Transcriptomic analysis of albendazole resistance in human diarrheal parasite Giardia duodenalis

Abstract

Benzimidazole-2-carbamates (BZ, e.g., albendazole; ALB), which bind β-tubulin to disrupt microtubule polymerization, are one of two primary compound classes used to treat giardiasis. In most parasitic nematodes and fungi, BZ-resistance is caused by β-tubulin mutations and its molecular mode of action (MOA) is well studied. In contrast, in Giardia duodenalis BZ MOA or resistance is less well understood, may involve target-specific and broader impacts including cellular damage and oxidative stress, and its underlying cause is not clearly determined. Previously, we identified acquisition of a single nucleotide polymorphism, E198K, in β-tubulin in ALB-resistant (ALB-R) G. duodenalis WB-1B relative to ALB-sensitive (ALB-S) parental controls. E198K is linked to BZ-resistance in fungi and its allelic frequency correlated with the magnitude of BZ-resistance in G. duodenalis WB-1B. Here, we undertook detailed transcriptomic comparisons of these ALB-S and ALB-R G. duodenalis WB-1B cultures. The primary transcriptional changes with ALB-R in G. duodenalis WB-1B indicated increased protein degradation and turnover, and up-regulation of tubulin, and related genes, associated with the adhesive disc and basal bodies. These findings are consistent with previous observations noting focused disintegration of the disc and associated structures in Giardia duodenalis upon ALB exposure. We also saw transcriptional changes with ALB-R in G. duodenalis WB-1B consistent with prior observations of a shift from glycolysis to arginine metabolism for ATP production and possible changes to aspects of the vesicular trafficking system that require further investigation. Finally, we saw mixed transcriptional changes associated with DNA repair and oxidative stress responses in the G. duodenalis WB-1B line. These changes may be indicative of a role for H₂O₂ degradation in ALB-R, as has been observed in other G. duodenalis cell cultures. However, they were below the transcriptional fold-change threshold (log₂FC > 1) typically employed in transcriptomic analyses and appear to be contradicted in ALB-R G. duodenalis WB-1B by down-regulation of the NAD scavenging and conversion pathways required to support these stress pathways and up-regulation of many highly oxidation sensitive iron-sulphur (FeS) cluster based metabolic enzymes.

Keywords: Albendazole; Drug-resistance; Giardia duodenalis.

Graphical abstract

Fig. 1

Differentially expressed genes between ALB-S and ALB-R lines. (A) 155 up- and 135 down-regulated genes under FDR <0.05 and absolute log₂-FC > 1. The top/bottom five genes are labelled. (B) Significantly enriched gene sets of molecular and domain function in DTGs under a P value less than 0.01. Gene numbers over the whole genome are shown in bars. Gene sets containing overlapped genes or related functions have been shaded into different clusters. (C) Log2 fold change distribution of varying gene sets clusters and representative gene sets.

Fig. 2

Unbiased global gene sets enrichment analysis summarized in chord plot. (A) Twenty-five gene ontology terms and 6 KEGG metabolism pathways are significantly enriched in ALBR lines, according to Romer. Directed enrichment p-value of all 31 gene sets are plotted in the upper grid where the positive value represents up-regulated gene sets, and the negative value represents down-regulated gene sets. More absolute log10 p-value represents more significance. Terms have further been classified into translation (translational initiation GO:0006413, translation GO:0006412, ribosome GO:0005840, small ribosomal subunit GO:0015935, intracellular GO:0043229. Translation initiation factor activity GO:0003743. RNA binding GO:0003723, structural constituent of ribosome GO:0003735), protein degradation (proteolysis involved in cellular protein catabolic process GO:0051603, proteasome core complex, alpha-subunit complex GO:0019773, proteasome core complex GO:0005839, threonine-type endopeptidase activity GO:0004298, endopeptidase activity GO:0004175, hydrolase activity GO:0016787), genomic regulation (nucleosome GO:0000786), carbohydrate metabolism (Amino sugar and nucleotide sugar metabolism PATH:gla00520, Glycerolipid metabolism PATH:gla00010, Pentose phosphate pathway PATH:gla00030, Starch and sucrose metabolism PATH:gla00500, Pyruvate metabolism PATH:gla00620), oxidation-reduction (oxidation-reduction process GO:1990204, oxidoreductase activity GO:0016491, Nicotinate and nicotinamide metabolism PATH:gla00760), GTPase related (GTP binding GO:0005525, GTPase activity GO:0003924, protein transport GO:0015031), cytoskeleton and calcium (calcium ion binding GO:0005509, calcium-dependent phospholipid binding GO:0005544, pyridoxal phosphate binding GO:0030170), and nucleoside metabolism (nucleoside metabolic process GO:0009116, DNA repair GO:0006281). (B) Barcode plot of t-statistics for representative GO terms.

Fig. 3

Mapping transcript variations on glycolysis, PPP, pyruvate, and arginine metabolic pathway. 6PGL, 6-phosphogluconolactonase, NO homolog; ACYP, acylyphosphatase, GL_5359, GL_7871; ADI, arginine deiminase, GL_112103; ARG-S, arginyl-tRNA synthetase, GL_10521; CK, carbamate kinase, GL_16453; DERA, deoxyribose-phosphate aldolase, GL_15127; ENO, enolase, GL_11118; FBA, fructose-bisphosphate aldolase, GL_11043; G6PD, glucose-6-phosphate dehydrogenase, GL_8682; GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GL_17043, GL_ 6687; GCK, glucokinase, GL_8826; GNPDA, glucosamine-6-phosphate deaminase, GL_10829, GL_8245; GNPNAT, glucosamine 6-phosphate N-acetyltransferase, GL_14651; GPI, glucose-6-phosphate isomerase, GL_9115; NOS, nitric oxide synthase, GL_91252; OCD, ornithine cyclodeaminase, GL_2452; OCT, ornithine carbamoyltransferase, GL_10311; ODC, ornithine decarboxylase, GL_94582; PFK, phosphofructokinase (pyrophosphate-based), GL_14993; PGAM, phosphoglycerate mutase, GL_8822; PGD, phosphogluconate dehydrogenase, GL_14759; PGK, phosphoglycerate kinase, GL_90872; PGM, phosphoglucomutase, GL_16069, GL_17254; PGM3, phosphoacetylglucosamine mutase, GL_16069; PK, pyruvatekinase, GL_3206, GL_17143; PRO-S, prolyl-tRNAsynthetase, GL_15983; PRPPS, phosphoribosylpyrophosphate synthetase, GL_21750; RBKS, ribokinase, GL_15297; RPE, ribulose-phosphate 3 epimerase, GL_ 10324; RPI, ribose-5-phosphate isomerase, GL_27614; TKT, transketolase, GL_9704; TPI, triose phosphate isomerase, GL_93938; UAE, UDP-N-acetylglucosamine 4-epimerase, 5.1.3.7; UAP, UDP-N-acetylglucosamine diphosphorylase, GL_16217; PFOR, pyruvate:ferredoxin oxidoreductase (Pyruvate-flavodoxin oxidoreductase), GL_17063, GL_114609; GDH, NADP-specific glutamate dehydrogenase, GL_21942; MAL, malic enzyme, GL_14285; HYD, hydrogenase, GL_6304; ACoAS, acetyl-CoA synthetase, GL_16667, GL_13608; AAT, alanine aminotransferase, GL_12150, GL_16353.