Abstract

Giardia lamblia (also known as G. duodenalis or G. intestinalis), a fl agellated protozoan, is the most common causative agent of persistent diarrhea world-wide. The life cycle includes motile, fl agellated tro-phozoites parasitizing the upper intestine and thick-walled cysts forming in the lower intestine, which are subsequently shed with the feces. Chemo-therapy is directed against the trophozoite stage. The current antigiardial therapy of choice is metronida-zole. In the case of resistance, alternatives are other nitroimidazoles, quinacrine, albendazole, and nita-zoxanide. Here, we present a review of current anti-giardial drugs and of their modes of action.

Introduction

For the treatment of giardiasis, chemotherapy is the method of choice. After the introduction of quinacrine as the fi rst antigiardial drug in the 1940s, metronida-zole (commercially known as Flagyl®) and other ni-troimidazoles such as tinidazole have been used as a therapy of choice for more than fi ve decades. In the case of resistance formation, substitution therapies include benzimidazole albendazole, the acridine de-rivative quinacrine – historically the fi rst antigiardial drug –, or the aminoglycoside paromomycin alone or in combination with metronidazole (see for review Upcroft and Upcroft, 2001 and for a recent clinical study Mørch et al., 2008). In the mid-1990s, a new antiparasitic agent, the thiazolide nitazoxanide (com-mercially known as Alinia®), has been introduced in the market. Nitazoxanide has been approved in the

USA for the treatment of persistent diarrhea due to cryptosporidiosis and giardiasis in people older than 12 years (reviewed by Hemphill et al., 2006). More-over, in vitro effects of natural compounds such as isofl avones, polyenes or saturated fatty acids against trophozoites have been described, but clinical data are lacking. An overview of antigiardial compounds with references is given in Table 1.

In this chapter, we will focus on the mode of action of these drug families (so far as known) and put some new light on resistance mechanisms.

Mode of Action and Drug Targets

General Remarks

The ideal situation for the mode of action of antipara-site drugs consists of binding to a target protein (en-zyme or receptor) resulting in the inhibition of essential cellular functions. Such targets are identi-fi ed by analyzing structural and metabolic alterations of the parasite due to the drug followed by the isola-tion of drug binding proteins or the analysis of resis-tant vs. sensitive parasites. The isolated targets are then validated by inhibition studies in vitro, structural analysis of protein-ligand complexes, and – if possi-ble – knock-out and overexpression studies of the corresponding gene in the target organism (see e.g. Wang, 1997).

In an ideal situation, the drug-target interaction de-pends on distinct structural features of the target. In this case, point mutations causing the presence or absence of a single amino acid or nucleotide (in the case of rRNA as a target) discriminate between resis-tance or susceptibility. In reality, the situation is more complex. In resistant organisms, the “ideal” target may still be present, but the drug cannot access it due

Treatment of Giardiasis and Drug Resistance

Joachim Müller, Andrew Hemphill and Norbert Müller

Norbert Müller ( ) Institute of Parasitology, University of Bern, Länggass-Strasse 122, 3012 Bern, Switzerland

J. Müller et al.

Methods for the Determination of Drug Effi cacy

In order to determine the effi cacy of antigiardial drugs (e.g. in terms of IC50 values), a couple of methods are currently in use. The most classical way is counting trophozoites grown axenically in the presence of drugs (see e.g. Müller et al., 2006). The incorporation of 3H-thymidine is less subjective (see e. g. Adagu et al., 2002). Both methods are time-consuming or need high-level equipment. An elegant alternative is the use of the vital staining resazurin (Alamar Blue; Bénéré et al., 2007). Viable cells reduce resazurin forming a pink compound absorbing at 550 nm and fl uorescing at >590 nm. Although it is possible to use spectrophotometry for quantifying the product, the best sensitivity is obtained with fl uorimetry. Fluorim-eters are, however, expensive and therefore not present in many labs. Moreover, all these methods cannot be

to uptake limitation or detoxifi cation mechanisms. A search for drug targets (e.g. by protein binding studies) would thus yield detoxifi cation enzymes, transporters or even irrelevant drug binding proteins besides the “true” target. Conversely, susceptibility to a drug may be triggered by the ability to convert a (ineffective) prodrug to the (effective) drug. In this case, search for targets have to be performed with the effective mole-cule, otherways, the transforming enzyme would be identifi ed rather than the target itself.

In the case of antiparasite drugs (as well as other antibiotics), the situation is rendered more compli-cated by the fact that the drug acts in the vicinity or even inside the host cell (in the case of intracellular pathogens). Therefore, the mode of action of the drug may be modulated if not depend on the susceptibility of the host cell and not the parasite itself. In the fol-lowing paragraphs, we will discuss some examples within the framework of antigiardial drugs.

Table 1 Overview of antigiardial drugs. With regard to the effi cacy, the order of magnitude of the IC50 from in vitro studies and – if available – the dose for patients are given

Class Compound Effi cacy Reference

Acridines Quinacrine In vitro (10–7

M) Bell et al., 1991; Bénéré et al., 2007

In vivo (100 mg tid + MET 750 mg tid for 2–3 weeks)

Nash et al., 2001; Mørch et al., 2008

Aminoglycosides Paromomycin In vitro (10–4

–10–5

M) Edlind, 1989

In vivo: 500 mg tid for 1 week Mørch et al., 2008

Benzimidazoles Albendazole In vitro (10–8

M) Katiyar et al., 1994, Upcroft and Upcroft, 2001; Cruz et al., 2003

In vivo (400 mg bid + MET 250 mg bid or tid for 1 week)

Mørch et al., 2008

Bis-benzimidazoles In vitro (10–8

–10–6

M) Bell et al., 1993

Fatty acids Dodecanoic acid In vitro (10–5

M) Rayan et al., 2005

Fluoroquinolones Ciprofl oxacine In vitro (10–4

M) Sousa and Poiares-da-Silva, 2001

Isofl avones Genistein Formononetin

In vitro (10–6

M)In vitro (10

–8 M)

