Two-hybrid analysis and functional annotation of Bm86 and ATAQ from Rhipicephalus microplus

Abstract

Vaccination with recombinant Bm86 was found to protect against tick infestation, however, the efficacy of the vaccine varies against different tick species and geographical strains. An improvement of current vaccines could be achieved by identifying homologs of the current Bm86 antigen. A novel protein ATAQ was previously identified during a study that aimed to identify Bm86 homologs. It was shown that these two proteins do not share identical antigenic and immunogenic regions and that they also have different expression patterns. This indicates that ATAQ is not a Bm86 homolog, but rather an entirely different protein. Hence it is reasonable to assume that Bm86 antibodies would not cross-react with ATAQ antigens. Determination of the function of Bm86 and its structurally related protein ATAQ would provide invaluable insight into an unexplored biological system in ticks and potentially lead to the development of an improved vaccine formulation. Therefore, this study sets out to analyze protein-protein interactions by means of the yeast two-hybrid screening system using Bm86 and ATAQ from R. microplus as bait proteins. We were able to obtain that Bm86 and ATAQ have two common (aldehyde and retinol dehydrogenases), as well as a unique (Kunitz-like protein) interacting partners, respectively, via yeast two-hybrid screening. Further confirmation was achieved by immunoprecipitation, western blot and LC/MS analysis. The aldehyde and retinol dehydrogenases, as well as the Kunitz-like protein are involved in retinol metabolism. Therefore, as Bm86 and ATAQ share common binding partners, we hypothesized that they both are involved in the same metabolic pathway in the ticks. Future studies, would involve further confirmation of this pathway in R. microplus and evaluation of a mixture of both Bm86 and its interacting partners in animal vaccination trials.