To kill a piroplasm: genetic technologies to advance drug discovery and target identification in Babesia

Keroack, Caroline D.; Elsworth, Brendan; Duraisingh, Manoj T.

doi:10.1016/j.ijpara.2018.09.005

Cited by 17 publications

(19 citation statements)

References 204 publications

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“…To date, the tools developed for the genetic manipulation of B . bovis are limited and remain most appropriate for use in characterizing individual targets, although there is a need for high-throughput methodologies [8]. The expression of exogenous genes has been demonstrated, using babesial promoter sequences to drive transcription of the target sequences.…”

Section: Introductionmentioning

confidence: 99%

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

2019

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Babesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bb rad51 gene encoding the B . bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bb rad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bb rad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B . bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bb rad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.

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Section: Introductionmentioning

confidence: 99%

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

2019

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“…Implementation of inducible Cas9 or Cas13 targeting RNA will further develop the capabilities of this tool kit for use in analysis of the functional genomics of B. bovis (19). Considering the limited number of selectable markers for B.…”

Section: Discussionmentioning

confidence: 99%

“…; therefore, classical genetic manipulation technologies based on homologous recombination have been used for gene functional studies in B. bovis during the erythrocytic and tick stages (6, 17–19). Functional studies of this parasite have been hampered by the limited breadth of available tools and inefficiency of transfection.…”

Section: Introductionmentioning

confidence: 99%

Genome Editing of Babesia bovis Using the CRISPR/Cas9 System

Hakimi

Ishizaki

Kegawa

et al. 2019

mSphere

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Babesia bovis, the most virulent causative agent of bovine babesiosis, is prevalent in tropical and subtropical regions of the world. Although the whole-genome sequence was released more than a decade ago, functional analysis of the genomics of this parasite is hampered by the limited breadth of genetic engineering tools. In this study, we implemented the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system for B. bovis and demonstrated its potential for genome editing. Cas9 and human dihydrofolate reductase (hDHFR) were simultaneously expressed by the B. bovis elongation factor-1α bidirectional promoter, and a single guide RNA was expressed via the B. bovis U6 spliceosomal RNA promoter. Using a single plasmid construct, we were able to add an epitope tag to spherical body protein 3 (SBP3), introduce a point mutation into thioredoxin peroxidase 1 (tpx-1) to impair the function of the product, and replace the tpx-1 open reading frame with the other protein. Epitope tagging of SBP3 was efficient using this system, with a negligible number of remaining wild-type parasites and a pure transgenic population produced by allelic replacement of tpx-1. This advancement in genetic engineering tools for B. bovis will aid functional analysis of the genome and underpin characterization of candidate drug and vaccine targets. IMPORTANCE Babesia bovis is the most virulent cause of bovine babesiosis worldwide. The disease consequences are death, abortion, and economical loss due to reduced milk and meat production. Available vaccines are not effective, treatment options are limited, and emergence of drug and acaricide resistance has been reported from different regions. There is an urgent need to identify new drug and vaccine targets. Greater than half of the genes in B. bovis genome, including several expanded gene families which are unique for Babesia spp., have no predicted function. The available genetic engineering tools are based on conventional homologous recombination, which is time-consuming and inefficient. In this study, we adapted the CRISPR/Cas9 system as a robust genetic engineering tool for B. bovis. This advancement will aid future functional studies of uncharacterized genes.

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“…The genus Babesia contains a very diverse group of piroplasmid organisms, such as Babesia bovis, Babesia bigemina, Babesia microti and Babesia caballi [1]. Babesiosis caused by these Babesia species represents a veterinary and medical threat.…”

Section: Introductionmentioning

confidence: 99%

Activities of artesunate-based combinations and tafenoquine against Babesia bovis in vitro and Babesia microti in vivo

et al. 2020

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Background: Babesiosis represents a veterinary and medical threat, with a need for novel drugs. Artemisinin-based combination therapies (ACT) have been successfully implemented for malaria, a human disease caused by related parasites, Plasmodium spp. The aim of this study was to investigate whether ACT is active against Babesia in vitro and in vivo. Methods: Mefloquine, tafenoquine, primaquine, methylene blue and lumefantrine, alone or in combination with artesunate, were tested in vitro against Babesia bovis. Parasite growth was verified using a SYBR green I-based fluorescence assay. Mice infected with Babesia microti were treated with mefloquine or tafenoquine, alone or in combination with artesunate, and parasitemia was verified by microscopy and PCR. Results: All drugs, except lumefantrine, showed in vitro activity against B. bovis, with methylene blue showing the most potent activity (concentration 0.2 μM). Combination with artesunate led to improved activity, with mefloquine showing a striking 20-fold increase in activity. Tafenoquine (10 mg/kg, base), combined or not with artesunate, but not mefloquine, induced rapid clearance of B. microti in vivo by microscopy, but mice remained PCR-positive. Blood from mice treated with tafenoquine alone, but not with tafenoquine-artesunate, was infective for naive mice upon sub-inoculation. Conclusions: Tafenoquine, and most likely other 8-aminoquinoline compounds, are promising compounds for the development of ACT for babesiosis.

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To kill a piroplasm: genetic technologies to advance drug discovery and target identification in Babesia

Cited by 17 publications

References 204 publications

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

Genome Editing of Babesia bovis Using the CRISPR/Cas9 System

Activities of artesunate-based combinations and tafenoquine against Babesia bovis in vitro and Babesia microti in vivo

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