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Institute of Parasitology, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland
Correspondence
Norbert Müller
nmueller{at}ipa.unibe.ch
Joachim Müller
joachim.mueller{at}ipa.unibe.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors equally contributed to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Infection by this parasite is one of the most important causes of diarrhoea in the world, and is especially prevalent in children in developing countries. Giardiasis is considered as a disease of the poor and has been included in the ‘Drugs for Neglected Diseases Initiative’ list (Savioli et al., 2006). This initiative is focused, among others, on the urgent need for effective drugs with minor side effects. In the past, 5-nitroimidazole metronidazole (MET) (commercially known as Flagyl) and a few other drugs such as thiazolide nitazoxanide (NTZ) (commercially known as Alinia) have been used as therapy against giardiasis (reviewed by Gardner & Hill, 2001; Upcroft & Upcroft, 2001). Although these compounds are able to efficiently kill Giardia trophozoites, treatment of giardiasis with these drugs may be associated with recurrence of symptoms (e.g reviewed by Upcroft & Upcroft, 2001). Furthermore, drug resistance is one of the reasons for treatment failure. Resistance to MET and other nitroimidazoles has mostly been demonstrated or induced in vitro, but various genotypically distinct isolates with reduced drug susceptibility have also been isolated from human patients (reviewed by Upcroft & Upcroft, 2001; Upcroft et al., 1999, 2006). In contrast, clinical isolates resistant to NTZ have not yet been found. However, our recent in vitro studies revealed that exposure of G. lamblia trophozoites to increasing concentrations of NTZ resulted in resistance formation, and at least one of the highly resistant clones isolated exhibited cross-resistance to MET (Müller et al., 2007). This observation suggests a careful re-evaluation of NTZ regarding its suitability as an alternative drug for treatment of MET-resistant giardiasis.
In order to improve the situation in chemotherapy of resistant giardiasis, new potential pharmaceuticals from natural sources – such as isoflavones isolated from the bark of Dalbergia frutescens – have been evaluated regarding their antigiardial activity in vitro (Khan et al., 2000). Isoflavones are mainly found in Leguminosae where they have anti-oxidant, anti-microbial and signalling functions (reviewed by Dakora & Phillips, 1996; Dixon & Steele, 1999). Within this group of compounds, Khan et al. (2000) identified formononetin (FOR), a major isoflavone of Ononis sp., as the most potent antigiardial agent (Fig. 1). In vitro, FOR has an IC50 value more than 5 times lower than MET, and more than 150 times lower than genistein (GEN), a major isoflavone of soybean (Khan et al., 2000). Furthermore, even at high concentrations this compound did not exhibit a cytotoxic effect in a Vero cell culture and had no detectable adverse effects in mice (Khan et al., 2000). Due to the observations that FOR and MET have differential kinetics of antigiardial activity in vitro and possess a differential spectrum of antiprotozoal activity, the mode of action of these two drugs was suggested to be completely different (Khan et al., 2000).
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) coupling the simplest isoflavone, daidzein (DAI) to agarose and succeeded in isolating a polypeptide of approximately 40 kDa, which was identified by mass spectrometry as a nucleoside hydrolase (NH) homologue (EAA37551.1). The resistant clone was analysed regarding NH gene expression. Furthermore, since upon selective drug pressure G. lamblia trophozoite populations may undergo antigenic variation (Müller et al., 2007), variant surface protein (VSP) gene expression was also analysed in this study. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. They were kept as 100 mM (FOR, GEN) or 40 mM (DAI) stock solutions in DMSO at 4 °C.
Drug treatment assays.
Trophozoites from G. lamblia WB C6 were grown under anaerobic conditions in 10 ml culture tubes (Nunc) containing modified TYI-S-33 medium as described (Keister, 1983). The effects of drugs in axenic G. lamblia trophozoite cultures were assayed as previously described (Müller et al., 2007).
Generation of FOR-resistant Giardia lamblia trophozoites.
