Accepted Manuscript

Invited Review

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

identification in Babesia

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

PII: S0020-7519(18)30248-0
DOI: https://doi.org/10.1016/j.ijpara.2018.09.005
Reference: PARA 4114

To appear in: International Journal for Parasitology

Received Date: 20 June 2018

Revised Date: 7 September 2018
Accepted Date: 19 September 2018

Please cite this article as: Keroack, C.D., Elsworth, B., Duraisingh, M.T., To kill a piroplasm: genetic technologies
to advance drug discovery and target identification in Babesia, International Journal for Parasitology (2018), doi:
https://doi.org/10.1016/j.ijpara.2018.09.005

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1 Invited Review

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

3 Babesia

5 Caroline D. Keroacka,1, Brendan Elswortha,b,1, Manoj T. Duraisingha,*

7 aHarvard T. H. Chan School of Public Health, 651 Huntington Ave, Boston, MA, 02115, USA.

8 bUniversity of Melbourne, School of Biosciences, Royal Parade, Parkville VIC 3052, Australia.

9 1These authors contributed equally.

10 *Corresponding author.

11 E-mail address: mduraisi@hsph.harvard.edu

12

13
14 Abstract

15 Babesia parasites infect a diverse range of vertebrate hosts, from penguins to pigs. Recently, the

16 emergence of zoonotic Babesia infection has been increasing, and the list of species reported to

17 infect humans continues to grow. Babesiosis represents a burgeoning veterinary and medical

18 threat, and the need for novel therapeutic drugs to effectively target this diverse group of

19 parasites is pressing. Here, we review the current culture systems that exist to study and

20 manipulate Babesia parasites, and identify the scope and methods for target discovery and

21 validation to identify novel, potent anti-babesial inhibitors. Challenges exist including difficulties in

22 the culture systems of important zoonotic parasites, and there is a lack of integrated

23 morphological and molecular data. While molecular approaches in several Babesia spp. has

24 become a reality, the ability to rapidly identify and validate drug targets is hindered by a lack of

25 sophisticated genetic tools to probe parasite biology. The minimal genome size and haploid nature

26 of blood-stage Babesia parasites presents an opportunity to adapt techniques from related

27 systems and characterize the druggable genomic space in a high-throughput way. The

28 considerable diversity of parasites within the genus suggests the existence of highly divergent

29 biology and polymorphism that could present a formidable barrier to the development of a pan-

30 babesiacidal therapeutic strategy.

31

32 Keywords: Babesia; Apicomplexan; Drug discovery; Drug target identification; Target validation;

33 Genetics

34

35

36
37 1. Introduction

38 The genus Babesia contains an incredibly diverse group of piroplasmid organisms (Fig. 1).

39 Babesia is one of the most common blood-borne parasites in vertebrate animals, second only to

40 the trypanosomes (Vannier et al., 2008). To date, over 100 species of Babesia have been

41 described, as well as many more related piroplasmid organisms such as Theileria spp. (Vannier et

42 al., 2008). Babesiosis is a significant cause of veterinary disease from canines to cattle (Bock et al.,

43 2004; Solano-Gallego et al., 2016; Eichenberger et al., 2017). More recently, zoonotic infection has

44 been emerging in humans. Human babesiosis is caused by an increasingly diverse array of

45 parasites including Babesia microti, Babesia duncani, Babesia divergens, Babesia venatorum,

46 Babesia crassa-like and many related, undescribed parasites (Conrad et al., 2006; Bloch et al.,

47 2012; Yabsley and Shock, 2013; Ord and Lobo, 2015; Rajkumari, 2015; Vannier et al., 2015; Jia et

48 al., 2018) (Fig. 1). Indeed, infections in humans have been described from Babesia odocoilei-like, B.

49 divergens-like, and B. microti-like parasites. The recent detection of these new pathogens

50 highlights the potential for emerging infections and the wide-spread nature of the parasite

51 (Herwaldt et al., 1996, 2003; Holman et al., 2005). The emergence of a diverse set of zoonotic

52 parasites, compounded with the abundance of veterinary piroplasmids, presents a unique

53 opportunity to identify conserved biology which can be exploited to identify novel pan-

54 piroplasmid compounds. However, the five recognized clades of Babesia parasites display unique

55 biology between them (Schreeg et al., 2016). This poses a challenge in identification of conserved

56 biological mechanisms which can be targeted with small molecules, due to divergence within and

57 between species. This is exemplified by the variation in efficacy of different compounds identified

58 in screening multiple species with the Medicines for Malaria Ventures Malaria Box (Rizk et al.,

59 2015; Hostettler et al., 2016; Paul et al., 2016; Van Voorhis et al., 2016). This poses a challenge in

60 identification of conserved biological mechanisms which can be targeted with small molecules due
61 to the likely existence of high polymorphism within and between species. Conversely, the smaller

62 genome sizes of Babesia spp., together with high-throughput genomics, facilitates the

63 identification of core apicomplexan biology which can be exploited for therapeutic development

64 through comparative approaches.

