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Antimicrobial Agents and Chemotherapy, January 2006, p. 162-170, Vol. 50, No. 1
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.1.162-170.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vitro Effects of Thiazolides on Giardia lamblia WB Clone C6 Cultured Axenically and in Coculture with Caco2 Cells

Joachim Müller,1* Géraldine Rühle,1 Norbert Müller,1 Jean-François Rossignol,2 and Andrew Hemphill1,2*

Institute of Parasitology, University of Bern, Länggass-Strasse 122, CH-3012 Bern, Switzerland,1 The Romark Institute for Medical Research, Tampa, Florida 336072

Received 21 September 2005/ Returned for modification 12 October 2005/ Accepted 18 October 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC50) of 18.8 µM; in comparison, NTZ and tizoxanide had IC50s of 2.4 µM, and MTZ had an IC50 of 7.8 µM. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Giardia lamblia (also known as G. duodenalis or G. intestinalis), a flagellated protozoan, is the most common causative agent of persistent diarrhea worldwide. The life cycle includes motile, flagellated trophozoites parasitizing the upper intestine and thick-walled cysts forming in the lower intestine, which are subsequently shed with the feces (29). Antigiardial chemotherapy is directed against the trophozoite stage. Metronidazole (MTZ) and other nitroimidazoles have been used against giardiasis since the late 1950s (12, 30). These compounds can cause, however, a number of side effects, including headache, vertigo, nausea, and pancreatitis, as well as central nervous system toxicity at high doses. Moreover, MTZ has been shown to be mutagenic in bacteria and carcinogenic in mice (2). Other treatments include quinolones (28), benzimidazoles such as albendazole (7), nitrofurans, paromomycin, and bacitracin zinc, all compounds that can exhibit notorious side effects (12).

Originally developed as a veterinary antihelminthic (24), 2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide (NTZ), also known as Alinia, has been shown to exhibit a wide spectrum of in vivo activity against a broad spectrum of intestinal parasites, such as Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis (1, 5), the apicomplexan Cryptosporidium parvum (11), and enteric bacteria infecting animals and humans (13, 33). NTZ has been postulated to exhibit a mode of action based upon reduction of its nitro group by nitroreductases, including pyruvate ferredoxin oxidoreductase. Thus, in this model, the nitro group would play a crucial role similar to the one postulated for MTZ. Experimental evidence for this mode of action, however, is lacking. In contrast to MTZ, NTZ has been shown to be nonmutagenic (27). In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ) (4), a compound with equal effectiveness (1, 33). In the liver, TIZ is then transformed to tizoxanide glucuronide, an inactive form, and excreted via bile or urine (4).

In this study, we have used two culture systems to investigate the efficacy and mechanisms of action of NTZ, other thiazolides (11), and MTZ against Giardia trophozoites.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture media, biochemicals, and drugs. If not otherwise stated, all tissue culture media were purchased from Gibco-BRL (Zürich, Switzerland), and biochemical reagents were from Sigma (St. Louis, MO). All thiazolides (Table 1) were synthesized at the Romark Center for Drug Discovery at the Department of Chemistry, University of Liverpool, and were obtained from Romark Laboratories (Tampa, FL). They were kept as 100 mM stock solutions in dimethyl sulfoxide (DMSO) at –20°C.


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  		TABLE 1. Overview of compounds used in this study

 
Axenic culture of Giardia trophozoites and drug treatment assays. Trophozoites from Giardia lamblia WB clone C6 were grown under anaerobic conditions in 10-ml culture tubes (Nunc, Roskilde, Denmark) containing modified TYI-S-33 medium as described previously (8). In order to initiate subcultures, cultures with confluent trophozoite lawns were incubated on ice for 15 min. Suspended motile trophozoites were counted (Neubauer chamber, 200x magnification). Subcultures were initiated by adding 104 trophozoites to a new culture tube.

The effects of drugs were assayed by adding increasing concentrations of various thiazolides or MTZ to trophozoite subcultures. Proliferation was monitored by counting attached trophozoites during the growth phase (reverse microscope, 200x magnification) and by counting trophozoites after the final harvest at day 5 postincubation. For this final harvest, medium and unattached trophozoites were removed and replaced with ice-cold phosphate-buffered saline (PBS) in order to count only attached trophozoites. Fifty percent inhibitory concentration (IC50) values were obtained by extrapolating trophozoite numbers from the DMSO control.

