Gene Expression Profile of Selected Fasciola hepatica Gene Families Implicated in Triclabendazole Resistance

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. 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.片形吸虫病（Fasciolosis）由肝片形吸虫（liver flukes）——肝片形吸虫（F. hepatica）及其姊妹物种巨片形吸虫（F. gigantica）——引发。当前该病防控中日益受到关注的问题是针对三氯苯达唑（triclabendazole, TCBZ）的耐药性，而该药物是目前唯一被证实可同时杀灭成虫与未成熟肝片形吸虫的药剂。目前，学界对肝片形吸虫产生三氯苯达唑耐药性的分子机制尚未明确，且尚无获批上市的有效疫苗。既往研究提出了与三氯苯达唑作用模式及耐药性相关的三种机制：微管蛋白（tubulin）结合活性、药物摄取机制以及药物代谢机制。探究作用于与三氯苯达唑作用模式及耐药性相关的肝片形吸虫基因的进化压力，或可阐明该寄生虫产生药物耐药性的机制，助力潜在药物靶点的筛选，并推动新型抗寄生虫药物的研发。 研究人员针对与三氯苯达唑作用模式相关的基因，在肝片形吸虫的各个生活史阶段开展了表达谱分析。结果显示，微管蛋白基因在囊蚴（metacercariae）与新脱囊幼虫（newly excysted juveniles, NEJs）中表达量升高，且在脱囊后1小时的新脱囊幼虫中达到表达峰值，至脱囊后21天的虫体中表达水平逐渐降低。类似地，与三氯苯达唑摄取相关的基因，其表达水平同样主要在囊蚴和新脱囊幼虫中升高，随后随虫体成熟逐渐下调。 研究人员在实验感染的绵羊体内探究了三氯苯达唑对肝片形吸虫的作用效果。他们比较了三氯苯达唑耐药株与敏感株肝片形吸虫分离株在感染并经药物处理的绵羊体内的应答反应。在排除未表达基因与组成型表达基因（constitutively expressed genes）后，研究人员在72%（125个基因中的90个，P < 0.05）的被检测基因中观察到了三氯苯达唑处理诱导的基因表达模式。本研究结果表明，三氯苯达唑可影响寄生虫的多种生理机制，由此证实，既往提出的肝片形吸虫针对三氯苯达唑的三种作用模式与耐药性机制，均可能参与了该寄生虫的药物耐药过程。