Khan et al., 2000; Sterck et al., 2008

Nitrofuranes Furazolidone In vitro (10–5

–10–6

M) Cedillo-Rivera and Munoz, 1992

Nitroimidazoles Metronidazole Tinidazole

In vitro (10–6

M); in vivo Gardner and Hill, 2001

Diaminidines Pentamidine and derivatives In vitro (10–8

–10–6

M) Bell et al., 1991

Polyenes Allicin and derivatives In vitro Anthony et al., 2005

Thiazolides Nitazoxanide In vitro (10–6

M)In vivo

Fox and Saravolatz, 2005; Hemphill et al., 2006

Treatment of Giardiasis and Drug Resistance

Fluoroquinolones such as ciprofl oxacine inhibit DNA-gyrases and topoisomerases from prokaryonts (Kidwai et al., 1998) and are highly effective against G. lamblia as shown in a descriptive study (Sousa and Poiares-da-Silva, 2001). It is, however, unclear, which type of enzyme is inhibited there since more pro-nounced studies are lacking. A phylogeny analysis of type II topoisomerases shows that the gene of G. lamblia (GlTop2) is eukaryont-like and not closely related to prokaryont topoisomerases (He et al., 2005).

Aminoglycosides bind to the small subunit of ribo-somes, more exactly to distinct features of the 16S-rRNA secondary structure and thereby inhibit protein biosynthesis (see e.g. Brodersen et al., 2000 for a de-tailed structural analysis). Therefore, specifi c muta-tions in this region confer resistance to various aminoglycosides in various prokaryonts. G. lamblia has a 16S-rRNA structure similar to prokaryonts with a primary sequence suggesting that only paromomycin and hygromycin are effective, other well known amin-oglycosides such as kanamycin are not. This pattern correlates well with observed susceptibility or resis-tance to a panel of aminoglycosides (Edlind, 1989).

Cytoskeleton Proteins as Targets

Early studies with benzimidazoles such as albenda-zole showed effects on the cytoskeleton, especially in the region of the ventral disk (Fig. 1; see e.g. Chavez et al., 1992; Hemphill et al., 1996). Molecular genet-ics has revealed that sensitivity to albendazole (or other benzimidazoles) in evolutionary distant organ-isms such as fungi, nematodes, platyhelminthes, and various protozoa including G. lamblia is correlated to the presence of specifi c alleles of β-tubulin-genes (Kirk-Mason et al., 1988; Driscoll et al., 1989; Katiyar et al., 1994; Kwa et al., 1995; Henriquez et al., 2008), the substitution of Phe to Tyr in position 200 being suffi cient for switching from benomyl sensitivity to resistance as shown in nematodes (Kwa et al., 1995). Molecular modeling of the putative albendazole bind-ing site in albendazole resistant Acanthamoeba β-tubulin indicates that 4 of 13 residues are different from tubulins of sensitive organisms. Resistance is conferred when, besides Phe200, Phe167 is replaced (Henriquez et al., 2008).

used when trophozoites are not grown axenically, but in a coculture system with intestinal cells (e.g. Caco 2). In this system, trophozoites may be quantifi ed us-ing real-time PCR with a Giardia-specifi c target (Müller et al., 2006).

A suitable alternative would be the use of a report-er strain as shown for instance for Toxoplasma gondii expressing β-galactosidase (McFadden et al., 1997; see for a recent application Müller et al., 2009a). In the case of Giardia, a reporter strain expressing glucuronidase A from E. coli has been created (Müller et al., 2009b). This strain is well suited for the deter-mination of IC50 values and for high-throughput drug screening using simple colorimetric assays. More-over, it can be used in a coculture system with host cells thus allowing the determination of the “thera-peutical window”, i.e. the gap between drug effi cacy and host cell toxicity in one screen.

Nucleic Acids as Targets

Early studies have shown that the acridine derivative quinacrine, the fi rst antigiardial drug, interacts with DNA inhibiting replication and RNA and protein bio-synthesis in prokaryonts (Ciak and Hahn, 1967). Like other acridine derivatives, quinacrine is an intercalat-ing agent binding to DNA with a preference for (A+T)-rich regions and thereby blocking DNA repli-cation and RNA biosynthesis (Wilson et al., 1994). In studies concerning pentamidine derivatives (Bell et al., 1991) and bis-benzimidazoles (Bell et al., 1993), very nice correlations between the binding af-fi nities of these compounds to calf thymus DNA and their effi cacies against Giardia lamblia have been pointed out. Since DNA binding is by far not species specifi c and therefore prone to side effects on host cells, the relevance of a DNA binding agent as an an-timicrobial drug resides in other – more species spe-cifi c – properties like a differential uptake or metabolism or the presence of other targets besides DNA. For instance, pentamidines and a variety of other DNA binding compounds such as etoposide in-duce the cleavage of Trypanosoma DNA minicircles in a pattern that resembles the action of topoisomerase II inhibitors (Shapiro and Englund, 1990). DNA mod-ifying enzymes may thus constitute more selective targets for antimicrobial agents.