FOR-resistant G. lamblia trophozoites were generated using parasites with the same genetic background (i. e. WB C6). In order to obtain drug resistant trophozoites, G. lamblia WB C6 wild-type parasites grown to confluency were initially cultured in the presence of sublethal concentrations (100 nM) of FOR. After 3 days, the medium was removed, viable trophozoites remained attached to the tube walls, and fresh medium was added, containing 250 nM FOR. Living (attached) trophozoites were counted under the microscope as previously described (Müller et al., 2006). When the respective numbers remained constant or increased, fresh medium was added, containing similar or slightly higher FOR concentrations. In case the number of living trophozoites decreased, medium was replaced by fresh medium without the corresponding drug (see Megraud et al., 1998). This operation was repeated during eight passages until levels of 500 nM FOR were reached. Clones from the resistant cell line were generated by two rounds of limited dilution of trophozoites (Müller et al., 2007). In total, 12 clones having maintained their resistance were subcultivated. Among these clones, clone 3 (C3) exhibited the shortest generation time and no visible morphological alterations in the presence of the drug, and was selected for detailed characterization. C3 was kept in long-term cultures by periodic passaging in modified TYI-S-33 medium containing 500 nM FOR.
Preparation of crude extracts from G. lamblia trophozoites.
Crude extracts were prepared by suspending 108 trophozoites in 2 ml extraction buffer, i.e. PBS containing 0.1 % Triton X-100 and 1 mM phenyl-methyl-sulfonyl fluoride followed by vigorous vortexing and centrifugation (10 000 g, 4 °C). The pellet was re-extracted once with 2 ml and once with 1 ml of extraction buffer. The supernatants were pooled, yielding approximately 5 ml of crude extract.
Affinity chromatography using DAI-agarose.
In order to produce DAI-agarose, 0.75 g lyophilized epoxy-agarose with a C-12 spacer was suspended in 15 ml H2O and centrifuged at 300 g for 5 min. Washes in water were repeated twice, and once using coupling buffer (NaHCO3 0.1 M, pH 9.5). After the last wash, 20 mg DAI was added and coupling buffer was added to a maximum volume of 5 ml. The mixture was incubated for 3 days at 37 °C under slow but continuous shaking in order to allow coupling of the epoxy group to DAI via one of its OH groups. The resulting column medium (approx. 2 ml) was then transferred to a chromatography column (Novagen, Merck) and the column was washed with coupling buffer (20 ml). This was followed by ethanolamine (1 M, pH 9.5) for 4 h at 20 °C in the absence of light in order to block residual reactive groups. Finally, the column was extensively washed with PBS and PBS/DMSO (1 : 1) in order to remove unbound DAI. The DAI column was stored in PBS containing 0.02 % NaN3 at 4 °C.
Prior to affinity chromatography, the DAI column was washed with 50 ml PBS equilibrated at 20 °C. Crude extracts (5 ml) of Giardia trophozoites prepared as described above were loaded with a flow rate of approximately 0.25 ml min–1. The column was washed with PBS until the baseline was flat (8 column volumes, corresponding to about 24 ml). Proteins binding to the DAI column were eluted with 0.1 mM FOR in PBS followed by elution with a pH shift (glycine Cl– 100 mM, pH 2.9) in order to remove non-specifically bound proteins. Moreover, fractions were taken before elutions with FOR (pre-FOR) or pH shift (pre-pH shift). Sizes of these fractions ranged between 3 and 5 ml. From all fractions, 0.05–0.2 ml aliquots were taken for analysis by SDS-PAGE. SDS-PAGE was performed according to Laemmli (1970) using a Hoefer Minigel 250 apparatus (Amersham). Silver staining was performed according to Blum et al. (1987).
Protein sequencing by mass spectrometry.
For protein sequencing, the FOR eluates with the highest amounts of binding protein were pooled and dialysed against ammonium bicarbonate (1 g l–1) for 4 h, then against 0.4 g l–1 overnight at 4 °C in the dark. The dialysed fraction was then lyophilized. Aliquots of the lyophilized binding protein (approx. 200 ng) were suspended in SDS-PAGE sample buffer, loaded on a 12 % acrylamide gel and subjected to electrophoresis. After staining with colloidal Coomassie (0.1 % Coomassie brilliant blue G 250 in 34 % methanol with 0.5 % acetic acid and 17 % ammonium sulfate), a band of approximately 40 kDa was excised and processed for mass spectrometry analysis performed by Core Facility Proteomics (Centre Médical Universitaire, Geneva, Switzerland).
Cloning and heterologous expression of Giardia lamblia nucleoside hydrolase (NH).
In order to clone NH into the His-tag-expression vector pET151 directional TOPO (Invitrogen), a forward (NHfullF; see Table 1) and reverse (NHfullR, see Table 1) primer (MWG Biotech) were created for the amplification of gene fragments encoding the NH polypeptide. CACC at the 5' end of NHfullF was added in order to allow directional cloning. The cyclic NH gene amplification reaction, cloning of the NH gene amplification product into pET151 directional TOPO, as well as bacterial expression and subsequent His-tag purification of recombinant NH was done according to the procedure previously applied for production of recombinant protein disulfide isomerase (PDI) 4 of G. lamblia WB clone C6 (Müller et al., 2007). Purified recombinant G. lamblia NH protein (recGlNH) was stored in 50 % glycerol at –20 °C.
