In vitro selection of Giardia duodenalis for Albendazole resistance identifies a β-tubulin mutation at amino acid E198K

Abstract

Benzimidazole-2-carbamate (BZ) compounds, including Albendazole (Alb), are one of just two drug classes approved to treat the gastrointestinal protist Giardia duodenalis. Benzimidazoles bind to the tubulin dimer interface overlapping the colchicine binding site (CBS) of β-tubulin, thereby inhibiting microtubule polymerisation and disrupting microtubule networks. These BZ compounds are widely used as anthelmintic, anti-fungal and anti-giardial drugs. However, in helminths and fungi, BZ-resistance is widespread and caused by specific point mutations primarily occurring at F167, E198 and F200 in β-tubulin isoform 1. BZ-resistance in Giardia is reported clinically and readily generated in vitro, with significant implications for Giardia control. In Giardia, BZ mode of action (MOA) and resistance mechanisms are presumed but not proven, and no mutations in β-tubulin have been reported in association with Alb resistance (AlbR). Herein, we undertook detailed in vitro drug-susceptibility screens of 13 BZ compounds and 7 Alb structural analogues in isogenic G. duodenalis isolates selected for AlbR and podophyllotoxin, another β-tubulin inhibitor, as well as explored cross-resistance to structurally unrelated, metronidazole (Mtz). AlbR lines exhibited co-resistance to many structural variants in the BZ-pharmacophore, and cross-resistance to podophyllotoxin. AlbR lines were not cross-resistant to Mtz, but MtzR lines had enhanced survival in Alb. Lastly, Alb analogues with longer thioether substituents had decreased potency against our AlbR lines. In silico modelling indicated the Alb-β-tubulin interaction in Giardia partially overlaps the CBS and corresponds to residues associated with BZ-resistance in helminths and fungi (F167, E198, F200). Sequencing of Giardia β-tubulin identified a single nucleotide polymorphism resulting in a mutation from glutamic acid to lysine at amino acid 198 (E198K). To our knowledge, this is the first β-tubulin mutation reported for protistan BZ-resistance. This study provides insight into BZ mode of action and resistance in Giardia, and presents a potential avenue for a genetic test for clinically resistance isolates.

Keywords: Albendazole; Benzimidazoles; Drug-resistance; Giardia duodenalis; Tubulin.

Graphical abstract

Fig. 1

Generation of an AlbR line and BZ compound screens: A) Time in culture and induction of AlbR in a WB-1B line starting at 0.1 μM and finishing at 0.5 μM over 400 days. B) IC₅₀ dose response curves for albendazole, podophyllotoxin and metronidazole in AlbS and AlbR isogenic lines, demonstrating a substantial shift in IC₅₀ in the AlbR for albendazole and podophyllotoxin but not for metronidazole. C) Bar chart of IC₅₀ values for AlbS and AlbR isogenic lines (left axis) as well as the calculated RF for the AlbR (right axis) for BZ compounds. Compounds are listed from left to right in order of potency. The full dose response curves of all BZ compounds are in Supplementary Fig. S1. Thiabendazole, albendazole sulfone and reduced mebendazole were not sufficiently active in both lines at 10 μM to calculate an IC₅₀, and therefore has not been shown.

Fig. 2

Albendazole susceptibility in MtzR lines: IC₅₀ dose response curves for albendazole and metronidazole in MtzR isogenic lines (Ansell et al., 2017) derived from WB-1B, BRIS/83/HEPU/106 and BRIS/87/HEPU/713. These demonstrate the expected shift in dose response for Mtz in all 3 isolates (above), and an enhanced survival in MtzR lines in Alb between substantial shift 2–0.25 μM, but no change in IC₅₀ or shift in the dose response, indicative of enhanced survival at higher Alb concentration.

Fig. 3

Screening of BZ analogues and Albendazole thioesters: A) Change in potency for BZ analogues based on the Alb IC₅₀ (0.16 μM). Compounds include albendazole and 7 additional albendazole thioethers. B) Structures of albendazole carbamate thioethers (methylthio-to octylthio-) C) Table of IC₅₀ in WB AlbS and AlbR lines and their respective resistance factors. albendazole (*) corresponds to the R group -SC₃H₇ in the thiolether series.