J. Müller et al.

A mode of action similar to nitroimidazoles has been postulated for nitrothiazolides. Upon oral up-take, nitazoxanide, the best studied nitrothiazolide, is rapidly deacetylated to tizoxanide and further me-tabolized to tizoxanide-glucuronide (Fox and Saravolatz, 2005). Tizoxanide has been reported to display antimicrobial activity similar to nitazoxanide, while tizoxanide-glucuronide is largely inactive against a number of pathogens including Giardia. Thiazolides without the thiazole-associated nitro group have been shown to exhibit decreased or no effi cacy against Giardia (Adagu, et al., 2001; Müller et al., 2006). Therefore, an involvement of the nitro group in the mechanism of action, with the participa-tion of PFOR similar to metronidazole, is likely (Wright et al., 2003). The nitro group is also required for in vitro activity against anaerobic bacteria (Pankuch and Apelbaum, 2006). Moreover, nitazox-anide inhibits activities of recombinant PFOR from various bacteria and parasites including G. lamblia (Sisson et al., 2002). A reduction of nitazoxanide, however, has never been observed in vivo. Instead, there is some evidence that unreduced nitazoxanide blocks an intermediate step of the reaction thus inhib-iting PFOR in a non-competitive manner (Hoffman et al., 2007). When G. lamblia trophozoites are treat-ed with nitrothiazolides, ultrastructural analyses

Drugs Interfering with Intermediary Metabolism

The nitroimidazole metronidazole is clearly the best studied compound affecting intermediary metabo-lism. When trophozoites are treated with metronida-zole the cell looses motility within a few hours followed by appearance of large zones of lesions on the dorsal shield and swelling of the cell body (Fig. 2; see e.g. Müller et al., 2006). According to a wi dely referred theory, metronidazole (as a prodrug) is reduced to a nitro radical by electrons coming from the enzyme pyruvate:fl avodoxin/ferredoxin oxi-doreductase (PFOR), a protein lacking in higher eu-karyotic cells (Horner et al., 1999). The radical causes irreversible damages. Results obtained with the mi-croaerophilic parasite Trichomonas vaginalis suggest, however, another mode of action. By comparing 2-D-protein electrophoresis patterns from treated and un-treated cells, the authors suggest that metronidazole binds to the sulfhydryl group in the active center of various enzymes including thioredoxin reductase thereby impairing essential cellular functions (Leitsch et al., 2009). Also in this model, an activated form of metronidazole would act on multiple targets, the number being limited to enzymes with sulfhydryl groups in their active center.

A: Albendazole B: Control

Fig. 1 Examples of ultrastructural changes induced by albendazole and visualized by transmission electron microscopy, A, albendazole; B, control; N, nucleus

Treatment of Giardiasis and Drug Resistance

Recent studies have shown that not only nitazox-anide, but also derivatives lacking the nitro group (re-viewed in Hemphill et al., 2007) exhibit in vitro activity against the intracellular apicomplexan para-sites N. caninum, Cryptosporidium parvum and Besnoitia besnoiti (Hemphill et al., 2006; Cortes et al., 2007) and as well as proliferating host cells (Müller et al., 2008c). Therefore, it cannot be exclud-ed that the effi cacy of nitazoxanide against giardiasis and cryptosporidiosis in patients is partly due to ef-fects on intestinal cells. In vivo studies on these dis-eases with non-nitro-thiazolides are lacking. In proliferating host cells (Caco2), inhibition of gluta-thione-S-Transferase P1 (GSTP1) by nitazoxanide

reveal severe damages of the ventral disk instead of damages of the dorsal surface membrane as observed with metronidazole (Fig. 2; see e.g. Müller et al., 2006). In G. lamblia, a nitroreductase as a nitazox-anide-binding protein was identifi ed and character-ized. Nitroreductase is inhibited by nitazoxanide, and, as for PFOR, there is no evidence for a reduction by nitroreductase (Müller et al., 2007b). Overexpres-sion of this nitroreductase is, however, correlated with increased sensitivity to metronidazole and nita-zoxanide (Nillius et al., 2011). Besides nitrore-ductase, protein disulfi de isomerases from G. lamblia (Müller et al., 2007a) and Neospora caninum (Müller et al., 2008b) are potential targets for nitazoxanide.

A: Control B: NTZ

C: MTZ D: MTZ

Fig. 2 Examples of ultrastructural changes induced by nitazoxanide and metronidazole and visualized by scanning electron micro-scopy, A, control; B, nitazoxanide (NTZ); C, D, metronidazole (MTZ)

J. Müller et al.

protein. Nucleoside hydrolase activity is inhibited by the three isofl avones mentioned above in a range of concentrations where daidzein and genistein inhibit trophozoite growth in vivo. In Crithidia (Parkin et al., 1991), Leishmania (Shi et al., 1999) or Trypanosoma (Parkin, 1996), the function of nucleoside hydrolase is central in salvaging purine nucleosides from the host and essential since the parasites lack purine de novo synthesis. Inhibition would thus lead ultimately to a block of DNA synthesis and thus cell division. This effect would be rather slow. Formononetin acts faster by stopping motility and inducing detachment within less than 2 h (Lauwaet et al., 2010, and own, unpublished data) and is inhibitory at ten times lower concentrations suggesting other mechanisms to be in-volved in addition such as depolarization of the mem-brane due to uncoupling effects or direct inhibition of membrane ATPases. However, experimental evidence for any of these possibilities is lacking.

Resistance Formation

Besides the search for drug binding proteins, another way to elucidate the action of drugs is the comparison of resistant vs. non-resistant trophozoites. Resistant trophozoites are created by selecting growing cells in the presence of increasing drug concentrations fol-lowed by cloning and characterization of the resistant clones vs. wildtype clones. Moreover, in some cases, drug (mainly metronidazole) resistant strains have been isolated from patients and compared to non-re-sistant strains. Table 2 gives an overview of selected studies.

Resistance to metronidazole and other nitroimida-zoles has been induced in vitro; and various genotypi-cally distinct isolates with reduced drug susceptibility have also been found in human patients (Gardner and Hill, 2001; Upcroft and Upcroft, 1993). Formation of resistance to metronidazole is associated with an al-tered uptake of the drug (Boreham et al., 1988; see also Upcroft et al., 1996 for quinacrine), downregula-tion of the PFOR activity (Upcroft and Upcroft 2001). This is consistent with the model claiming an involve-ment of PFOR in metronidazole activation (Townson et al., 1996; Sisson et al., 2002). Recent fi ndings in T. vaginalis suggest, however, that metronidazole and other nitroimidazoles covalently bind and thereby

and some non-nitro-thiazolides is well correlated with their effi cacy to induce apoptosis. Overexpres-sion and downregulation of GSTP1 is correlated with an increase, or decrease, of sensitivity, respectively (Müller et al., 2008c).

