Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/14/2016
Publication Date: 9/26/2016
Citation: Alzan, H.F., Lau, A., Knowles, D.P., Herndon, D.R., Ueti, M.W., Scoles, G.A., Kappmeyer, L.S., Suarez, C.E. 2016. Expression of 6-Cys gene superfamily defines babesia bovis sexual stage development within rhipicephalus microplus. PLoS One. doi:10.1371/journal.pone.0163791.

Interpretive Summary: Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. The parasite undergo sexual replication in the midgut of Rhiphicephalus microplus, its most common vector, a step leading to transovarial transmission of the parasite. We aim at targeting the process of development of Babesia parasites in the tick vector as a strategy to prevent subsequent transmission of the parasite to naive cattle by larva from Babesia infected ticks. Our approach is based in the identification of sexual stage specific antigens that can be targeted by vaccines. In this study we identify four new members of the 6-cys supergene family of B. bovis, encoding for proteins known to be involved in sexual stage development in the Babesia related Plasmodium parasites. In addition, we defined the pattern of expression of the Babesia 6-cys genes and demonstrated that the genes are all well conserved among distinct strains and not expressed in the blood stages of the parasites. Interestingly, we detected expression of the 6-cys genes A and B both at the transcriptional and translational levels in the midgut stages of the parasite, and defined these two gens as markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis in the tick vector.

Technical Abstract: Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) and their orthologues in Plasmodium parasite have shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while A, B and E genes were also transcribed in the subsequent B. bovis kinete stage. Transcription of C gene was found exclusively in the kinete. In contrast, transcription of D, F and G genes in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis in the tick vector.