Coculture of Giardia trophozoites and Caco2 intestinal cells. Caco2 cells (ATTC HTB-37) were kindly provided by Daniel Lottaz, Institute of Biochemistry and Molecular Biology, University of Bern, and were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 50 U/ml of penicillin, and 50 µg/ml of streptomycin (Caco2 growth medium) at 37°C and 5% CO2. For coculture experiments, confluent lawns of Caco2 cells were trypsinized, resuspended in Caco2 growth medium, and added to the wells of a 24-well plate at a volume of 1 ml containing 2.5 x 106 cells/well. After formation of a confluent lawn (48 h postinfection), medium was exchanged for fresh medium. Axenically grown Giardia lamblia trophozoites were harvested by immersing the culture tubes for 15 min in ice, which was followed by transfer of the parasites to 15-ml Falcon tubes and centrifugation (13 min at 1,300 x g and 4°C). Parasites were then resuspended in Caco2 growth medium, and 0.1-ml portions of this suspension containing increasing numbers of trophozoites (from 5 x 103 up to 106) were added per well. The compounds to be tested were dissolved in DMSO and were added at the desired concentrations. For each experiment, control incubations were performed with DMSO alone. At different time points following the initiation of drug treatment, the medium was removed, and cultures were carefully washed with PBS (prewarmed at 37°C) and, finally, processed for DNA purification. For scanning electron microscopy (SEM), cells were grown in 24-well tissue culture plates on glass slides coated with poly-L-lysine (100 µg/ml).

Processing of DNA samples and LightCycler-based quantitative PCR. DNA purification was performed using a DNeasy kit (QIAGEN, Basel, Switzerland) according to the standard protocol suitable for tissue samples. DNA was eluted in 2 x 50 µl of AE buffer (elution buffer from the kit) and subsequently boiled for 5 min. For quantitative real-time PCR, forward cwp primer 2 (H7CWF2; 5'-GGC GAT ATT CCC GAG TGC ATG TG -3') and reverse cwp primer 2 (NH7CWR2; 5'-GTG AGG CAG TAC TCT CCG CAG T -3') were used (32). The primers were specific for cwp genes in strain GS/M-83-H7 but had the same amplification efficacy in strain WB clone C6. Detection of DNA amplification products and quantification of parasite numbers through fluorescence resonance energy transfer on a LightCycler instrument (Roche Diagnostics, Basel, Switzerland) were done as previously described (32) by assessing mean values (plus standard deviations) from triplicate determinations. As external standards, samples containing DNA equivalents from 100 trophozoites, 10 trophozoites, and 1 trophozoite were included. Reproducibility of the test system was demonstrated by proving an overall low variation within three independent runs of the standard reactions.

Scanning and transmission electron microscopy. For SEM or transmission electron microscopy (TEM), confluent lawns of trophozoites were treated with NTZ or MTZ (50 µM) or with DMSO as a solvent control. Parasites were harvested as described above and resuspended in 1 ml ice-cold PBS, transferred to 1.5 ml Eppendorf tubes, and centrifuged (5,000 x g, 5 min, 4°C). Pellets were resuspended in 100 mM cacodylate (pH 7.3) containing 2.5% glutaraldehyde and fixed overnight at 4°C. Pellets were then washed three times in 100 mM cacodylate buffer and postfixed in 100 mM cacodylate containing 1% OsO4 for 2 h. Pellets were then washed three times in distilled water and contrasted in saturated uranyl acetate for 30 min (for TEM) or directly dehydrated in an ethanol series (50%, 70%, 90%, 3 x 100%). For SEM of trophozoites in coculture with Caco2 cells, fixation, postfixation, and dehydration were performed with coated slides directly inside 24-well plates.

Samples for TEM were embedded in Epon 820 resin (16). The resin was polymerized at 65°C over a period of 48 h. Ultrathin sections were cut on a Reichert and Jung ultramicrotome and loaded onto 300-mesh copper grids (Plano GmbH, Marburg, Germany). Staining with uranyl acetate and lead citrate was performed as described previously (16). Finally, grids were viewed on a Phillips CM12 transmission electron microscope operating at 80 kV.