Compounds of Plant Origin with Miscellaneous Targets

In order to improve the situation in chemotherapy of metronidazole resistant giardiasis, new potential pharmaceuticals from natural sources have been eval-uated regarding their antigiardial activity in vitro. In general, crude extracts from traditional medicinal herbs are tested with respect to antigiardial activities and then fractionated in order to identify the active principles (see for review Anthony et al., 2005). Essential oils are a well explored source for antimi-crobial compounds. A good example for a study on antigiardial properties in oregano extracts is given by Ponce-Macotela et al. (2006). Often, essential oils have a broad spectrum of effi cacy and therefore a po-tential toxicity to host cells. A good example is a study where active compounds have been identifi ed from Allium extracts (Harris et al., 2000). In this study, the compound with the lowest IC50 is allyl alco-hol, a compound with an unspecifi c toxicity and a po-tential cancerogene. Allicin, another molecule effective against Giardia and other parasites such as Entamoeba, bind to SH-groups from cysteine pro-teases and other SH-proteins including thioredoxin reductase (see Ankri et al., 1997; Ankri and Mire-lman, 1999), a mode of action recently postulated for metronidazole (Leitsch et al., 2009).

Another well studied group are isofl avones. Isofl a-vones are mainly found in Leguminosae where they have anti-oxidant, anti-microbial and signalling func-tions (e. g. reviewed in Dakora and Phillips, 1996; Dixon and Steele, 1999). Within this group of com-pounds, Khan et al. (2000) identifi ed formononetin, a major isofl avone of Ononis sp, as the most potent an-tigiardial agent. In vitro, formononetin has an IC50

value in the range of 10–7 M, thus one order of magni-tude lower than the non-methylated daidzein and genistein, two isofl avones of soybean (Sterk et al. 2008). By daidzein-affi nity chromatography, a nucle-oside hydrolase was identifi ed as daidzein-binding

Treatment of Giardiasis and Drug Resistance

Table 2 Overview of studies on resistance formation in G. lamblia

Drug Source of resistance Method Important fi ndings Reference

Albendazole Induction of resistance in vitro in WB

Comparison to wildtype with respect to growth

Resistance at 4.5 µM for several weeks, normal cyst formation

Lindquist, 1996

Induction of resistance in vitro in WB 1B

Molecular genetics, ultrastructural analysis

Induced resistance not related to β-tubulin modifi cations

Upcroft et al., 1996

Patient isolates Comparison of isolates with respect to growth

Isolates exhibit variations in their sensitivities prior to drug exposition

Argüello-García et al., 2004

Induction of resistance in vitro in WB 1

Representational difference analysis of gene expression

Induced resistance not related to β-tubulin modifi cations; antigenic variation

Argüello-García et al., 2009

Quinacrine Induction of resistance in vitro in 7 different lines

Comparison to sensitive lines with respect to growth, investigation of quinacrine uptake by fl uorescence

Quinacrine excluded from resistant lines, multidrug resistance in some lines

Upcroft et al., 1996

Metronidazole Patient isolates with different susceptibilities

Analysis of uptakegrowth

Resistant isolates have a lower uptake

Boreham et al., 1988

Patient isolates, induction of resistance formation in vitro

PFOR assay PFOR activity is lower in resistant isolates

Upcroft and Upcroft 1993, 2001 and references therein

Patient isolates Comparison of isolates with respect to growth

Isolates exhibit variations in their sensitivities prior to drug exposition

Argüello-García et al., 2004

Induction of resistance in vitro in WB 1

Representational difference analysis of gene expression

PFOR expression higher in resistant clones; antigenic variation

Argüello-García et al., 2009

Induction in vitroT. vaginalis

Comparison to wildtype by proteomics

Metronidazole binds to proteins of thioredoxin pathway

Leitsch et al., 2009

Nitazoxanide Induction of resistance formation in vitro in WB C6

Comparison to wildtype with respect to expression of selected genes and by microarray

Cross-resistance to metronidazole. Complex changes of gene expression including antigenic variation and overexpression of chaperones in resistant clone

Müller et al., 2007a, 2008a

Isofl avones Induction of resistance formation in vitro in WB C6

Comparison to wildtype with respect to expression of selected genes and by microarray

Most pronounced changes of gene expression concern antigenic variation

Sterk et al., 2007Unpublished data

J. Müller et al.

expression patterns has been found upon expression of neomycin phosphotransferase as a resistance mark-er and a subsequent selection for puromycin resistant trophozoites (Su et al., 2007).

As mentioned above, nitazoxanide inhibits protein disulfi de isomerases (PDIs) from G. lamblia (Müller et al., 2007a) and N. caninum (Müller et al., 2008b). Inhibition of PDIs in vivo could lead to the formation of badly folded proteins and thus to an impairment of central metabolic and regulatory processes. Cells that overexpress chaperonins and proteins involved in the stabilisation of subcellular structures such as ankyrins could overcome the deleterious effects due to PDI inhibition.

Such indirect, stabilizing effects due to chaper-onins or related proteins could also explain why arti-fi cially generated albendazole resistance (Upcroft et al., 1996; Argüello-Garcia et al., 2009) is not cor-related to specifi c mutations of the β-tubulin gene as found in other albendazole resistant organisms. In this context, it is noteworthy that antigenic variation was also found to be a striking phenomenon associ-ated with in vitro resistance formation against al-bendazole (Argüello-Garcia et al., 2009).