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). The assay was performed in a quartz cuvette containing HEPES 50 mM, pH 7.3 with inosine in concentrations ranging from 0 to 2 mM in a dual-beam spectrophotometer (UV 1700; Shimadzu). After initiation of the reaction by adding recGlNH (1 to 5 µM), absorption was continuously read at 280 nm. The values were corrected by a corresponding substrate blank inserted into the second beam. The values obtained were fitted to the Lineweaver–Burk transformation of the Michaelis–Menten law in order to calculate the Km.
For the determination of substrate specificity and for inhibition studies, recGlNH was assayed using an indirect colorimetric assay of the ribose released based on the cuproin method as described previously (Parkin et al., 1991). Eppendorf tubes containing 0.1 ml HEPES 50 mM, pH 7.3 with substrates and/or isoflavones as indicated were incubated for 30 min to 1 h at 37 °C in a water bath. Reaction was stopped by adding 0.4 ml water, 0.15 ml cuproin reagent I (Na2CO3 4 %, glycine 1.6 %, CuSO4 . 5 H2O 0.05 %) and 0.15 ml cuproin reagent II (cuproin, 2,9-dimethyl-1,10-phenanthrolin 0.12 % suspended in water) followed by 10 min incubation at 95 °C. After cooling to room temperature, absorption was measured at 450 nm. Blanks for enzyme, substrate and isoflavones were included and subtracted from the reaction. A standard curve (glucose from 0 to 100 nmol) made in HEPES buffer under reaction conditions was included in each assay.
Processing of RNA samples and quantitative reverse transcription (RT)-PCR.
For quantification of gene expression by real-time RT-PCR, trophozoites of wild-type WB C6 and isoflavone resistant clone C3 were grown in the absence of drugs until near confluency was reached. Cells were harvested as described, and RNA was extracted using a Qiagen RNeasy kit (Qiagen) and including a DNase I digestion (to remove residual genomic DNA) according to the instructions provided by the manufacturer. RNA was eluted with 50 µl RNase free water and stored at –80 °C.
First-strand cDNA synthesis using a polyT-ANC primer (von Allmen et al., 2004; primer sequences, see Table 1) and subsequent quantitative ACT-, VSPtot- and TSA417-PCR (primer sequences, see Table 1) were done as previously described (Müller et al., 2007). In parallel runs, forward (NHquantF) and reverse (NHquantR) primers (see Table 1) were applied for the quantitative NH-PCR. From the quantitative RT-PCR, mean values (±SEM) from triplicate determinations were assessed and expression levels of NH, VSPtot and TSA417 were given as values in arbitrary units relative to the constitutively expressed ‘house keeping’ gene ACT.
Statistical methods.
The significance of the differences of the NH, VSPtot and TSA417 gene, and gene expression between wild-type clone WB C6 and isoflavone resistant clone C3 were determined by Student's t-test using Microsoft's Excel program. P values of <0.01 were considered statistically highly significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Upon prolonged growth of C3 in drug-free medium for more than 10 generations, resistance was maintained. The clone C3 encysted as well as the wild-type when put on encystation medium (not shown).
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). At 0.5 µM FOR, C3 had about 75 % of control growth, whereas the wild-type was decreased to approximately 1.5 % (Fig. 3a). On 10 µM DAI, C3 reached approximately 58 % of the control level whereas the wild-type was decreased to approximately 1 % (Fig. 3b). Growth on GEN essentially corresponded to growth on DAI (growth curves not shown). C3 could thus be regarded as isoflavone-resistant.
