Knockdown of phosphoethanolamine transmethylation enzymes decreases viability of Haemonchus contortus

• Phosphorodiamidate morpholino oligomer (PPMO) antisense approach was adapted as a reverse genetic tool in Haemonchus contortus.
• Significant down-regulation of HcPMT1 and HcPMT2 expression was attained using PPMOs targeting HcPMT1 and HcPMT2.
• Knockdown of HcPMT1 and HcPMT2 demonstrated that they are essential for survival of H. contortus adult worms.

The phosphobase methylation pathway, in which phosphoethanolamine N-methyltransferases (PMTs) successively catalyze the methylation of phosphoethanolamine to phosphocholine, is essential in the free-living nematode Caenorhabditis elegans. Two PMT-encoding genes (HcPMT1 and HcPMT2) cloned from Haemonchus contortus have been shown, by in vitro assays, to possess enzymatic characteristics similar to those of C. elegans PMTs, but their physiological significance in H. contortus is yet to be elucidated. Therefore, in this study, we endeavored to determine the importance of HcPMT1 and HcPMT2 in the survival of H. contortus by adapting the use of phosphorodiamidate morpholino oligomers (PPMO) antisense approach to block the translation of HcPMT1 and HcPMT2 in the worms. We found that PPMOs targeting HcPMT1 and HcPMT2 down-regulated the expression of HcPMT1 and HcPMT2 proteins in adult H. contortus. Analysis of the effect of HcPMT1 and HcPMT2 knockdown showed that it significantly decreased worm motility and viability, thus validating HcPMT1 and HcPMT2 as essential enzymes for survival of H. contortus. Studies of gene function in H. contortus have been constrained by limited forward and reverse genetic technologies for use in H. contortus. Thus, our success in adaptation of use of PPMO antisense approach in H. contortus provides an important reverse genetic technological advance for studying this parasitic nematode of veterinary significance.

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