Giardia populations seem to have very fl exible mechanisms in order to respond to changes in the en-vironment such as drug pressure, presence of antibod-ies or even changes in medium composition and to changes of the internal milieu by introduction of transgenes (Su et al., 2007) in a way that complex gene expression patterns seem to be fi ne-tuned in or-der to fi nd an optimal compromise between speed of growth and survival. How can these changes be explained? Individual trophozoites in cultures of G. lamblia (clone WB C6) exhibit substantial varia-tions in their degree of susceptibility/resistance to dif-ferent drugs even in absence of any previous drug pressure (Argüello-Garcia et al., 2004). This suggests that resistance formation within a G. lamblia in vitro culture seems to have its origin in a drug-independent process of continuous growth competition between relatively drug-susceptible and drug-resistant tropho-zoite subpopulations. The differences in these sub-populations may be explained by genetic effects, i.e. random point mutations all over the genome and se-lection of resistant phenotypes upon drug application or by epigenetic effects (transcriptional or posttran-scriptional) resulting in subpopulations with different

inactivate proteins related to the thioredoxin reductase pathway. Resistant cells overcome this blocking by reregulating other enzymes involved in oxidoreduc-tive processes, such as PFORs. In this model, down-regulation of PFOR would be a consequence rather than a prerequisite of resistance formation (Leitsch et al., 2009). In another study, even a positive correla-tion between metronidazole-resistance formation and PFOR gene transcription activity has been observed (Argüello-Garcia et al., 2009). Furthermore, possible giardial metronidazole-resistance mechanisms in-volving reduced uptake of the drug (Boreham et al., 1988) or complex changes in the gene expression pat-tern including alterations of those genes (encoding variant surface proteins, VSPs) that mediate antigenic variation of the parasite (Müller et al., 2007; 2008; see also below) have been described.

In order to elucidate the biochemical nature of re-sistance formation against nitazoxanide metronida-zole, and formononetin, G. lamblia WB C6 clones resistant to these drugs were generated (Müller et al., 2007a, 2008a; Sterck et al., 2007) and compared to the wildtype WB C6 with respect to their growth be-havior and to the expression pattern of genes that are potentially involved in resistance formation. In all cases, resistance formation occurred after a small number of selection cycles. The pattern of up- and downregulated mRNAs was completely different in a nitazoxanide/metronidazole double resistant line, a line resistant for metronidazole only and a formonon-etin resistant line. The only annotated ORFs down-regulated in all screens encoded the major surface-labeled trophozoite antigen 417 encoding the major surface antigen C6 (TSA417; Müller et al. 2007a, 2008a; Sterk et al., 2007 and unpublished data). In the nitazoxanide/metronidazole double re-sistant line, genes potentially involved in protein phosphorylation and network formations, especially ankyrins, and chaperonins (especially the cytosolic heat shock protein 70) are upregulated. The roles of these chaperonins in Giardia are not well understood. In human cells, molecular chaperones such as HSP72 and HSP27 prevent cells from apoptosis induced by heat shock (Gabai and Sherman, 2002). If similar mechanisms occur in Giardia, a general response to stress via induction of chaperonins could be respon-sible for thermotolerance as well as for drug resis-tance. Interestingly, a similar change in gene

Treatment of Giardiasis and Drug Resistance

a common, underlying mechanism far from being un-derstood. Future approaches aimed at transgenic up- or down-regulation of putative drug target proteins in G. lamblia are expected to provide conclusive insights into both the mode of action of anti-giardial drugs and the mechanisms that mediate resistance against these drugs.

References

Adagu IS, Nolder D, Warhurst D, and Rossignol JF (2002) In vitro activity of nitazoxanide and related compounds against isolates of Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. J Antimicrob Chemother 49: 103–111

Adam M, Murali B, Glenn N, and Potter SS (2008) Epigenetic inheritance based evolution of antibiotic resistance in bac-teria. BMC Evol Biol 8: 52

Argüello-García R, Cruz-Soto M, Romero-Montoya L, and Ortega-Pierres G (2004) Variability and variation in drug susceptibility among Giardia duodenalis isolates and clones exposed to 5-nitroimidazoles and benzimidazoles in vitro. J Antimicrob Chemother 54: 711–721

Argüello-García R, Cruz-Soto M, Romero-Montoya L, and Ortega-Pierres G (2009). In vitro resistance to 5-nitroimida-zoles and benzimidazoles in Giardia duodenalis: variability and variation in gene expression. Infect Genet Evol 9: 1057–1064

Ankri S and Mirelman D (1999) Antimicrobial properties of allicin from garlic. Microbes Infect 1: 125–129

Ankri S, Miron T, Rabinkov A, Wilchek M, and Mirelman D (1997) Allicin from garlic strongly inhibits cysteine protei-nases and cytopathic effects of Entamoeba histolytica. An-timicrob Agents Chemother 41: 2286–2288

Anthony J-P, Fyfe L, and Smith H (2005) Plant active compo-nents – a resource for antiparasitic agents? Trends Parasitol 21: 462–468

Bell CA, Dykstra CC, Naiman NA, Cory M, Fairley TA, and Tidwell RR (1993) Structure-activity studies of dicationi-cally substituted bis-benzimidazoles against Giardia lam-blia: correlation of antigiardial activity with DNA binding affi nity and giardial topoisomerase II inhibition. Antimi-crob Agents Chemother 37: 2668–2673

Bell CA, Hall JE, Cory M, Fairley T, and Tidwell RR (1991) Structure-activity relationships of pentamidine analogs against Giardia lamblia and correlation of antigiardial activity with DNA binding affi nity. Antimicrob Agents Chemo ther 35: 1099–1107

Bénéré E, Inocencio da Luz RA, Vermeesch M, Cos P, and Maes L (2007) A new quantitative in vitro microculture method for Giardia duodenalis trophozoites. J Microbiol Methods 71: 101–106

Boreham PF, Phillips RE, and Shepherd RW (1988) Altered uptake of metronidazole in vitro by stocks of Giardia intes-tinalis with different drug sensitivities. Trans R Soc Trop Med Hyg 82: 104–106

gene expression patterns. In the latter case, exposure of the trophozoites to a drug in sublethal concentra-tions might even enhance conversion from a sensitive to a resistant phenotype.