Samples for SEM were resuspended in 500 µl of hexamethyldisilazane, centrifuged (5,000 x g, 5 min, room temperature), resuspended in 20 µl of hexamethyldisilazane, and spotted onto glass coverslips pretreated with hexamethyldisilazane. Coverslips were air dried, sputter coated with gold, and inspected on a JEOL 840 scanning electron microscope operating at 25 kV.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of thiazolides on G. lamblia trophozoite proliferation in axenic culture. The best activity of thiazolides in axenic cultures of G. lamblia trophozoites was observed with compounds containing a nitro group in position 5 of the thiazole ring, such as NTZ, TIZ, MTZ, RM4802, and RM4805, with IC50s ranging from 2.4 to 14.7 µM (Table 1). However, the nitro group is not the only determinant of antiparasitic activity. We found that modification of the benzene ring at position 3 by either methylation (such as in RM4802) or methoxylation (such as in RM4805) significantly reduced drug efficacies by about five- to sixfold, and methylation of tizoxanide at position 5 (RM4807 in Table 1) completely abolished the antigiardial activity (IC50 of >50 µM). RM4808, with a nitro group on an additional benzene ring attached with a sulfone group to the thiazole moiety, was also inactive.

NTZ derivatives containing a bromo instead of a nitro group (Table 1) were all inactive against Giardia trophozoites (IC50s of >50 µM), with the exception of RM4820, which showed a moderate inhibitory activity (IC50 of 18.8 µM) that was lower than the ones for RM4802 and RM4805. The efficacy of MTZ was lower than those of NTZ and TIZ but still higher than that of any other drug tested in this study.

In order to visualize the morphological alterations of G. lamblia trophozoites induced by NTZ and compare them with those induced by MTZ, confluent axenic trophozoite cultures were treated with 50 µM NTZ or MTZ or with DMSO as a control. Observations with the light microscope showed that after 3 h of treatment with NTZ, approximately 50% of trophozoites were immobile; at 5 h of treatment, over 95% of trophozoites were immobile and formed large multicellular aggregates; and at 24 h of NTZ treatment, no motile trophozoites were found (data not shown). They all exhibited aberrant vacuolar cytoplasmic structures. Inspection by TEM (Fig. 1) largely confirmed these findings and showed that at 1 h of NTZ treatment, a considerable number of trophozoites already exhibited relatively small, aberrant cytoplasmic inclusions within their cytoplasm (Fig. 1A and B). At 3 h of treatment, larger vacuoles containing membranous inclusions or membrane stacks could be observed in a large number of parasites (Fig. 1C), and at 24 h of treatment, these parasites were seriously damaged, as exhibited either by a dissociation of their cytoplasmic organization (Fig. 1D) or, in many instances, by large vacuoles containing membrane residues (Fig. 1E). Besides this, however, the cytoskeletal elements of trophozoites, such as filaments associated with the ventral disk or the flagella and basal bodies, were not notably altered. In control preparations incubated in DMSO alone, no obvious changes in parasite ultrastructure could be detected (Fig. 1F).



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  		FIG. 1. Transmission electron microscopy of G. lamblia trophozoites treated with NTZ (50 µM) and fixed and processed at 1 h (A and B), 3 h (C), and 24 h (D and E) of treatment. Controls (F) were treated with the corresponding amount of DMSO. vd, ventral disk; nu, nucleus; f, flagellum. Bars: 0.5 µm (A and C), 2 µm (B), 1 µm (D, E, and F).

 
Further examination of NTZ-treated trophozoites by SEM was performed in order to visualize possible alterations on the surface. Indeed, in NTZ- and TIZ-treated parasites, SEM revealed the formation of distinct lesions on the surface membrane of the ventral disk, which first appeared between 1 and 3 h after commencement of treatment. These membrane alterations were more noticeable after 5 h (Fig. 2B to D; compare to Fig. 2A). After 24 h, the ventral disk was severely damaged, while, generally, the dorsal surface membrane was not obviously affected (Fig. 2E). In contrast to treatment with NTZ/TIZ, MTZ treatment did not result in the formation of multicellular aggregates of trophozoites (data not shown), and SEM showed that MTZ-treated parasites did not show these pronounced alterations of the ventral disk surface membrane to the extent seen with NTZ-treated trophozoites (Fig. 2F and G). After 24 h of MTZ treatment, the alterations on the ventral surface were barely visible, while the dorsal surface membrane was affected (Fig. 2H).