65

66 2. Cultivation systems for piroplasmids of veterinary and zoonotic importance

67 A major advantage of piroplasmid diversity is the abundance of experimental systems

68 which can facilitate translational discoveries. Despite recent efforts to describe Babesia spp. using

69 microscopy, much remains to be understood about the life cycle, including the molecular

70 progression of development and differentiation between sexual and asexual stages (Park et al.,

71 2015; Cursino-Santos et al., 2016). Such discoveries, using the various culture systems discussed,

72 will facilitate future therapeutic development. In vitro culture systems enable drug discovery by

73 increasing the throughput of screening and ease of experimental systems for understanding the

74 biology of these organisms (Astashkina et al., 2012). Many species from the various piroplasmid

75 clades can be propagated in vitro. Indeed, many of the most relevant veterinary parasites

76 including Babesia bovis, Babesia bigemina, B. divergens, Babesia major, Babesia ovata, Babesia

77 ovis, Babesia gibsoni, Babesia canis, Babesia caballi, Theileria equi (Babesia equi) and Theileria

78 annulata can be cultured in vitro (Thomson and Fantham, 1914; Irvin et al., 1979; Levy and Ristic,

79 1980; Molinar et al., 1982; Vayrynen and Tuomi, 1982; Vega et al., 1985a, 1985b; Goff and Yunker,

80 1986, 1988; Ben Musa and Phillips, 1991; Holman et al., 1994a; Igarashi et al., 1994; Zweygarth et

81 al., 1995, 1999; Van Niekerk and Zweygarth, 1996; Grande et al., 1997; Viseras et al., 1997;

82 Posnett et al., 1998; Yamasaki et al., 2000; Zweygarth and Lopez-Rebollar, 2000; Musa and Abdel

83 Gawad, 2004; Adaszek and Winiarczyk, 2011; Gharbi et al., 2012; de Rezende et al., 2015) (Fig. 1).

84 Additionally, other animal and wildlife Babesia parasite in vitro culture systems have been
85 developed for a few undescribed species, as well as B. odocoilei, Babesia occultans and Babesia

86 orientalis (Holman et al., 1988, 1994b, 1994c, 2005; Thomford et al., 1993; Van Niekerk and

87 Zweygarth, 1996; Zhao et al., 2002) (Fig. 1). While many of these culture systems remain to be

88 fully optimized and may be limited by the appropriate blood source etc., the methods of culture

89 for many species of Babesia parasites are relatively simple, and conducive to high-throughput

90 experimentation (reviewed in Schuster, 2002). The existence of such a broad and diverse set of

91 cultivable Babesia parasites offers a unique opportunity to explore conserved, essential biology

92 through parallel experimentation in multiple species.

93 Unfortunately, despite major effort, most human zoonotic parasites cannot be

94 continuously cultured in vitro. However, the WA-1 strain of B. duncani has been reported to be

95 maintained continuously in culture (Thomford et al., 1994). Additionally, in vitro and in vivo (i.e.

96 hamsters) models exist to cultivate the rare zoonotic parasite B. divergens and related species

97 (Irvin et al., 1979; Vayrynen and Tuomi, 1982; Ben Musa and Phillips, 1991; Grande et al., 1997;

98 Musa and Abdel Gawad, 2004; Holman et al., 2005). Of the more recently identified zoonoses, B.

99 venatorum and B. crassa, an in vitro culture system has only been described for the former

100 (Bonnet et al., 2009). Recently, an in vitro culture system for B. microti, the major etiological agent

101 of human babesiosis, was patented by Fuller Laboratories (USA), but exact details of this system

102 remain to be fully disclosed (Fuller, L., 2018. In vitro propagation of Babesia microti, US Patent

103 20180080004, U.S.A.P., U.S.A.). As such, the main avenue to study B. microti (and the related

104 parasite Babesia rodhaini) relies on short-term ex vivo culture or in vivo models, which limits the

105 scale of drug discovery efforts and functional validation (Shikano et al., 1995; Lawres et al., 2016;

106 Saito-Ito et al., 2016). However, in vivo models are valuable for understanding physiological

107 dynamics of infection and facilitate downstream drug and vaccine validation and discovery
108 (Gardner and Molyneux, 1987; Penzhorn et al., 2000; Lawres et al., 2016; Saito-Ito et al., 2016)

109 (Fig. 1).

110

111 3. Identification of new anti-babesial inhibitors

112 The existence of model systems allows for the development of high-throughput screening

113 (HTS) to identify novel anti-piroplasmid compounds. The need for novel drugs to treat veterinary

114 and human babesiosis is pressing. Current treatments in humans have been reported to result in

115 de novo resistance generation, and reports of resistance exist for veterinary parasites as well

116 (Yeruham et al., 1985; Sakuma et al., 2009; Wormser et al., 2010; Lemieux et al., 2016; Simon et

117 al., 2017). Additionally, the targets and mechanisms of action of some current treatments are not

118 fully understood. While the targets of atovaquone (cytB) and azithromycin (rpl4) are known

119 (Simon et al., 2017), those for imidocarb and others remain unknown (reviewed in Mosqueda et

120 al. (2012). Recently there has been effort to repurpose many compounds to combat piroplasmid

121 infection such as epoxomicin, artesunate, triclosan, and others (reviewed in Mosqueda et al.

122 (2012). Efforts to repurpose antibiotics such as Draxxin®, enoxacin, and clofazamine have shown

123 promise (Omar et al., 2016; Tuvshintulga et al., 2017; Silva et al., 2018b). Novel anti-babesial

124 compounds have also been identified, such as the endochin-like quinolones and a series of

125 analogues of the antimitotic herbicide trifluralin (Silva et al., 2013; Lawres et al., 2016). While

126 there are several novel anti-babesials of interest emerging, many of their targets are the same as

127 those for currents treatments (i.e. cytochrome bc1 complex for atovaquone and the new

128 endochin-like quinolones) (Kessl et al., 2003; Lawres et al., 2016). In contrast, trifluralin analogues

129 have been shown to target α-tubulin, a novel target, showcasing the strength of such comparative

130 approaches in identifying lead compounds (Silva et al., 2013). Cysteine proteases, lactate

131 dehydrogenase, dihydroorotate dehydrogenase, apicoplast pathways, and kinases have been
132 proposed as novel targets with some evidence that inhibition of these pathways can inhibit

133 parasite growth (Bork et al., 2004; Okubo et al., 2007; AbouLaila et al., 2012; Kamyingkird et al.,

134 2014; Pedroni et al., 2016). However, the need remains to advance research into these targets,

135 and further identify novel druggable targets.