The only common fi nding in all resistant lines is the change in variable surface protein gene expres-sion (where investigated) resulting in antigenic varia-tion (Müller and Gottstein, 1998; Svärd et al., 1998; Prucca and Lujan, 2009). Antigenic variation in G. lamblia allows the parasite to escape the host im-mune response (Müller and Gottstein, 1998; Müller, and von Allmen, 2005; Roxström-Lindquist et al., 2006). Epigenetic mechanisms at the transcriptional level involving histone acetylation (Kulakova et al., 2006) and at the post-transcriptional level involving RNA interference (Prucca et al., 2008) are considered as controlling antigenic variation (Prucca and Lujan, 2009). Thus, common epigenetic mechanisms may control both antigenic variation and drug resistance in Giardia.

The role of epigenetics in antigenic variation and drug resistance in Giardia and other parasites is – of course – far from being understood, but may be of a broader interest since evolution of antibiotic resis-tance initiated by increasing sublethal doses of vari-ous antibiotics has been attributed to epigenetic inheritance also in bacteria (Adam et al., 2008). The overview in Table 2 shows that most studies have been performed with the strain WB C6 showing a high degree of antigenic variation (Nash et al., 1990b). Drug resistance studies from other strains with little variation such as GS-M-83-H7 or WB A6 (Nash et al., 1990a) are lacking but would be most useful in put-ting the light on the relationship between antigenic variation and drug resistance formation.

Conclusions

After more than 7 decades of antigiardial chemother-apy, metronidazole is still the treatment of choice. Novel drugs such as nitazoxanide or plant-derived compounds do not offer obvious advantages with re-spect to effi cacy or resistance formation. A couple of studies concerning resistance formation suggest a more complex mechanism than the mere mutation of a target protein. Where investigated, resistance forma-tion is correlated with antigenic variation suggesting

J. Müller et al.

ment of gastrointestinal infections. Expert Opin Pharmaco-ther 7: 953–964

Hemphill A, Müller N, and Müller J (2007) Structure-function relationship of thiazolides, a novel class of anti-parasitic drugs, investigated in intracellular and extracellular proto-zoan parasites and larval-stage cestodes. Antiinf Agents Med Chem 6: 273–282

Hemphill A, Stäger S, Gottstein B, and Müller N (1996) Elec-tron microscopical investigation of surface alterations on Giardia lamblia trophozoites after exposure to a cytotoxic monoclonal antibody. Parasitol Res 82: 206–210

Henriquez F, Ingram PR, Muench SP, Rice DW, and Roberts CW (2008) Molecular basis for the resistance of Acan-thamoeba tubulins to all major classes of antitubulin com-pounds. Antimicrob Agents Chemother 52: 1133–1135

Hoffman PS, Sisson G, Croxen MA, Welch K, Harman WD, Cremades N, and Morash MG (2007) Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of Heli-cobacter pylori and selected anaerobic bacteria and para-sites, and Campylobacter jejuni. Antimicrob Agents Chemother 51: 867–876

Horner DS, Hirt RP, and Embley TM (1999) A Single Eubacte-rial Origin of Eukaryotic Pyruvate: Ferredoxin Oxi-doreductase Genes: implications for the Evolution of Anaerobic Eukaryotes. Mol Biol Evol 16: 1280–1291

Katiyar SK, Gordon VR, McLaughlin GL, and Edlind TD (1994) Antiprotozoal activities of benzimidazoles and cor-relations with β-tubulin sequence. Antimicrob Agents Chemother 38: 2086–2090

Khan IA, Avery MA, Burandt CL, Goins DK, Mikell JR, Nash TE, Azadegan A, and Walker LA (2000) Antigiardial activ-ity of isofl avones from Dalbergia frutescens bark. J Nat Prod 63: 1414–1416

Kidwai M, Misra P, Kumar P (1998) The fl uorinated quinolo-nes. Curr Pharmaceut Design 4: 101–118

Kirk-Mason KE, Turner MJ, and Chakraborty PR (1988) Clon-ing and sequence of β-tubulin cDNA from Giardia lamblia. Nucleic acids Res 16: 2733

Kulakova L, Singer SM, Conrad J and Nash TE (2006) Epige-netic mechanisms are involved in the control of Giardia lamblia antigenic variation. Mol Microbiol 61: 1533–1542

Kwa MSG, Veenstra JG, Van Dijk, M, and Roos MH (1995) β-tubulin genes from the parasite nematode Haemonchus contortus modulate drug resistance in Caenorhabditis ele-gans. J Mol Biol 246: 500–516

Lauwaet T, Andersen Y, Van de Ven L, Eckmann L, and Gillin FD (2010) Rapid detachment of Giardia lamblia trophozo-ites as a mechanism of antimicrobial action of the isofl a-vone formononetin. J Antimicrob Chemother. doi:10.1093/jac/dkp501

Leitsch D, Kolarich D, Binder M, Stadlmann J, Altmann F, and Duchêne M (2009) Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the fl avin enzyme thioredoxin reductase and disrupt the cellular re-dox system. Implications for nitroimidazole toxicity and resistance. Mol Microbiol 72: 518–536

Lindquist AHD ( 1996) Induction of albendazole resistance in Giardia lamblia. Microb Drug Resist 2: 433–434

McFadden DC, Seeber F, and Boothroyd JC (1997) Use of Toxoplasma gondii expressing β-galactosidase for colori-

Brodersen DE, Clemons WM Jr, Carter AP, Morgan-Warren RJ, Wimberly BT, and Ramakrishnan V (2000) The struc-tural basis for the action of the antibiotics tetracycline, pac-tamycin, and hygromycin B on the 30S ribosomal subunit. Cell 103: 1143–1154

Cedillo-Rivera R, and Muñoz O (1992) In-vitro susceptibility of Giardia lamblia to albendazole, mebendazole and other chemotherapeutic agents. J Med Microbiol 37: 221–224