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  		FIG. 2. Scanning electron microscopy of G. lamblia trophozoites at different time points of NTZ or MTZ treatment. (A) Trophozoites treated with DMSO. (B to E) Trophozoites treated with 50 µM NTZ. (F to H) Trophozoites treated with 50 µM MTZ. vd, ventral disk; ds, dorsal shield. Arrows indicate distinct zones of lesions.

 
Effects of thiazolides on trophozoite survival and proliferation during coculture with Caco2 cells. Analysis by SEM revealed that after 24 h in coculture with Caco2 cells, trophozoites were attached via the ventral disk on Caco2 cells only, and none were found on the polylysine-coated glass surface of the slide (Fig. 3A). The trophozoites formed short but distinct filopodiumlike surface membrane protrusions that were not found in axenic culture (Fig. 3B and C).



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  		FIG. 3. Scanning electron microscopy of G. lamblia trophozoites in coculture with Caco2 cells. Note the presence of small filopodiumlike surface membrane protrusions (arrows).

 
In a first series of experiments, Caco2 cells were incubated with increasing numbers of trophozoites (103 to 106 parasites per well) in the presence of DMSO as a solvent control or in the presence of 30 µM NTZ. The parasites attaching to Caco2 cells were then quantified by real-time PCR (Fig. 4). In the absence of any drug and at an initial inoculum density of 105 parasites per well, 70 to 90% of the trophozoites remained attached to the Caco2 cells for a period of 24 to 48 h. At an inoculum density 10 times higher (106 parasites/well), this value was decreased to nearly 50%, indicating that binding to the Caco2 cell surface is saturable and dependent on the presence of suitable binding sites and/or host cell surface receptors. In the presence of 30 µM NTZ, with an inoculum density of 105 trophozoites, the number of parasites still attached to Caco2 cells after 24 h decreased to less than 20% of the control value. Based upon these findings, the effects of different thiazolides compared to those of MTZ were investigated. Confluent Caco2 cells were supplemented with fresh Caco2 growth medium. Trophozoites (105) were added to each well, and a number of thiazolides, MTZ, and DMSO were added (Fig. 5). Cells were harvested after 24 h, and the attached trophozoites were quantified by real-time PCR. Only those compounds that had exhibited strong inhibitory effects in axenic cultures (NTZ, TIZ, RM4802, RM4805, and MTZ) interfered with trophozoite attachment in the Caco2 coculture system. In contrast to what was seen with axenic culture, the efficacies of RM4802, RM4805, and MTZ in Caco2 coculture were comparable to those of NTZ and TIZ (Fig. 5). Concentrations lower than 15 µM did not show any significant inhibitory effects for any of the drugs tested.



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  		FIG. 4. Trophozoite numbers in coculture with Caco2 cells. Trophozoites were inoculated to the cells in various densities in the presence of 30 µM NTZ or DMSO. Cells were harvested 24 h after infection, and trophozoites were quantified using real-time PCR. Mean values and standard errors correspond to quadruplicates.

 


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  		FIG. 5. Trophozoite numbers in coculture with Caco2 cells. Trophozoites (trophos; 105) were inoculated to the cells in the presence of 15 µM (white bars) or 30 µM (black bars) of various thiazolides, MTZ, or DMSO (control [C]). Cells were harvested at various time points, and trophozoites were quantified using real-time PCR. Mean values and standard errors correspond to quadruplicates. TIG, tizoxanide glucuronide.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Effects of thiazolides and metronidazole on Giardia trophozoites in axenic culture. Previous studies on the intracellular parasite Neospora caninum have shown that, besides NTZ and TIZ, the corresponding bromo derivatives also exhibited profound antiparasitic activities (10). In this study with Giardia, only thiazolide compounds with a nitrothiazole moiety exhibited IC50 values below 10 µM. The IC50 values of NTZ, TIZ, and MTZ for trophozoite proliferation were in the order of magnitude of previously published results, using the inhibition of thymidine incorporation as an assessment parameter (1). Conversely, of those compounds containing a bromo instead of a nitro group, only RM4820 showed a moderate inhibitory activity, indicating that the nitro group has an essential function with regard to the efficacy against Giardia. This is in contrast to what has been recently found for the apicomplexan parasite Neospora caninum, which has been shown to be susceptible to NTZ and TIZ as well as to the bromo derivatives RM4820, RM4821, and RM4822, demonstrating that the nitrothiazole was not required for anti-Neospora efficacy (10). In vitro findings similar to those for Neospora have been recently obtained with another intracellular apicomplexan parasite, Cryptosporidium parvum (unpublished results). These differences in susceptibilities against different thiazolides of Giardia and Neospora can be attributed to the metabolisms of these two protozoans (extracellular versus intracellular). The results also suggest that the broad-spectrum activities of NTZ and TIZ could be based on the presence of more than one active site on these drugs, namely, the nitrothiazole ring and the benzene moiety, respectively.