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A nucleoside hydrolase homologue is a major DAI-binding protein in Giardia lamblia
Following DAI-affinity chromatography, a major Giardia protein of approximately 40 kDa was eluted with 0.1 mM FOR (Fig. 4a, lane FOR). FOR was used as eluent since this compound showed the best growth effects on Giardia trophozoites and since the blocking of one OH group corresponded best to the ligand DAI bound via a C12-spacer to one of its OH groups to the matrix. By mass spectrometry, the 40 kDa protein was identified as a nucleoside hydrolase (NH) homologue (accession EAA37551.1; orf 13272 in the Giardia genome database; approximately 30 % coverage of the sequence by peptides from MS sequencing). Protein–protein BLAST (Altschul et al., 1997) revealed that the closest homologues of EAA37551.1 were NHs from Trichomonas vaginalis (two enzymes), Entamoeba histolytica, Danio rerio and Xenopus tropicalis. Besides the conserved domains, EAA37551.1 possessed, however, short peptide motives (between aa residues 61 and 226) not found in the other NHs (Fig. 4b), a phenomenon that has been observed in many other Giardia orthologues (see Giardia genome database). Like its homologues, EAA37551.1 had no signal peptide and was considered as cytosolic.
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). The purified protein was subjected to an indirect nucleoside hydrolase assay based on the determination of riboside released from various nucleosides. The recombinant protein hydrolysed the purine nucleosides adenosine, guanosine and inosine much better than the pyrimidine nucleosides uridine or cytidine (Fig. 5a).
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. These values were in good agreement with previously published values from NHs from Crithidia fasciculata (Parkin et al., 1991) and Trichomonas vaginalis Gero et al. (2000). Thus, we propose that EAA37551.1 should be named G. lamblia purine nucleoside-preferring nucleoside hydrolase (GlNH).
Recombinant nucleoside hydrolase (NH) is inhibited by isoflavones
When an indirect NH assay was performed in the presence of DAI and FOR, NH activity decreased in a concentration-dependent manner. The inhibition was more pronounced at 0.1 mM than at 0.5 mM of the substrate inosine (Fig. 5c). GEN behaved similarly to DAI (data not shown). Assuming a competitive inhibition, the Ki values were 3.8±0.7 µM for DAI and 11±2.8 µM for FOR as calculated according to Parkin (1996).
Gene expression
In order to determine gene expression of NH identified as potential target of isoflavones (see above) and thus potentially involved in resistance formation, we developed a real-time RT-PCR assay quantifying reverse transcripts of the NH gene. In this assay, reverse transcripts of ACT gene were also quantified as reference for the amount of total cDNA. ACT gene expression had been previously revealed to be constant in WB C6 wild-type and drug-resistant strains (Müller et al., 2007). Respective analysis demonstrated that gene expression of NH was significantly decreased in C3 (approx. 41 %) compared with the corresponding wild-type WB C6 expression level (Fig. 6). However, the differences between the wild-type and the resistant G. lamblia clone were much more pronounced when expression of VSP genes was investigated. For respective analyses, cDNA was synthesized issuing from polyT-ANC primers (see Methods). This experimental strategy allowed RT-PCRs to be performed with a general forward primer (MM16 primer) targeted to a highly conserved VSP gene region and a reverse primer targeted to the 3' terminal anchor (ANC) sequence adjacent to the polyT stretch of the cDNA molecules (Table 1). By comparative quantitative RT-PCR, the cDNA synthesized with polyT-ANC was also amplified by including a primer pair specific for the gene TSA417 that encodes the major VSP in WB C6 (Fig. 6). While wild-type clone WB C6 exhibited TSA417 gene expression, corresponding expression levels were dramatically reduced and virtually non-detectable in FORr clone C3. Conversely, the overall expression of VSP genes (VSPtot) remained constant, suggesting that resistance was associated with an antigenic switch from TSA417 to one, or several unknown, VSPs
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Upcroft & Upcroft, 2001). In the field of antigiardial drug development, the possibility of resistance formation has been recognized as an important problem and was the subject of various preclinical and clinical investigations (reviewed by Upcroft & Upcroft, 2001). Formation of resistance to MET is a major constraint for efficient treatment of human giardiasis, and therapeutic failure due to giardial resistance to this compound is occurring at an increasing frequency (Townson et al., 1994; Wright et al., 2003). Furthermore, our recent investigations revealed that G. lamblia trophozoites grown in vitro were able to develop resistance to NTZ, and at least one of the resistant clones exhibited cross-resistance to MET (Müller et al., 2007).