Chavez B, Cedillo-Rivera R, and Martinez-Palomo A (1992) Giardia lamblia: ultrastructural study of the in vitro effect of benzimidazoles. J Protozool 39: 510–515

Ciak J and Hahn FE (1967) Quinacrine (atebrin): mode of ac-tion. Science 156: 655–656

Cortes HCE, Müller N, Esposito M, Leitão A, Naguleswaran A, and Hemphill A (2007) In vitro effi cacy of nitro- and bromo-thiazolyl-salicylamide compounds (thiazolides) against Besnoitia besnoiti infection in Vero cells. Parasitol-ogy 134: 975–985

Dakora FD and Phillips DA (1996) Diverse functions of isofl a-vonoids in legumes transcend anti-microbial defi nitions of phytoalexins. Physiol Mol Plant Pathol 49: 1–20

Dixon RA and Steele CL (1999) Flavonoids and isofl avonoids – a good mine for metabolic engineering. Trends Plant Sci 4, 394–400.

Driscoll M, Dean E, Reilly E, Bergholz E, and Chalfi e, M. (1989) Genetic and molecular analysis of a Caenorhabditis elegans β-tubulin that conveys benzimidazole sensitivity. J Cell Biol 109: 2993–3003

Edlind TD (1989) Susceptibility of Giardia lamblia to amino-glycoside protein synthesis inhibitors: correlation with rRNA structure. Antimicrob Agents Chemother 33: 484–488

Edlind TD, Hang TL, and Chakraborty PR (1990) Activity of the anthelmintic benzimidazoles against Giardia lamblia in vitro. J Infect Dis 162: 1408–1411

Escobedo AA and Cimerman S (2007) Giardiasis: a pharmaco-therapy review. Expert Opin Pharmacother 8: 1885–1902

Fox LM and Saravolatz LD (2005) Nitazoxanide: a new thia-zolide antiparasitic agent. Reviews of Anti-infective Agents 40: 1173–1180

Gabai VL and Sherman MY (2002) Interplay between molecu-lar chaperones and signaling pathways in survival of heat shock. J Appl Physiol 92: 1743–1748

Gardner TB and Hill DR (2001) Treatment of Giardiasis. Clin Microbiol Rev 14: 114–128

Harris JC, Plummer S, and Lloyd D (2001) Antigiardial drugs. Appl Microbiol Biotechnol 57: 614–619

Harris JC, Plummer S, Turner MP, and Lloyd D (2000) The microaerophilic fl agellate Giardia intestinalis: Allium sati-vum (garlic) is an effective antigiardial. Microbiology 146: 3119–3127

Hausen MA, Freitas JC Jr, and Monteiro-Leal LH (2006) The effects of metronidazole and furazolidone during Giardia differentiation into cysts. 1: Exp Parasitol 113: 135–141

He D, Wen JF, Chen WQ, Lu SQ, and Xin DD (2005) Identifi -cation, characteristic and phylogenetic analysis of type II DNA topoisomerase gene in Giardia lamblia. Cell Res 15: 474–482

Hemphill A, Müller J, and Esposito M (2006) Nitazoxanide, a broad-spectrum thiazolide anti-infective agent for the treat-

Treatment of Giardiasis and Drug Resistance

Nillius D, Müller J, and Müller N (2011) Nitroreductase (GlNR1) increases susceptibility of Giardia lamblia and Escherichia coli to nitro drugs. J Antimicrob Chemother 66: 1029–1035

Olekhnovich IN, Goodwin A and Hoffman PS (2009) Charac-terization of the NAD(P)H oxidase and metronidazole re-ductase activities of the RdxA nitroreductase of Helicobacter pylori. FEBS J 276: 3354–3364

Pankuch GA and Appelbaum PC (2006) Activities of tizoxa-nide and nitazoxanide compared to those of fi ve other thia-zolides and three other agents against anaerobic species. Antimicrob Agents Chemother 50: 1112–1117

Parkin DP (1996) Purine-specifi c Nucleoside N-Ribohydrolase from Trypanosoma brucei brucei. J Biol Chem 271: 21713–21719

Parkin DP, Horenstein BA, Abdulah DA, Estupiian B, and Schramm VL (1991) Nucleoside hydrolase from Crithidia fasciculuta. J Biol Chem 266: 20658–20665

Ponce-Macotela M, Rufi no-González Y, González-Maciel A, Reynoso-Robles R, and Martínez-Gordillo MN (2006) Oregano (Lippia spp.) kills Giardia intestinalis tropho-zoites in vitro: antigiardiasic activity and ultrastructural damage. Parasitol Res 98: 557–560

Prucca CG, Slavin I, Quiroga R, Elias EV, Rivero FD, Saura A, Carranza PG, and Lujan H (2008) Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456: 750–754

Rayan P, Stenzel D, and McDonnell PA (2005) The effects of saturated fatty acids on Giardia duodenalis trophozoites in vitro. Parasitol Res 9: 191–200

Roxström-Lindquist K, Palm D, Reiner D, Ringqvist E, and Svärd SG (2006) Giardia immunity – an update. Trends Parasitol 22: 26–31

Shapiro TA, and Englund PT (1990) Selective cleavage of ki-netoplast DNA minicircles promoted by antitrypanosomal drugs. Proc Natl Acad Sci USA 87: 950–954

Shi W, Schramm VL, and Almo SC (1999) Nucleoside hydrolase from Leishmania major. J Biol Chem 274: 21114–21120

Sisson G, Goodwin A, Raudonikiene A, Hughes NJ, Mukho-padhyay AK, Berg DA, and Hoffman PS (2002) Enzymes associated with reductive activation and action of nitazoxa-nide, nitrofurans, and metronidazole in Helicobacter pilori. Antimicrob Agents Chemother 46: 2116–2123

Sousa MC and Poiares-da-Silva J (2001) The cytotoxic effects of ciprofl oxacin in Giardia lamblia trophozoites. Toxicol in vitro 15: 297–301

