Functional characterization of a novel gene, Hc-dhs-28 and its role in protecting the host after Haemonchus contortus infection through regulation of diapause formation.

• Haemonchus contortus protein Hc-DHS-28 has 3-hydroxyacyl CoA dehydrogenase activity. • Hc-dhs-28 is highly transcribed in the diapause worm and located in the peroxisome. • Hc-dhs-28 shares high similarity in gene function with Caenorhabditis elegans (Ce) -dhs-28. • Hc-dhs-28 might regulate the formation of diapause in H. contortus. • Immunisation with recombinant Hc-DHS-28 could protect the host after H. contortus infection. Haemonchus contortus could enter the diapause stage to avoid hostile conditions, however the inducing mechanism still remains poorly understood. A similar dauer strategy exists in Caenorhabditis elegans , and dauer phenomones, which are produced through a four step cycle of peroxisomal fatty acid β-oxidation, are essential in this stage. In this study, a novel gene, Hc-dhs-28 , was identified and characterised. Hc-DHS-28 was the homologue of Ce-DHS-28, a key enzyme in the oxidation cycle, and the protein contained a short chain dehydrogenase domain and a peroxisomal targeting signal 1. The expression pattern of Hc-DHS-28 detected by quantitative real-time PCR and indirect immunofluorescence assay revealed that this protein was mainly expressed in the intestine and subdermal regions of larvae at diapause and in free-living stages. Enzyme activity analysis confirmed its 3-hydroxyacyl CoA dehydrogenase activity with 121, 149, 162 and 166 as key functional sites; meanwhile co-localization in human embryonic kidney 293 cells indicated that Hc-DHS-28 was targeted to the peroxisome of cytoplasm under the guide of peroxisomal targeting signal 1, which was consistent with the functional domain prediction of Hc-dhs-28. Overexpression, rescue and RNA interference experiments were carried out to explore the function of Hc-dhs-28. Our results showed that Hc-dhs-28 was very similar to Ce-dhs-28 and partially rescued its function in C. elegans. RNAi with Hc-dhs-28 in C. elegans led to decreased transcription of genes in the peroxisomal fatty acid β-oxidation cycle, considerable fat accumulation and dauer formation defects. Furthermore, immunisation with recombinant Hc-DHS-28 protein in sheep was able to maintain the body weight of the host after infection and reduce the worm burden. In conclusion, Hc-DHS-28 is most likely involved in the peroxisome fatty acid β-oxidation as the third 3-hydroxyacyl CoA dehydrogenase to regulate the production of diapause-related pheromones, and then influence the formation of diapause in H. contortus. [ABSTRACT FROM AUTHOR]