INTRODUCTION

The intestinal anaerobic protozoan parasite Giardia-lamblia is a major cause of persistent diarrhea. The treatment of Giardia infections is typically performed with nitro-compounds¹. Most likely the mechanism of action relies on the reduction of the nitro group via enzymatic pathways in G.lamblia thereby activating the prodrugs. Enzymes such as thioredoxin reductase (TrxR) and the nitroreductases NR1, NR2, and NR3 with weak NR activity have been identified in G.lamblia and may be involved in the activation of or the resistance towards nitro-prodrugs^2,3. Overexpression of NR1 results in an increased susceptibility to nitro-compounds in G.lamblia, whereas overexpression of NR2 results in an increased resistance in E.coli^2,3. TrxR was found to have only little impact on nitro-drug activity and TrxR overexpression in resistant strains may be related to its role as radical scavenger⁴. The physiologic functions of these enzymes TrxR and NR1-3 in G.lamblia are not yet known.
Previously, we have introduced HR-MAS NMR to study the metabolome of G.lamblia⁵. The aim of the current study was to compare the metabolomic patterns of G.lamblia strains with altered expression levels of the respective genes for getting insight into physiologic pathways.

EXPERIMENTAL PROCEDURES

G.lamblia strains -
(i) G.lamblia overexpressing either the wildtype thioredoxin reductase (TrxR, n=10), or a dominant negative allele (TrxR-DN, n=10) and WBC6 wildtype as controls (WT1, n=10);
(ii) G.lamblia overexpressing the nitroreductases NR1 (n=5), NR2 (n=6), and NR3 (n=5), control strains overexpressing glucuronidase A (GusA, n=6) and WBC6 wildtype (WT2, n=4);
(iii) G.lamblia nitro-drug resistant strain (C4, n=10) and WBC6 wildtype (WT3, n=4).
Preparation of cell samples - G.lamblia trophozoites were grown to confluence in modified TYI-S33 medium and harvested. After centrifugation, washing, and shock freezing, samples were stored at -70°C until measurement.
NMR Spectroscopy - NMR was performed on a 500 MHz NMR spectrometer. 1H HR-MAS NMR experiments (3kHz and 279K) were recorded using a 1D-PROJECT sequence⁶ with water-presaturation.
Data Analysis - The NMR spectra were subdivided into 138 individually sized buckets. Multivariate data analysis was performed applying probabilistic quotient normalization (PQN), mean centering and pareto scaling. PCA (for unsupervised detection of group clustering) and orthogonal partial least squares discriminant analysis (oPLS-DA) were performed, cross-validated, and subjected to permutation testing.
For univariate statistical analysis representative spectral regions for 27 metabolites were integrated, normalized and mean±sd calculated. Single factor ANOVA and t-tests were performed. p-values were multiplied with 27 to correct for multiple comparisons (Bonferroni-correction). Metabolites that were significantly different between the independent wildtype populations were excluded from the discussion (Ala, Asn, Glu, Leu, Thr, Val, Glc-1-P, and TMA).

RESULTS

Three independent sample cohorts of G.lamblia trophozoites were compared:
(i) TrxR, TrxR-DN, and WT1;
(ii) NR1, NR2, NR3, GusA, and WT2;
(iii) C4 and WT3.
Twenty-seven metabolites were identified based on our previous assignment⁵. PCA and oPLS-DA score plots and average normalized peak integrals for each of the 27 metabolites are displayed in Fig.1 for the first and in Fig.2 for the second sample cohort. The two transgenic strains TrxR and TrxR-DN were well separated from WT1 in both, PCA and oPLS with TrxR scores lying furthest away from WT1 (Fig.1A,B). Main metabolite contributions to distinguishing TrxR and TrxR-DN derived from increased acetate and decreased lysine levels in TrxR (Fig.1C).
When comparing trophozoites overexpressing nitroreductases NR1, NR2, and NR3 to GusA and WT2, PCA and oPLS-DA resulted in complete separation of NR1 and NR2 classes whereas NR3, GusA and WT2 overlapped (Fig.2A,B). The plot shows greatest distance for NR1 from WT and there is the order NR1 -> NR2 -> NR3 clustering with GusA -> WT. Specifically, nine metabolites were significantly altered in NR1 versus GusA (Fig.2C), namely acetate, cystine, glutamine, methionine, phenylalanine, tyrosine, citrulline, ornithine and pipecolic-acid. A typical NMR spectrum representing peaks for ornithine and citrulline from NR1 and GusA trophozoites is shown in Fig.3. To probe, whether the metabolic pattern obtained by comparing NR1 versus GusA could be linked to the nitro drug susceptibility, an oPLS-DA model calculated for discrimination between NR1 and GusA (Fig.4A) was applied to the nitro drug resistant C4 trophozoites with reduced NR1 activity and WT (Fig.4B). The predictive value of the model was very good, leading to complete separation of C4 versus WT, i.e. suggesting a correlation between nitroreductase activity and the metabolome. This was underlined by a significant correlation between the ratios WT/C4 vs. NR1/GusA of eight metabolites (Fig.4C).

DISCUSSION

Trophozoites expressing the nitroreductase NR1 exhibited a distinct pattern of nine differentially regulated metabolites. This pattern inversely correlated with a pattern of the same metabolites in the nitro drug resistant strain C4. The catabolism of arginine, a major energy source for G.lamblia, is of particular interest. Citrulline and ornithine, the first and the second catabolites of arginine were positively, glutamine, an indirect catabolite of ornithine, was negatively correlated with NR1 levels, suggesting that NR1 interferes with the catabolism of arginine. Since the ATP levels are unaltered in NR1 as well as in all other transgenic trophozoites, a direct interference with energy supply is unlikely.

CONCLUSION

The study demonstrates significant metabolic differences between G.lamblia wildtype and transgenic strains that are likely related to nitroreductase activity.

Acknowledgements

This work was supported by the Swiss National Science Foundation (grant No. 31003A_163230) and grant No. 206021-128736 for the purchase of the HR-MAS probe.