Interestingly, methylation of a bromothiazolide at position 3 on the benzene (RM4820) completely abolished the efficacy against N. caninum (10). Our studies with Giardia also indicate that the nitro group is not the only determinant of antigiardial activity. Methylation or methoxylation on position 3 of the benzene moiety had a negative impact on the efficacy compared to that of the unmodified compounds (for example, compare RM4802 to NTZ, RM4805 to TIZ, or RM4803 to RM4820). Moreover, methylation of the benzene ring at position 5, as in RM4807, had an even more pronounced negative impact, rendering the compound completely inactive. These changes in activity may reflect transport of the drug or other reactions within the cell and may not be directly related to the mechanism of action. The 5-methyl group of RM4807 may impede the reduction of the nitro group without being involved in the mechanism itself. RM4808, with a nitro group on an additional benzene ring attached with a sulfone to the thiazole moiety, was also completely inactive. Whether these changes in antiparasitic efficacy upon modification of the benzene ring are related to changes in the rate of drug uptake or whether different targets could be affected by different domains of the molecule remains to be investigated. Additional studies employing a range of other thiazolide derivatives are in progress.

In order to investigate how NTZ affects G. lamblia trophozoites under axenic culture conditions, SEM and TEM studies were performed comparatively with MTZ-treated parasites. Trophozoites treated with NTZ showed distinct alterations on the ventral disk surface membrane, which were already evident after 1 h of NTZ treatment and actually, during the following 24 h, remained the main cellular effect seen by SEM on the surface of NTZ-treated Giardia trophozoites. At present, we do not know what is causing these ventral disk surface membrane alterations. Intracellularly, distinct alterations, including the formation of cytoplasmic vacuoles and structural disintegration of the cytoplasmic compartments, were observed by TEM, showing that the parasite is seriously impaired both metabolically and structurally as early as after 1 to 5 h of NTZ treatment. We therefore conclude that the damage imposed on the ventral disk membrane is a specific mechanism leading to parasite death. If the ventral disk is responsible not only for attachment (14, 19, 21) but also for the absorption of nutrients, corresponding membrane damage will inevitably be followed by nutrient starvation of the trophozoites, resulting in a negative energy balance (18), detachment, and, finally, cell death.

Interestingly, light microscopic inspection and SEM demonstrated that trophozoites treated with MTZ show a different pattern of alterations. Parasites exhibited a tendency to swell, as described earlier (26), and in contrast to NTZ-treated parasites, trophozoites did not exhibit these alterations on the ventral disk membrane, but significant lesions were visible on the dorsal surface, most notably at 24 h of MTZ treatment. Similar alterations have been described previously on trophozoites treated with saturated fatty acids (23). Studies on the ultrastructural effects of these drugs towards cysts have revealed that NTZ and MTZ exhibit different effects, and they also differentially affect cyst viability (3). This clearly demonstrates significant differences in the mechanisms by which MTZ and NTZ kill G. lamblia trophozoites. Preliminary studies with MTZ- and NTZ-resistant strains show clear differences in the facilities to generate MTZ-resistant strains and NTZ-resistant strains (data not shown), a finding which is in agreement with findings with Helicobacter pylori (15, 22) and Cryptosporidium (11).