136 Thanks to the robustness of several in vitro and in vivo culture systems, screening for

137 novel, effective anti-piroplasmid compounds is possible. Small scale screens have been used to

138 test the efficacy of compounds against several species, often in parallel experiments, such as

139 Babesia felis, T. equi (B. equi), Babesia caballi, B. bovis, and B. bigemina (Penzhorn et al., 2000;

140 Nagai et al., 2003; Okubo et al., 2007; AbouLaila et al., 2012). Recently, fluorescence based tools

141 have been developed which have enabled larger scale screening for anti-piroplasmid compounds

142 both in vitro (T. equi, B. caballi, B. bovis, B. bigemina, B. divergens) and in vivo (B. microti)

143 (Guswanto et al., 2014; Rizk et al., 2015, 2016, 2017). These advances have allowed screening of

144 larger libraries such as the Medicines for Malaria Ventures ‘Malaria Box’ in several Babesia and

145 Theileria spp. in vitro, which have identified many novel compounds with higher potency than

146 previously identified lead compounds of interest (Hostettler et al., 2016; Paul et al., 2016). With

147 the ability to perform HTS in piroplasmids, the next major hurdle lies in the elucidation of the

148 mechanism of action. This relies on a combination of phenotypic characterization and genetic

149 validation. Thus, robust systems for assessment of drug phenotypes and genetic tools to

150 manipulate the parasite are necessary.

151

152 3.1. Biochemical and phenotypic assessment as a method to characterize mechanism of action

153 There are many ways of identifying potent compounds and parasite targets for further hit

154 to lead optimization to develop novel effective drugs for Babesia. Targeted screening approaches

155 such as recombinant protein assays (ie. DHFR, DHODH, Kinases), can determine direct inhibition of
156 the parasite protein (Brobey et al., 1996; Baldwin et al., 2005; Biftu et al., 2005; Qian et al., 2006;

157 Zhang et al., 2006). These assays in Babesia would generally rely on identification of orthologous

158 genes in related parasites. These assays have the advantage of being suitable to HTS of millions of

159 compounds and being specific to the target of interest, being able to counter screen against the

160 host homologs and aid structure based design. However, compounds discovered by this process

161 have several limitations. Firstly, the ability to kill the parasite is dependent on complex properties

162 such as membrane permeability, which is not addressed in these screens. Secondly, the specificity

163 of the compound within the parasite does not always correlate with that seen with recombinant

164 protein. An example of this was with kinase inhibitors, where the activity against recombinant

165 PfCDPK1 protein did not correlate to the anti-Plasmodium activity, most likely due to the primary

166 target within the parasite being a different kinase (Ansell et al., 2014). Other widely used methods

167 rely on phenotypic screening, which may help provide a potential pathway that is being inhibited

168 (i.e., apicoplast function, new permeability pathways, calcium signaling, egress, invasion etc.) but

169 generally do not identify a single parasite molecule as the target (Pillai et al., 2010; Salmon et al.,

170 2001; Boyle et al., 2010; Wu et al., 2015; Dickerman et al., 2016; Sidik et al., 2016).

171 Alternatively, many compounds have been tested in Babesia based on their known target

172 in other systems. As described above, many of these compounds have proven to be potent

173 inhibitors of Babesia, including two of the most common treatments for human babesiosis,

174 atovaquone and azithromycin. However, the strong reliance of screening compounds that are

175 active against related parasites, mainly Plasmodium, will likely not encompass the full chemical

176 space and may miss the most potent anti-babesial compounds. The large evolutionary distance

177 between apicomplexan parasites and more widely studied organisms may lead to false

178 assumptions about a compound’s activity and target. This is exemplified by the mTOR inhibitor,

179 Torin 2, which is an extremely potent inhibitor of Plasmodium falciparum growth, however, no
180 mTOR homolog is present in Plasmodium parasites (Hanson et al., 2013; Sun et al., 2014b). More

181 recently, significant advancements have been developed in metabolomics and proteomic methods

182 leading to the identification of several drug-target pairs (Sun et al., 2014a; Allman et al., 2016;

183 Creek et al., 2016; Dickerman et al., 2016). While each of these methods are valuable for

184 identifying potential inhibitors and their broad mode of action, genetic methods are still required

185 to validate specific targets within the parasite, and as previously mentioned can be used to

186 identify targets.

187 A critical step in the characterization of a novel compound is elucidation of the phenotypic

188 effect generated upon treatment. Stage specificity has been critical in understanding the

189 mechanism of action in the related Plasmodium parasites (i.e. Skinner et al., 1996; Sriwilaijareon

190 et al., 2002). In comparison to Plasmodium, Babesia spp. have a relatively simple asexual life cycle

191 by which parasites invade a red blood cell, egress from the parasitophorous vacuole, grow and

192 divide by binary fission, and finally egress from the host cell (Mackenstedt et al., 1990; Hunfeld et

193 al., 2008; Chauvin et al., 2009; Eisen and Gage, 2009; Mosqueda et al., 2012; Rossouw et al., 2015;

194 Cursino-Santos et al., 2017). For several species such as B. bovis, only one division occurs per host

195 cell prior to egress (Hunfeld et al., 2008). However, in other species such as B. divergens and B.

196 microti, the parasite is able to undergo multiple rounds of binary fission or budding prior to egress

197 (Rossouw et al., 2015; Cursino-Santos et al., 2016, 2017). The differences in asexual cycles

198 between different species present challenges in identifying compounds which have conserved

199 modes of action based on phenotyping alone. However, the unifying developmental steps

200 between all Babesia parasites, such as invasion, egress, and motility, provide novel avenues of

201 attack for future compounds. For example, compounds which block egress would lead to an

202 accumulation of merozoites within a host cell, likely regardless of species. Indeed, in B. bovis

203 treatment with bumped kinase inhibitors halts egress and leads to an accumulation of merozoites
204 (Pedroni et al., 2016). Similarly, upon treatment with EGTA, B. divergens fails to egress and a

205 marked accumulation of merozoites is observed (Cursino-Santos et al., 2017). As egress is a

206 uniquely parasitic process with a clear phenotype, it presents a promising potential target.

