Functional genomics of Rhipicephalus bursa and Babesia ovis interactions towards disease control.

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Advances in the development of a stable transfection system for Babesia ovis

Publication . Rosa, Catarina Maria da Silva; Antunes, Sandra Isabel da Conceição; Pimentel, Madalena Maria Vilela

Babesia species, etiological agents of babesiosis, a recognized emerging vector-borne zoonose, are a significant animal and human health concern with a worldwide socio-economic impact. Genetic manipulation methods are pivotal to improve knowledge regarding the biology of these poorly studied parasites towards better disease control strategies. For Babesia ovis, responsible for ovine babesiosis, a tick-borne disease of small ruminants, these tools are not yet available. Transfection technologies have a wide range of applications, being particularly useful in the study of the parasite-host cell interactions. The establishment of parasites expressing a fluorescent marker has allowed imaging of tick vector colonization, an approach that can also be applied to clarify B. ovis and related Babesia spp. life cycle events in the tick vector tissues. Thus, the main goal of this study was to set up basis for the development of a stable transfection system, driving expression of a fluorescent marker for B. ovis. The present work has four objectives, (1) screening of regulatory regions in B. ovis genome and/or identification of heterologous promoters from other Babesia species, (2) development of transient transfection systems with the previously selected regions and evaluation of transfection conditions, promoter activity, transfection efficiency, (3) identification of a proper selection system for upcoming stable transfection of B. ovis and (4) the development of the plasmid construct for B. ovis stable transfection. The study was based on the existence of interchangeable cross-species functional promoters between Babesia species. Therefore, first steps consisted in the development of transient transfection constructs with elongation factor 1-alpha (ef-1α) promoter regions from B. bovis and B. ovata to drive expression of the luciferase reporter gene. A promoterless plasmid was also developed to use as negative control in the luminescence assays. Herein, we describe for the first time B. ovis transient transfection using heterologous promoters, the ef-1α-B intergenic regions from B. bovis and B. ovata. Their ability to drive expression of a reporter luciferase in B. ovis supports their cross-species functionality. The ef-1α-B promoter region from B. ovata resulted in the expression of high luciferase levels, thus an appropriate promoter for stable gene expression. Transfection efficiency was evaluated through qPCR to preclude the hypothesis that higher luminescence values were related with a higher transfection efficiency. Based on available sequences from B. bovis, B. bigemina and B. divergens, PCR experiments were performed aiming the search for regulatory elements in B. ovis genome. Analysis of the actin 5’ flanking region revealed homology with B. bovis actin promoter but curiously the absence of a transcriptional start site consensus motif common between B. bovis and Theileria species. Regarding ef-1α and rhoptry associated protein -1 (rap-1) locus, their organization in B. ovis remains unclear but it was possible to retrieve a rap-1 intergenic region with transcription termination signals, essential for the development of future autologous transfection systems. Also, a potential region for stable construct integration has been sequenced, the ef-1α gene. Evaluation of B. ovis sensitivity to WR99210 and blasticidin-S, antibiotics commonly used to select apicomplexan transfectants, has been performed and blasticidin-S was identified as a selectable marker for future stably transfected B. ovis. Currently, efforts are being conducted in the construction of a stable transfection plasmid with the ef-1α-B promoter region from B. ovata driving expression of a fluorescent reporter gene and an antibiotic resistance gene. The establishment of a B. ovis lineage expressing a fluorescent reporter gene will be applied to study B. ovis life cycle, enlightening interactions with the host cells.

2018-12-13Dissertação de mestrado

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

3599-PPCDT

Número da atribuição

PTDC/CVT-EPI/4339/2012