Two-hybrid analysis and attempted expression of elongation factor 1α from the cattle tick, Rhipicephalus microplus.

Abstract

Control of Rhipicephalus microplus is predominantly mediated by the application of acaricides, but the rapid acquisition of resistance by this species and environmental pollution resulting from discarded acaricides, necessitates the discovery of new control measures. Due to the fact that Rhipicephalus spp. are genetically diverse and often have more than one host, it has been difficult to identify a common protective vaccine candidate able to target all species of this genus. Only one anti-tick antigen, Bm86, has been commercialized to date and is sold as GAVAC® and GAVACPlus® in South America. In an attempt to identify protective antigens, a protein termed subolesin was identified using expression library immunisation. RNAi studies showed that subolesin knockdown causes the degeneration of tick guts, salivary glands, reproductive tissues and embryos. Subolesin additionally mediates tick gene expression, impacts the innate immune response and affects tick infection by Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. The R. microplus EF-1α homolog was identified as a subolesin-interacting protein via yeast two-hybrid and co-affinity purification experiments. RNAi experiments have suggested that EF-1α is another possible anti-tick vaccine candidate since it exhibits a similar phenotype as subolesin upon knockdown. The aim of the present research was to express R. microplus EF-1α in the yeast, Pichia pastoris and to exploit the yeast two-hybrid system in an attempt to identify its protein-binding partners. This will provide insight into understanding the translational machinery of this species and of ixodid ticks. Recombinant EF-1α was expressed as a 24 kDa protein, validated by western blotting. A highly representative cDNA library was produced from R. microplus mixed lifestages mRNA, fractionated and cloned into a two-hybrid prey vector. No definitive hits were obtained during the two-hybrid screen of reporter genes, as E-values attained after tblastx and PSI-BLAST analysis were higher than the required limit of 1 x 10-4.