207 Compounds which elicit similar or identical phenotypes in multiple species should be prioritized

208 for their pan-piroplasmid potential.

209 A current challenge in understanding mechanisms of action in a wholistic way is the

210 relative lack of transcriptomic and proteomic data to characterize the various cellular states of the

211 parasite as it progresses through the cell cycle. Transcriptomic studies that exist have mainly

212 focused on characterizing pathogenesis and host-pathogen interactions (Gohil et al., 2010;

213 Pedroni et al., 2013; Silva et al., 2016a; Eichenberger et al., 2017). Recently, more detailed

214 transcriptomic analyses attempting to characterize intraerythrocytic development and virulence

215 have been released for B. divergens, B. bovis, and Babesia canis (Pedroni et al., 2013; Rossouw et

216 al., 2015; Eichenberger et al., 2017). The availability of these data provides an important new

217 resource to be used in the identification of novel anti-babesials. Additional detailed transcriptomic

218 studies which fully characterize the cell cycle of both the sexual and asexual stages of Babesia

219 parasites will be essential moving forward with high-throughput drug discovery. Proteomic studies

220 have focused on identification of novel secreted antigens, mainly for diagnostic purposes. Smaller

221 studies which study expression of specific families of proteins have been undertaken, but fully

222 characterized proteomes remain to be generated (Alzan et al., 2016; Cornillot et al., 2016; Silva et

223 al., 2016a; Eichenberger et al., 2017; Johnson et al., 2017). Of Babesia parasites, B. bovis is the

224 most well characterized, and may serve as a model for future technological development

225 (reviewed in Gohil et al. (2010). Compounding these challenges is a relative lack of available

226 complete genome sequences, although culture systems exist for many species of Babesia.

227 Genomes exist for B. microti, Babesia ovata, B. bovis, B. bigemina, B. canis, and B. divergens
228 (Brayton et al., 2007b; Cornillot et al., 2012b; Cuesta et al., 2014a; Jackson et al., 2014b;

229 Eichenberger et al., 2017; Yamagishi et al., 2017). Additionally, genomic sequences for several

230 Theileria spp. are available, which could be useful in identifying essential piroplasmid pathways

231 (Gardner et al., 2005; Pain et al., 2005; Hayashida et al., 2012; Kappmeyer et al., 2012).

232

233 3.2. Target identification and validation

234 In order to accelerate drug development, effective means for identifying molecular targets

235 are essential. Due to the ease of phenotypic screening, often potent compounds are identified

236 without a hint as to the mechanism of action. Target identification is important in understanding

237 mechanisms of action and, more broadly, pharmacological properties of a novel compound

238 (reviewed in Schenone et al. (2013). Indeed, target identification is often the first step in the drug

239 discovery pathway. Understanding the target of a compound can aid in understanding potential

240 off target or toxic effects. This helps circumvent costly clinical failures (reviewed in Chan et al.

241 (2010). Simply identifying potential targets is insufficient for the drug discovery pipeline-

242 successful progression of novel compounds relies on both the identification and validation of their

243 interacting partners (Cong et al., 2012). Target discovery and validation can be long and arduous,

244 however for Babesia the small genome size (6-14 Mbp) and rapid replication cycle may facilitate

245 this process (Brayton et al., 2007a; Lau, 2009; Cornillot et al., 2012a, 2013; Cuesta et al., 2014b;

246 Jackson et al., 2014a; Eichenberger et al., 2017). The methods by which targets can be identified

247 and validated are subsequently discussed.

248

249 3.3. Chemical genomics as a method for target discovery

250 Using in vitro evolution followed by chemical genomics is an effective method for

251 identifying target-inhibitor pairs in many parasites. This is a strategy whereby resistant organisms
252 are generated against a compound of interest, potential causal mutations are identified through

253 whole genome sequencing, and validated downstream using reverse genetic techniques (Cowell et

254 al., 2018). This strategy was successful in identifying cytB as the target of the small molecule

255 inhibitor GNF7686 in Trypanosoma cruzi (Khare et al., 2015). In related apicomplexan parasites

256 Toxoplasma and Plasmodium, chemical genomics is one of the main strategies for target

257 identification (reviewed in McFadden et al. (2001); Luth et al. (2018)). For example, in vitro

258 evolution was used in Toxoplasma gondii to identify novel mutations in the dihydrofolate

259 reductase (DHFR) gene which confer resistance to pyrimethamine (Reynolds et al., 2001). Further,

260 this technique has been extensively used in the closely related parasite Plasmodium falciparum to

261 identify several novel target-inhibitor pairs (Ariey et al., 2014; Corey et al., 2016; Cowell et al.,

262 2018). The existence of many in vitro systems in Babesia and Theileria presents a unique

263 opportunity to take advantage of chemical genomics in a comparative fashion. Indeed, resistance

264 to several compounds has been generated in vitro in Babesia, such as diminazine aceturate in

265 Babesia gibsoni (Hwang et al., 2010). Unfortunately, a genome sequence for B. gibsoni remains to

266 be fully elucidated, thus identification of mutations in previously generated lines will be difficult

267 (Goo and Xuan, 2014). Imidocarb dipropionate has been an important treatment for veterinary

268 babesiosis. However, the mechanism of action for this compound has remained elusive (McHardy

269 et al., 1986; Coldham et al., 1995; Rodriguez and Trees, 1996; Belloli et al., 2006; Mosqueda et al.,

270 2012). Drug-adapted lines in B. bovis were generated against imidocarb over two decades ago,

271 prior to the genome being published. As the B. bovis genome is now available and annotated,

272 sequencing of those isolates may reveal insights into either the mechanism of action or

273 determinants of resistance (Rodriguez and Trees, 1996). Identification of targets for known and

274 novel compounds will be facilitated by the generation of genome sequence of wild-type and

275 previously generated resistant lines in the corresponding species. Once mutations are identified in
276 whole genome sequence, targets will need to be validated. This will require the development of

277 sophisticated genetic tools to probe Babesia parasites.