Fig. 4

Molecular modelling of Alb with Giardia β-tubulin A) Illustrations of the binding mode of albendazole with Giardia β-tubulin. Surface view to represent binding pocket for Alb (left) and with the ribbon structure demonstrating polar contact (middle), with C236 and its hydrogen bond interaction indicated with an arror. The predicted binding modes of Alb inside the proposed binding site with the amino acid residues involved in hydrogen bond formation and non-bonded interaction are shown on the right. B) Overlapped binding sites of the experimentally derived crystal structure of T2R-TTL-nocodazole complex (PDB ID: 5CA1_C/D) shown in grey and our model of G. duodenalis β-tubulin shown in green. A close-up inserted image in the top right compared the orientation of nocodazole (blue) from the crystal structure (5CA1_D) and the docked Alb (green) structure from our molecular modelling of Giardia, and demonstrates that nocodozole and Alb share the same binding position and direction. In contrast, the inserted structure on the bottom left of the experimentally derived crystal structure of the tubulin-colchicine complex (PDB ID: 4O2B_C/D) in grey shows that the colchicine (yellow) molecule partially overlaps with docked Alb (green) in our structure from Giardia. C) Ribbon structures demonstrating polar contact between Giardia β-tubulin and heptylthioether (BI-63-8; left) and octyl-thiol (BI-63-14; right) analogues associated with decreased potency in the AlbR line. Orientation of the molecular is the same as in Alb, but feature an additional hydrogen bond with E198 for both molecules as indicated by the arrow.

Fig. 5

Identification and modelling of an E198K mutation in tha AlbR line A) Representative sequence chromatograms from capillary sequencing of Giardia β-tubulin from AlbS and AlbR lines highlighting nucleotides corresponding to amino acids 165, 167, 198 and 200. A double peak at nucleotide position 440 G/A is highlighted with a yellow arrow in the AlbR isolate is indicative of a single nucleotide polymorphism which would correspond to a E198K mutation. Chromatograms from the three technical replicates in each AlbR and AlbS line can be seen in Supplementary Fig. S5. B) Results of amplicon sequencing analysing the G592A SNP in the AlbS and AlbR lines in replicates from in vitro culture (R1, R2, R3), as well as sequencing replicates (a, b, c). The figure shows both the number of reads aligned at this position for the G592A SNP (a), as well as the relative frequency of the G592A SNP (b). C) Orientation of Alb in the binding pocket where E198 is present (green) compared to K198 (blue), the latter which binds slightly shallower and in a twisted orientation. Side chains of other key residues are also shown, including cysteines which can be hydrogen bond donors, and amino acids implicated in anthelmintic resistance. D) Ribbon structures demonstrating polar contact between Giardia β-tubulin and albendazole with the E198K mutation. An additional hydrogen bond with K198 is observed, as is the hydrogen bond with C236, which was observed with Alb in models with E198.

fig. 6

BZ-resistance phenotypes and genotypes across AlbR induction. A) IC₅₀ dose response curves for albendazole, mebendazole, parbendazole and oxibendendazole in AlbS and AlbR isogenic lines grown at 0.21 μM, 0.35 μM and 0.5 μM drug, demonstrating a shift in IC₅₀ in all the AlbR lines relative to Alb concentration. B) Results of amplicon sequencing analysing the G592A SNP in the 0.21 μM and the 0.35 μM AlbR line in replicates from in vitro culture (R1, R2, R3), as well as sequencing replicates (a, b, c). The figure shows both the number of reads aligned at this position for the G592A SNP (a), as well as the relative allele frequency of the G592A SNP (b). C) Correlation between BZ-resistance phenotypes and the G592A SNP in the 0.21 μM, 0.35 μM and 0.5 μM lines. The left axis for the bar chart demonstrates the calculated RF for the AlbR lines, whereas the right axis for the violin plot demonstrates the allele frequency from in vitro culture as well as sequencing replicates, with statistical significance established via Wilcoxon signed-rank test.