Under the circumstances outlined above, the isoflavones previously described as a new group of antimicrobial compounds (reviewed by Dakora & Phillips, 1996) have been investigated as alternative drugs for the treatment of giardiasis (Khan et al., 2000). Among the different isoflavones tested in vitro, FOR turned out to be the most efficacious antigiardial compound (Khan et al., 2000) and was also demonstrated to efficiently inhibit growth of MET- and/or NTZ-resistant G. lamblia strains (Müller et al., 2007). This observed lack of cross-resistance in vitro certainly suggested a further evaluation of FOR regarding its applicability as an alternative antigiardial agent to overcome potential problems of resistance to conventional drugs. Conversely, it is evident that this concept of treatment may be complicated by the capacity of Giardia to develop high-level resistance to this drug. In fact, in our study, resistance to FOR was already detectable upon relatively short exposure of in vitro-cultivated trophozoites to the drug, suggesting that genetic predispositions allow the parasite to immediately adapt to an isoflavone-enriched host intestinal environment, e.g. emerging upon uptake of an isoflavone-rich vegetarian diet by the host.
In the present study, trophozoites from FORr clone C3 were found to be cross-resistant to other isoflavones DAI and GEN, but not to the nitro compounds MET or NTZ. This indicated that resistance was not caused by a general mechanism like multi-drug transporters, but rather by an as yet unknown mechanism that specifically concerns isoflavones. This conclusion was also supported by our previous observation demonstrating that NTZ-resistant G. lamblia trophozoites were not cross-resistant to isoflavones (Müller et al., 2007).
One, but not the only, effect of isoflavones could be the inhibition of the isoflavone-binding protein NH. This inhibition occurs in a range of concentrations where DAI and GEN inhibit trophozoite growth in vivo. FOR is inhibitory at much lower concentrations, suggesting other mechanisms to be involved. In Giardia, hydrolysis of exogenous nucleosides by NH has been previously described as the first step of one of two distinct salvage pathways to form purine (see e.g. Wang & Aldritt, 1983; Aldritt & Wang, 1986) and pyrimidine ribonucleotides (Aldritt et al., 1985; for review see Jarroll et al., 1989). Moreover, a low-affinity transporter for purine nucleobases has been described as a potential adenine and guanine donor for the second salvage pathway (Ey et al., 1992). In Crithidia, Leishmania or Trypanosoma, the function of NH is central in salvaging purine nucleosides from the host and essential since the parasites lack purine de novo synthesis (Parkin et al., 1991; Parkin, 1996; Shi et al., 1999). Inhibition of NH by a drug would thus lead ultimately to a block of DNA synthesis and thus cell division. Parasites could overcome this inhibition by synthesizing more NH. An evaluation of the gene expression pattern revealed, however, a significant reduction of NH transcripts in the isoflavone-resistant clone C3 rather than an increase. This pattern is expected if the target enzyme activates a (non-toxic) prodrug, e.g. pyruvate-oxidoreductase in the case of MET (see Müller et al., 2007 and references therein). One explanation could be that NH is downregulated and another, unrelated scavenging enzyme such as purine phosphorylase (Parkin et al., 1991; Parkin, 1996) is upregulated, thus compensating NH inhibition.
In comparison to NH gene expression, the gene regulatory effects associated with antigenic variation of the parasite were much more pronounced. Here, expression of TSA417 representing the major surface antigen (VSP C6) of WB C6 was massively downregulated in isoflavone-resistant clone C3. This finding is in agreement with results from our recent study that demonstrated a striking reduction of the TSA417 gene expression in trophozoites that had developed resistance to NTZ and/or MET (Müller et al., 2007). It is known from current literature that G. lamblia is able to escape host immunological defence mechanisms by changing the expression of its surface antigens (reviewed by e.g. Müller & Gottstein, 1998; Müller and von Allmen, 2005). Furthermore, it was found that, besides the immunological pressure, physiological factors such as intestinal proteases also act as driving forces for antigenic diversification of the parasite (Nash et al., 1991). Finally, we now realized that drug pressure is able to select for (a) new surface antigen variant(s) within a G. lamblia trophozoite population (Müller et al., 2007 and present study). Taking all these observations into account, antigenic variation seems to be part of a versatile adaptive mechanism that allows the parasite to respond to a broad spectrum of environmental stress factors. As reported recently, an epigenetic mechanism, probably including gene acetylation, seems to be responsible for antigenic switching of the parasite (Kulakova et al., 2006). Possibly, this epigenetic mechanism involves pleiotropic gene-regulatory effects that mediate, or at least participate in, antigiardial drug resistance formation. Future studies based on differential and functional genomics and proteomics approaches will reveal if surface antigen alterations per se or rather pleiotropic gene-regulatory processes associated with a surface antigen switch are responsible for in vitro-induced drug resistance in G. lamblia.
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ACKNOWLEDGEMENTS |
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Edited by: J. Tachezy
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Received 25 May 2007;
revised 29 August 2007;
accepted 7 September 2007.
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