278

279 4. Existing genetic technologies in Babesia for target validation

280 The development of genetic tools to study parasites in general has vastly expanded our

281 knowledge of cellular biology and has allowed for the identification and validation of small

282 molecule inhibitor - parasite target pairs: a challenge that remains for Babesia spp. Development

283 of transient transfection systems for B. bovis, B. bigemina, B. gibsoni and B. ovata has permitted

284 the assessment and optimization of different transcriptional elements and transfection methods

285 (Suarez et al., 2004, 2006; Suarez and McElwain, 2008; Hakimi et al., 2016; Silva et al., 2016b; Liu

286 et al., 2017a, 2017b). This has since led to the ability to stably transfect each of these organisms,

287 as well as B. divergens (Elsworth et al., unpublished data ) (Suarez and McElwain, 2009; Hakimi et

288 al., 2016; Liu et al., 2018; Silva et al., 2018a). Using these tools, the generation of parasites with

289 gene deletions, episomal and stable overexpression of two selection markers (BSD and hDHFR) as

290 well as reporter proteins, tick antigens and native Babesia proteins for gene complementation, is

291 now possible and has assisted in the elucidation of novel Babesia biology (Suarez and McElwain,

292 2009; Asada et al., 2012, 2015, 2018; Wang et al., 2012; Laughery et al., 2014; Pellé et al., 2015;

293 Hakimi et al., 2016; Oldiges et al., 2016; Alzan et al., 2017; Liu et al., 2018; Silva et al., 2018a;

294 Suarez et al., 2012, 2015).

295 Many aspects of Babesia biology make it highly suitable to genetic manipulation and drug

296 target identification. Babesia spp. have a minimalized genome in comparison to most parasites,

297 both in terms of genome size (6-14 MB) and number of genes (~3500-3800) and are also haploid

298 during the asexual cycle, reducing the number of potential targets (Brayton et al., 2007a; Lau,

299 2009; Cornillot et al., 2012a, 2013; Cuesta et al., 2014b; Jackson et al., 2014a; Eichenberger et al.,
300 2017). Babesia spp. have a relatively balanced GC content (~40%) and small intergenic regions –

301 and therefore untranslated regions (UTRs) – aiding in the development of plasmids and

302 sequencing (Brayton et al., 2007a; Lau, 2009; Cornillot et al., 2012a, 2013; Cuesta et al., 2014b;

303 Jackson et al., 2014a; Eichenberger et al., 2017). Homologous recombination is highly efficient and

304 specific, allowing the rapid transfection of linear constructs to manipulate the genome (Suarez et

305 al., 2015). However, the poorly characterized genomes, in terms of gene function and essentiality

306 (with ~50% of the genome having no predicted function), in Babesia parasites presents a challenge

307 moving forward (Brayton et al., 2007a; Lau, 2009; Cornillot et al., 2012a, 2013; Cuesta et al.,

308 2014b; Jackson et al., 2014a; Eichenberger et al., 2017). Furthermore, the relative lack of

309 experimental tools and knowledge (synchronization and purification methods, specific antibodies,

310 genomic, transcriptomic and proteomic datasets, etc.) compared with related parasites presents a

311 major barrier to understanding Babesia biology and identifying drug targets, especially in less

312 studied species. It also remains unclear how readily genetic and experimental tools will be

313 transferable between species, with the requirement for generating species-specific reagents likely,

314 and whether compounds will primarily target the same parasite molecule in all species.

315

316 5. Looking forward: new genetic approaches for drug target identification in Babesia

317 5.1. Novel genetic tools for understanding gene function

318 In recent years the genetic toolkit available in parasites has greatly expanded to include

319 methods for rapid and markerless gene editing, inducible knockdown and knockout as well as

320 genome-wide knockdown and knockout studies (a summary of available genetic tools in related

321 organisms is shown in Table 1). Currently no such technologies are available in any Babesia

322 species, however, the lessons learnt from related parasites could be used to accelerate the

323 adaptation of such genetic tools.

324 Currently, the only genetic method for determining gene essentiality in Babesia is through

325 attempting gene deletion by homologous recombination. Gene deletion has limited value for

326 studying essential genes as the inability to delete a gene does not strictly show essentiality and

327 provides no information on the function of the gene. For these reasons the parasite field has

328 widely adopted inducible knockdown and knockout strategies (reviewed in de Koning-Ward et al.

329 (2015). Inducible knockdown systems can broadly be grouped into those that alter gene

330 transcription (Tet-off, Cre-Lox) (Gossen et al., 1995; Wirtz and Clayton, 1995; Meissner et al., 2001,

331 2005; Pino et al., 2012; Collins et al., 2013), mRNA stability/translation (glmS, TetR, TetR-DOZI)

332 (Prommana et al., 2013; Goldfless et al., 2014; Ganesan et al., 2016), protein stability (DD and

333 DDD) (Banaszynski et al., 2006; Armstrong and Goldberg, 2007; Iwamoto et al., 2010;

334 Muralidharan et al., 2011; Beck et al., 2014) and protein localization (knock-sideways) (Robinson

335 et al., 2010; Birnbaum et al., 2017). The appropriateness of each system will depend on the gene

336 of interest (GOI) and intended use. For example, the protein targeting systems require a protein

337 tag that may interfere with the natural function of the GOI. On the other hand, the protein

338 targeting systems tend to act faster (knock-sideways acts within minutes) than the RNA systems,

339 which require natural turnover of the protein (Robinson et al., 2010; Birnbaum et al., 2017).