Effects of thiazolides on Giardia trophozoites in coculture with Caco2 cells. While the previous studies were undertaken under axenic culture conditions, a culture system in which G. lamblia trophozoites are maintained by attaching to Caco2 cells was established. Trophozoites were shown to adhere to Caco2 cells in a saturable manner, which argues for a limited number of binding sites on the Caco2 cell surface. In addition, we found that trophozoites can survive attached to Caco2 cells in Caco2 growth medium for a period of up to 48 h; during this time span, however, parasites do not proliferate.

Nevertheless, in contrast to those cell lines used for attachment assays in previous studies (6, 21), Caco2 cells are immortal and thus can be grown easily in a highly reproducible manner. For our studies with thiazolides and MTZ, a 24-h incubation period was the most useful. The differences in IC50 values observed in axenic cultures, were, however, obliterated in the coculture system, as only those compounds with low IC50 values in axenic culture—those containing nitro groups—showed significant inhibition of trophozoite attachment at a concentration of 30 µM. This is a drug concentration that kills all trophozoites in axenic cultures. One explanation for this observation could be that the thiazolides are inactivated by the Caco2 cells (e.g., by glycosylation) and/or stored in intracellular compartments; thus, the trophozoites would encounter at the cell surface thiazolide concentrations lower than in those in the medium. It is also possible that the presence of an additional intestinal component, provided by Caco2 cells as an adhesion matrix, interferes in the direct action of these drugs. Clearly, further investigations are needed to demonstrate the identities of the factors involved.

A model for the mode of action of thiazolides. The differences in the effects of NTZ and MTZ outlined above suggest differences in the modes of action of both compounds. The nitro group of MTZ is commonly admitted to be reduced to a toxic radical by nitroreductases that are present in microaerophilic microorganisms, including bacteria and protists, but not in host cells (17, 25). For NTZ and related compounds, evidence for an in vivo reduction is lacking. Although the nitro group is a prerequisite for thiazolides with low inhibitory constants in Giardia, it is not sufficient, since the efficacy of RM4805 is impaired and RM4807 is inactive. An alternative explanation would be that modification of the benzene ring, such as the 5-methyl group of RM4807, may impede the reduction of the nitro group. RM4820, an NTZ derivative with a bromo instead of a nitro group, shows a moderate inhibition of trophozoite growth in axenic culture. Moreover, in Neospora, an intracellular apicomplexan parasite, RM4820 exhibits a proliferation inhibitory effect comparable to that of NTZ (10). The same was observed for Cryptosporidium parvum (unpublished results). The fact that a modification of the salicylic acid moiety (by, e.g., methylation) reduces the activity or even renders the compounds ineffective suggests a more complex model for the mode of action of thiazolides. In this model, both the thiazole moiety with or without nitro group and the salicylic acid moiety of the molecule determine host specificity and efficacy. Besides the unique mode of action through the nitro group by formation of radicals and nitrosative stress (18, 26, 27), thiazolides could interact with specific signaling proteins via their salicylic acid moiety. Salicylic acid has been identified as a signal molecule in different kingdoms, with functions as widespread as the triggering of defense reactions in plants (for an example, see reference 9) and inhibiting cyclooxygenase in humans (31). Therefore, it is possible that also in Giardia and related protists, thiazolides interfere in intracellular signaling pathways through their salicylic acid moiety. For instance, the correct targeting of proteins destined to reach the extracellular space (20) could be affected, and this could lead to the observed proteolytic degradation of crucial components of the ventral disk membrane and ultimately to cell death.


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  		TABLE 1 Continued

 

   ACKNOWLEDGMENTS
 
We are indebted to Andrew Stachulski, Shelley Moores, Chandrakala Pidathala, and Neil G. Berry (Romark Center for Drug Discovery, Department of Chemistry, University of Liverpool) for the synthesis of the thiazolides. We thank Maja Suter (Institute of Animal Pathology) and Tony Wyler (Institute of Cell Biology, University of Bern) for access to their electron microscopy facilities.

This study was financially supported by Romark Research Laboratories.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Parasitology, University of Bern, Länggass-Strasse 122, CH-3012 Bern, Switzerland. Phone: 41 31 6312384. Fax: 41 31 6312477. E-mail for Andrew Hemphill: hemphill{at}ipa.unibe.ch. E-mail for Joachim Müller: joachim.mueller{at}ipa.unibe.ch. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Antimicrobial Agents and Chemotherapy, January 2006, p. 162-170, Vol. 50, No. 1
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.1.162-170.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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