Abstract
Fasciolosis is caused by liver flukes: F. hepatica, and a sister species - F. gigantica. A growing concern with controlling the disease is resistance to triclabendazole (TCBZ), the only drug shown to kill both adult and immature liver flukes. Currently, F. hepatica mechanism of resistance to TCBZ is not clearly understood and there is no effective commercially available vaccine. Previous work proposed three mechanisms associated with TCBZ mode of action and resistance: tubulin binding activity, drug uptake mechanisms, and drug metabolism mechanism. Exploring evolutionary forces acting on F. hepatica genes associated with TCBZ mode of action and resistance could explain how the parasite develops resistance to the drug, enable identification of potential drug targets, and facilitate development of new drugs. A re-annotation of the current F. hepatica genome was done using an updated version of the published F. hepatica draft genome (assembly GCA_000947175.1, BioProject PRJEB6687). Subsequently, the current annotation (Fasciola_10x_pilon, GCA_900302435.1 WormBase Parasite Version 15) was compared and critically assessed with the newly re-annotated version. Using coding sequences (CDS) of three well-described annotated gene families, manual validation of the annotation was done. A total of 15,879 F. hepatica genes were identified in this project compared to the 9,401 genes in the current annotation, while differences noticed in both annotations include gene fragmentation, missing exons, and missing genes. F. hepatica gene family members belonging to each of the three proposed mechanism of action of TCBZ action and resistance, and their trematode orthologous sequences were compiled. The gene families studied include tubulins, ATP-binding cassette transporters (ABC), AC, RAS, ADP ribosylation factor, cytochrome P450 (CYP450), GSTs, and Fatty Acid Binding Proteins (FABPs). Signals indicative of positive selection was identified using Phylogenetic Analysis by Maximum Likelihood (PAML) and McDonald and Kreitman test (MKtest). PAML branch-site model testing identified 1 alpha tubulin, 1 delta tubulin, 5 ABC genes, 9 RAS genes, and 4 ADP ribosylation factor genes with statistically significant sites under positive selection. While the MKtest analysis identified 2 RAS genes and 1 AC genes under positive selection. The expression profile of the genes associated with TCBZ mode of action was assessed across F. hepatica life stages. Findings indicate that tubulin gene expression was elevated in metacercariae and newly excysted juveniles (NEJs), with a peak expression pattern noticed in NEJs 1 hour post excystment, with levels reducing in flukes 21 days post excystment. Similarly, in genes associated with TCBZ uptake, expression was predominantly raised in metacercariae and NEJs, while gene expression gradually reduced towards fluke maturity. The effect of TCBZ on F. hepatica was investigated in experimentally infected sheep. Parasite response to the drug in TCBZ resistant and susceptible F. hepatica isolates was compared in sheep infected and treated with the drug. TCBZ treatment induced gene expression patterns were noticed in 72% (90 out 125 genes, P < 0.05) of all the genes assessed (excluding unexpressed genes and constitutively expressed genes). Findings in this study indicate TCBZ administration affects multiple mechanisms in the parasite. Therefore, this confirms that all the three proposed TCBZ mode of action and resistance mechanisms in F. hepatica could be implicated in drug TCBZ resistance.