340 Inducible gene deletion using the Cre-Lox system could also be used and is likely to produce a

341 stronger phenotype than knockdown systems (Andenmatten et al., 2013; Collins et al., 2013). A

342 single study has described the use of RNA interference (RNAi) in B. bovis, a technique used in

343 Trypanosoma brucei drug discovery, however, the relatively low success rate and lack of

344 identifiable RNAi machinery in the parasite will need to be investigated further before this

345 technique can be widely applied (Baker et al., 2011; Burkard et al., 2011; AbouLaila et al., 2016).

346

347 5.2. Genetic methods for drug discovery

348 Gene editing to introduce point mutations into a GOI can aid in validating the specificity of

349 a compound, for example to validate mutations observed after generation of resistant parasites or

350 through rational mutation based on homologs (Donald et al., 2006; Lourido et al., 2010; Chow et

351 al., 2016; LaMonte et al., 2016; Ng et al., 2016; Crawford et al., 2017; Dhingra et al., 2017; Sonoiki

352 et al., 2017). Parasites harboring point mutations can be generated by homologous

353 recombination, which is already possible in Babesia, or could be generated with a CRISPR/Cas9

354 system which would require less cumbersome plasmid design (Suarez and McElwain, 2009; Hakimi

355 et al., 2016; Liu et al., 2018; Silva et al., 2018a).

356 Synthetic lethal or overexpression studies, which rely on altered expression of the parasite

357 target causing increased or decreased sensitivity to an inhibitor, respectively, are widely used for

358 target validation or target orientated screening (i.e. Arakaki et al., 2008; Goldfless et al., 2014;

359 Sleebs et al., 2014; Aroonsri et al., 2016). Early Plasmodium studies utilized truncated 3’UTRs to

360 reduce the expression level of native genes (Waller et al., 2003; Nkrumah et al., 2009). While this

361 approach could be used with existing techniques in Babesia, development of the inducible systems

362 mentioned above would offer a tunable expression system that would be more widely applicable.

363 An alternative approach is to overexpress a GOI, which can either be the native GOI or a divergent

364 homolog with altered susceptibility to a compound, such as Saccharomyces cerevisiae DHODH

365 expression in P. falciparum (Baldwin et al., 2005; Gardiner et al., 2006; Painter et al., 2007;

366 Ganesan et al., 2011; Hoepfner et al., 2012; Sleebs et al., 2014; Phillips et al., 2015; Dickerman et

367 al., 2016). This can be achieved either by episomal expression of the gene, with the advantage of

368 multiple extra copies, or integration of a second copy of the gene into the genome, which will

369 consistently produce a single extra copy.

370

371 5.3. Prospect for forward genetic screens

372 The technologies available for Babesia are best suited to targeted approaches, with either

373 a hypothesized target of a compound or for screening compound libraries against a high priority

374 target of interest. Genome-wide screens that look for genes that are over- or under-represented

375 when exposed to an inhibitor have been widely used in drug discovery with no prior knowledge of

376 the inhibitor’s mechanism of action. This can be done with genome-wide overexpression libraries

377 as has been performed in Leishmania infantum and T. brucei, using methods which could be

378 adapted to Babesia, however, it would require significant effort to generate the library (Begolo et

379 al., 2014; Koushik et al., 2014; Gazanion et al., 2016; Tejera Nevado et al., 2016; Fernandez-Prada

380 et al., 2018). To date no large-scale knockdown screens have been performed in parasites lacking

381 RNAi, however, novel high-throughput methods have recently been developed for mammalian

382 cells and have been widely adopted in the mammalian drug screening field (Gilbert et al., 2013,

383 2014; Konermann et al., 2015; Joung et al., 2017). Cas13 is a newly described molecule that is able

384 to specifically cleave target mRNA, with the only requirement for activity being the presence of a

385 corresponding guide RNA, thus making library production more feasible (Abudayyeh et al., 2016,

386 2017; Cox et al., 2017; Konermann et al., 2018). The lack of non-homologous end joining (NHEJ)

387 repair mechanisms in Babesia precludes the use of CRISPR/Cas9 to perform genome-wide

388 knockout screens, however, the piggyBac transposon, which randomly inserts into TTAA sites in

389 the genome, has recently been used in P. falciparum, which can be used to help prioritize essential

390 genes (Zhang et al., 2018). An advantage of the piggyBac system is that insertion into the 5’ and 3’

391 UTR of genes can lead to altered gene expression (Pradhan et al., 2015; Zhang et al., 2018). Using

392 pools of piggyBac mutants has revealed novel genes that alter sensitivity to inhibitors as well as

393 networks of mutants that have similarly altered sensitivity to multiple compounds, suggesting a

394 related role in the parasite (Pradhan et al., 2015; Zhang et al., 2018).

395
396 6. Conclusions

397 Perhaps the greatest challenge in identifying novel anti-babesials is the extraordinary

398 diversity of the genus. The variability of the Babesia genus generates uncertainty about the ease

399 of adapting tools from orthologous systems. Yet, this diversity provides a unique opportunity to

400 study the parasites in a comparative manner to identify core, conserved biology. The large number

401 of piroplasmid species of medical and veterinary importance necessitates the development of a

402 species-transcendent compound. To date, no single compound shows broad range efficacy against

403 all species. Further, no target-inhibitor pairs have been conclusively validated in Babesia spp. using

404 genetic techniques. In related species, there has been rapid development of systems for genetic

405 manipulation to identify and validate druggable genes. This has lead to the discovery of lead

406 compounds and accelerated compound development (Phillips et al., 2015, 2016). The plethora of

407 in vitro and in vivo culture systems which are available for piroplasmids offers an unprecedented

408 opportunity to identify novel, conserved biology which could be leveraged in development of

409 species-transcendent compounds for treatment of medical and veterinary infections. The recent

410 success of transfection methods for Babesia spp. offers the prospect for development of more

411 advanced techniques. Such techniques would greatly accelerate drug discovery and development.

