investigated several modified versions of this drug. These modifications included on one hand the replacement of the nitro group on the thiazole ring with a bromide, thus removing the most reactive group, and on the other hand the differential positioning of methyl groups on the salicylate ring. We show that the thiazole-associated nitro group is not necessarily required for the action of the drug and that methylation of the salicylate ring can result in complete abrogation of the antiparasitic activity, depending on the positioning of the methyl group. These findings indicate that other mechanisms besides the proposed mode of action involving the pyruvate ferredoxin oxidoreductase enzyme could be responsible for the wide spectrum of antiparasitic activity of NTZ and that modifications in the benzene ring could be important in these alternative mechanisms.

FIG. 1.

Drugs investigated in this study. NTZ is composed of a nitrothiazole moiety and a salicylic acid moiety, Rm4820 is a de-nitro-NTZ with a bromide replacing the nitro group, Rm4803 has a methyl group at the ortho position on the salicylic acid ring, Rm4822 has a methyl group at position 4, and Rm4821 has a methyl group at position 5. TIZ, tizoxanide.

FIG. 2.

NTZ inhibits N. caninum proliferation in HFF monolayers. HFF monolayers were infected with N. caninum tachyzoites, and at 2 h p.i., 1, 5, or 10 μg/ml NTZ was added. Samples were collected at 1, 2, 4, 6, and 8 days p.i and were processed for the quantitative assessment of N. caninum proliferation by real-time PCR. Note the severe inhibition of proliferation with NTZ at 5 and 10 μg/ml.

FIG. 3.

Effects of several thiazolides on N. caninum tachyzoite proliferation in HFF. HFF monolayers were infected with N. caninum tachyzoites, and at 2 h p.i., 10 μg/ml NTZ, Rm4803, Rm4820, Rm4821, or Rm4822 was added and left for a period of 8 days. Note the severe inhibition of proliferation by Rm4820, Rm4821, and Rm4822, while Rm4803 has no effect.

FIG. 4.

Transmission electron microscopy of intracellular N. caninum tachyzoites at (A) 24 h p.i. and (B) 72 h p.i. Note that intracellular N. caninum tachyzoites (T) reside within a parasitophorous vacuole (PV), surrounded by a parasitophorous vacuole membrane (PVM). Bars, 0.9 μm (A) and 1 μm (B).

FIG. 5.

Transmission electron microscopy of NTZ-treated, N. caninum-infected HFF monolayers. (A) At 6 h p.i., intracellular tachyzoites (T) are characterized by the absence of a parasitophorous vacuole membrane (arrows). Bar, 0.6 μm. (B) At 24 h p.i., severe alterations within the infected host cell cytoplasm are evident. Bar, 2 μm. (C) Higher magnification reveals distinct changes within the parasites, including increased cytoplasmic vacuolization (arrows). con, conoid; mito, mitochondrion; mic, micronemes; rho, rhoptries; ld, lipid droplets. Bar, 0.5 μm. (D and E) At 48 h (D) and 72 h (E), distinct changes and considerable damage are evident in drug-treated tachyzoites. Lipid droplets often surround the parasites, and the tachyzoite cytoplasm is heavily compartmentalized by vesicles, the contents of which are granular or membranous and electron dense (arrows). Note also the fragmented mitochondrion. At 72 h, the plasma membrane has lost its characteristic electron-dense appearance, indicating that membrane disintegration has taken place (triangles). Bars, 0.5 μm.

FIG. 6.

Five days of continuous NTZ treatment is required to exert true parasiticidal activity. HFF monolayers were infected with N. caninum tachyzoites, and at 2 h p.i., 10 μg/ml NTZ was added. NTZ treatment was stopped after 2, 3, 4, or 5 days (arrows) by replacing the NTZ-containing medium with fresh medium devoid of NTZ. Note that parasite proliferation was irreversibly inhibited only after 5 days of continuous NTZ treatment.

FIG. 7.

NTZ inhibits proliferation at later stages of host cell infection. HFF monolayers were infected with N. caninum tachyzoites, and NTZ (10 μg/ml) was added at day 3 and day 4 p.i. (arrows). Note the immediate inhibition of proliferation of N. caninum tachyzoites.

FIG. 8.

NTZ acts on intracellular, but does not impair infectivity of extracellular, N. caninum tachyzoites. (A) HFF monolayers were infected with N. caninum tachyzoites. Four distinct conditions were assessed. First, no drug was added at any time (control). Second, NTZ (10 μg/ml) was present during the infection phase of 2 h, followed by further culture without the drug (+NTZinf/−NTZ). This was not effective at all. Third, NTZ was not present during infection but was added after 2 h upon completion of the infection phase (−NTZinf/+NTZ). Finally, NTZ was present during infection and also subsequently during the culture (+NTZinf/+NTZ). (B) Freshly purified N. caninum tachyzoites were incubated in medium in either the presence of absence of NTZ (10 μg/ml) for 2, 6, or 24 h. Parasites were then allowed to interact with HFF monolayers, and tachyzoites interacting with the monolayers (adhered/invaded) and the corresponding invaded parasites (invaded) were quantified by the PDTC adhesion/invasion assay. No significant impact on either adhesion or invasion was evident. Error bars indicate standard deviation.