Sterk M, Müller J, Hemphill A, and Müller N (2007) Charac-terization of a Giardia lamblia WB C6 clone resistant to the isofl avone formononetin. Microbiol 153: 4150–4158

Su LH, Lee GA, Huang YC, Chen YH, and Sun CH (2007) Neomycin and puromycin affect gene expression in Gia-rdia lamblia stable transfection, Mol Biochem Parasitol 156: 124–135

Svärd SG, Meng TC, and Hetsko ML, et al. (1998) Differenti-ation-associated surface antigen variation in the ancient eukaryote Giardia lamblia. Mol Microbiol 30: 979–989

Timmers LF, Pauli I, Barcellos GB, Rocha KB, Caceres RA, de Azevedo WF Jr, and Soares MB (2009) Genomic databases and the search of protein targets for protozoan parasites. Curr Drug Targets 10: 240–245

metric assessment of drug activity in vitro. Antimicrob Agents Chemother 41: 1849–1853

Morito K, Aomori T, and Hirose T (2002) Interaction of phy-toestrogens with estrogen receptors alpha and beta (II). Biol Pharm Bull 25: 48–52

Mørch K, Hanevik K, Robertson LJ, Strand EA and Langeland N (2008) Treatment-ladder and genetic characterisation of parasites in refractory giardiasis after an outbreak in Nor-way. J Infect 56: 268–273

Müller J, Ley S, Felger I, Hemphill A. and Müller N (2008a) Identifi cation of differentially expressed genes in a Giardia lamblia WB C6 clone resistant to nitazoxanide and metron-idazole. J Antimicrob Chemother 62: 72–82

Müller J, Limban C, Stadelmann B, Missir AV, Chirita IC, Chifi riuc MC, Nitulescu GM and Hemphill A (2009a) Thioureides of 2-(phenoxymethyl)benzoic acid 4-R substi-tuted: A novel class of anti-parasitic compounds. Parasitol Int 58: 128–135

Müller J, Naguleswaran A, Müller N, and Hemphill A (2008b) Neospora caninum: functional inhibition of protein disul-fi de isomerase by the broad-spectrum anti-parasitic drug nitazoxanide and other thiazolides. Exp Parasitol 118: 80–88

Müller J, Nillius D, Hehl A, Hemphill A, and Müller N (2009) Stable expression of Escherichia coli beta-glucuronidase A (GusA) in Giardia lamblia: application to high-throughput drug susceptibility testing. J Antimicrob Chemother 64: 1187–1191

Müller J, Rühle G, Müller N, Rossignol, J-F, and Hemphill A. (2006) In vitro effects of thiazolides on Giardia lamblia WB C6 cultured axenically and in co-culture with Caco2 cells. Antimicrob Agents Chemother 50: 162–170

Müller J, Sidler D, Nachbur U, Wastling J, Brunner T, and Hemphill A (2008c) Thiazolides inhibit growth and induce glutathione-S-transferase pi (GSTP1)-dependent cell death in human colon cancer cells. Int J Cancer 123: 1797–1806

Müller J, Sterck M, Hemphill A, and Müller, N (2007a) Char-acterization of Giardia lamblia WB C6 clones resistant to nitazoxanide and to metronidazole. J Antimicrob Chemo-ther 60: 280–287

Müller J, Wastling J, Sanderson S, Müller N, and Hemphill A (2007b) A novel Giardia lamblia nitroreductase, GlNR1, interacts with nitazoxanide and other thiazolides. Antimi-crob Agents Chemother 51: 1979–1986

Müller N and Gottstein B (1998) Antigenic variation and the murine immune response to Giardia lamblia. Int J Parasitol 28: 1829–1839

Müller N and von Allmen N (2005) Recent insights into the mucosal reactions associated with Giardia lamblia infec-tions. Int J Parasitol 35: 1339–1347

Nash TE, Banks SM, Alling DW, Merritt JW, and Conrad JT (1990a) Frequency of variant antigens in Giardia lamblia. Exp Parasitol 71: 415–421

Nash TE, Conrad JT, and Merritt JW (1990b) Variant specifi c epitopes of Giardia lamblia. Mol Biochem Parasitol 42: 125–132

Nash TE, Ohl CA, Thomas E, Subramanian G, Keiser P, and Moore TA (2001) Treatment of patients with refractory gia-rdiasis. Clin Infect Dis 33: 22–28

J. Müller et al.

Wang CC (1997) Validating targets for antiparasite chemother-apy. Parasitol 114: Suppl S31–S44

Wilson WD, Mizan S, Tanious FA, Yao S, and Zon G (1994) The interaction of intercalators and groove-binding agents with DNA triple-helical structures: the infl uence of ligand structure, DNA backbone modifi cations and sequence. J Mol Recognit 7: 89–98

Wright JM, Dunn LA, Upcroft P, and Upcroft JA (2003) Effi -cacy of antigiardial drugs. Expert Opin Drug Saf 2: 529–541

Townson SM, Upcroft JA, and Upcroft P (1996) Characterisa-tion and purifi cation of pyruvate:ferredoxin oxidoreductase from Giardia duodenalis. Mol Biochem Parasitol 79: 183–93

Upcroft JA, Campbell RW, and Upcroft P (1996) Quinacrine-resistant Giardia duodenalis. Parasitology 112: 309–313

Upcroft JA, Mitchell R, Chen N, and Upcroft P (1996) Alben-dazole resistance in Giardia is correlated with cytoskeletal changes but not with a mutation at amino acid 200 in β-tubulin. Microb. Drug Resist. 2: 303–308

Upcroft JA and Upcroft P (1993) Drug resistance and Giardia. Parasitol Today 9: 187–190

Upcroft P and Upcroft JA (2001) Drug targets and mechanisms of resistance in the anaerobic protozoa. Clin Microbiol Rev 14: 150–164