412

413 Acknowledgements

414 Brendan Elsworth was supported by an Australian National Health and Medical Research

415 Council CJ Martin fellowship (#1148392). Caroline D. Keroack was supported by a National

416 Institute of Health, USA, training grant (#5T32AI49928-13).

417

418
419 References

420 AbouLaila, M., Munkhjargal, T., Sivakumar, T., Ueno, A., Nakano, Y., Yokoyama, M., Yoshinari, T.,

421 Nagano, D., Katayama, K., El-Bahy, N., 2012. Apicoplast-targeting antibacterials inhibit the

422 growth of Babesia parasites. Antimicrob Agents Chemother 56, 3196-3206.

423 AbouLaila, M., Yokoyama, N., Igarashi, I., 2016 RNA interference (RNAi) for some genes from

424 Babesia bovis. Res J Applied Biotechnol (RJAB) ISSN 2356, 9433. 2, 81-92

425 Abudayyeh, O.O., Gootenberg, J.S., Essletzbichler, P., Han, S., Joung, J., Belanto, J.J., Verdine, V.,

426 Cox, D.B.T., Kellner, M.J., Regev, A., Lander, E.S., Voytas, D.F., Ting, A.Y., Zhang, F., 2017.

427 RNA targeting with CRISPR–Cas13. Nature 550, 280.

428 Abudayyeh, O.O., Gootenberg, J.S., Konermann, S., Joung, J., Slaymaker, I.M., Cox, D.B.T.,

429 Shmakov, S., Makarova, K.S., Semenova, E., Minakhin, L., Severinov, K., Regev, A., Lander,

430 E.S., Koonin, E.V., Zhang, F., 2016. C2c2 is a single-component programmable RNA-guided

431 RNA-targeting CRISPR effector. Science 353.

432 Adaszek, L., Winiarczyk, S., 2011. In vitro cultivation of Babesia canis canis parasites isolated from

433 dogs in Poland. Parasitol Res 108, 1303-1307.

434 Allman, E.L., Painter, H.J., Samra, J., Carrasquilla, M., Llinás, M., 2016. Metabolomic profiling of the

435 malaria box reveals antimalarial target pathways. Antimicrob Agents Chemother 60, 6635-

436 6649.

437 Alzan, H.F., Lau, A.O., Knowles, D.P., Herndon, D.R., Ueti, M.W., Scoles, G.A., Kappmeyer, L.S.,

438 Suarez, C.E., 2016. Expression of 6-Cys Gene Superfamily Defines Babesia bovis Sexual

439 Stage Development within Rhipicephalus microplus. PLoS One 11, e0163791.

440 Alzan, H.F., Silva, M.G., Davis, W.C., Herndon, D.R., Schneider, D.A., Suarez, C.E., 2017. Geno-and

441 phenotypic characteristics of a transfected Babesia bovis 6-Cys-E knockout clonal line.

442 Parasites Vectors 10, 214.

443 Andenmatten, N., Egarter, S., Jackson, A.J., Jullien, N., Herman, J.-P., Meissner, M., 2013.

444 Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion

445 mechanisms. Nature Methods 10, 125.

446 Ansell, K.H., Jones, H.M., Whalley, D., Hearn, A., Taylor, D.L., Patin, E.C., Chapman, T.M., Osborne,

447 S.A., Wallace, C., Birchall, K., 2014. Biochemical and antiparasitic properties of inhibitors of

448 the Plasmodium falciparum calcium-dependent protein kinase PfCDPK1. Antimicrob Agents

449 Chemother 58, 6032-6043.

450 Arakaki, T.L., Buckner, F.S., Gillespie, J.R., Malmquist, N.A., Phillips, M.A., Kalyuzhniy, O., Luft, J.R.,

451 DeTitta, G.T., Verlinde, C.L., Van Voorhis, W.C., 2008. Characterization of Trypanosoma

452 brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi

453 studies. Mol Microbiol 68, 37-50.

454 Ariey, F., Witkowski, B., Amaratunga, C., Beghain, J., Langlois, A.C., Khim, N., Kim, S., Duru, V.,

455 Bouchier, C., Ma, L., Lim, P., Leang, R., Duong, S., Sreng, S., Suon, S., Chuor, C.M., Bout,

456 D.M., Menard, S., Rogers, W.O., Genton, B., Fandeur, T., Miotto, O., Ringwald, P., Le Bras,

457 J., Berry, A., Barale, J.C., Fairhurst, R.M., Benoit-Vical, F., Mercereau-Puijalon, O., Menard,

458 D., 2014. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.

459 Nature 505, 50-55.

460 Armstrong, C.M., Goldberg, D.E., 2007. An FKBP destabilization domain modulates protein levels in

461 Plasmodium falciparum. Nature Methods 4, 1007.

462 Aroonsri, A., Akinola, O., Posayapisit, N., Songsungthong, W., Uthaipibull, C., Kamchonwongpaisan,

463 S., Gbotosho, G.O., Yuthavong, Y., Shaw, P.J., 2016. Identifying antimalarial compounds

464 targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic

465 profiling. Intl J Parasitol 46, 527-535.

466 Asada, M., Hakimi, H., Kawazu, S.-i., 2018. The application of the HyPer fluorescent sensor in the

467 real-time detection of H2O2 in Babesia bovis merozoites in vitro. Vet Parasitol 255, 78-82.

468 Asada, M., Tanaka, M., Goto, Y., Yokoyama, N., Inoue, N., Kawazu, S.-i., 2012. Stable expression of

469 green fluorescent protein and targeted disruption of thioredoxin peroxidase-1 gene in

470 Babesia bovis with the WR99210/dhfr selection system. Mol Biochem Parasitol 181, 162-

471 170.

472 Asada, M., Yahata, K., Hakimi, H., Yokoyama, N., Igarashi, I., Kaneko, O., Suarez, C.E., Kawazu, S.-i.,

473 2015. Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S

474 and Its Application for Functional Analysis of Thioredoxin Peroxidase-1. PLoS ONE 10,

475 e0125993.

476 Astashkina, A., Mann, B., Grainger, D.W., 2012. A critical evaluation of in vitro cell culture models

477 for high-throughput drug screening and toxicity. Pharmacol Therapeut 134, 82-106.

478 Baker, N., Alsford, S., Horn, D., 2011. Genome-wide RNAi screens in African trypanosomes identify

479 the nifurtimox activator NTR and the eflornithine transporter AAT6. Mol Biochem Parasitol

480 176, 55-57.

481 Baldwin, J., Michnoff, C.H., Malmquist, N.A., White, J., Roth, M.G., Rathod, P.K., Phillips, M.A.,

482 2005. High-throughput screening for potent and selective inhibitors of Plasmodium

483 falciparum dihydroorotate dehydrogenase. J Biol Chem 280, 21847-21853.

484 Banaszynski, L.A., Chen, L.-c., Maynard-Smith, L.A., Ooi, A.L., Wandless, T.J., 2006. A rapid,

485 reversible, and tunable method to regulate protein function in living cells using synthetic

486 small molecules. Cell 126, 995-1004.

487 Beck, J.R., Muralidharan, V., Oksman, A., Goldberg, D.E., 2014. PTEX component HSP101 mediates

488 export of diverse malaria effectors into host erythrocytes. Nature 511, 592.
489 Begolo, D., Erben, E., Clayton, C., 2014. Drug target identification using a trypanosome

490 overexpression library. Antimicrob Agents Chemother 58, 6260-6264.

491 Belloli, C., Lai, O.R., Ormas, P., Zizzadoro, C., Sasso, G., Crescenzo, G., 2006. Pharmacokinetics and

492 mammary elimination of imidocarb in sheep and goats. J Dairy Sci 89, 2465-2472.

493 Ben Musa, N., Phillips, R.S., 1991. The adaptation of three isolates of Babesia divergens to

494 continuous culture in rat erythrocytes. Parasitology 103 Pt 2, 165-170.

495 Biftu, T., Feng, D., Ponpipom, M., Girotra, N., Liang, G.-B., Qian, X., Bugianesi, R., Simeone, J.,

496 Chang, L., Gurnett, A., Liberator, P., Dulski, P., Leavitt, P.S., Crumley, T., Misura, A., Murphy,

497 T., Rattray, S., Samaras, S., Tamas, T., Mathew, J., Brown, C., Thompson, D., Schmatz, D.,

498 Fisher, M., Wyvratt, M., 2005. Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite

499 cGMP-dependent protein kinase as novel anticoccidial agents. Bioorgan Med Chem Lett 15,

500 3296-3301.

501 Birnbaum, J., Flemming, S., Reichard, N., Soares, A.B., Mesén-Ramírez, P., Jonscher, E., Bergmann,

502 B., Spielmann, T., 2017. A genetic system to study Plasmodium falciparum protein function.

503 Nature Methods 14, 450.

504 Bloch, E.M., Herwaldt, B.L., Leiby, D.A., Shaieb, A., Herron, R.M., Chervenak, M., Reed, W., Hunter,

505 R., Ryals, R., Hagar, W., Xayavong, M.V., Slemenda, S.B., Pieniazek, N.J., Wilkins, P.P.,

506 Kjemtrup, A.M., 2012. The third described case of transfusion-transmitted Babesia duncani.

507 Transfusion 52, 1517-1522.

508 Bock, R., Jackson, L., De Vos, A., Jorgensen, W., 2004. Babesiosis of cattle. Parasitology 129, S247-

509 S269.

510 Bonnet, S., Brisseau, N., Hermouet, A., Jouglin, M., Chauvin, A., 2009. Experimental in vitro

511 transmission of Babesia sp.(EU1) by Ixodes ricinus. Vet Res 40, 1-8.
512 Bork, S., Okamura, M., Boonchit, S., Hirata, H., Yokoyama, N., Igarashi, I., 2004. Identification of

513 Babesia bovis L-lactate dehydrogenase as a potential chemotherapeutical target against

514 bovine babesiosis. Mol Biochem Parasitol 136, 165-172.

515 Boyle, M. J., Wilson D. W., Richards J. S., Riglar D. T., Tetteh K. K., Conway D. J., Ralph S. A., Baum J.

516 and Beeson J. G., 2010. Isolation of viable Plasmodium falciparum merozoites to define

517 erythrocyte invasion events and advance vaccine and drug development. Proc Nat Acad

518 Sci, 107(32): 14378-14383.

519 Brayton, K.A., Lau, A.O., Herndon, D.R., Hannick, L., Kappmeyer, L.S., Berens, S.J., Bidwell, S.L.,

520 Brown, W.C., Crabtree, J., Fadrosh, D., 2007a. Genome sequence of Babesia bovis and

521 comparative analysis of apicomplexan hemoprotozoa. PLoS Pathog 3, e148.

522 Brayton, K.A., Lau, A.O., Herndon, D.R., Hannick, L., Kappmeyer, L.S., Berens, S.J., Bidwell, S.L.,

523 Brown, W.C., Crabtree, J., Fadrosh, D., Feldblum, T., Forberger, H.A., Haas, B.J., Howell,

524 J.M., Khouri, H., Koo, H., Mann, D.J., Norimine, J., Paulsen, I.T., Radune, D., Ren, Q., Smith,

525 R.K., Jr., Suarez, C.E., White, O., Wortman, J.R., Knowles, D.P., Jr., McElwain, T.F., Nene,

526 V.M., 2007b. Genome sequence of Babesia bovis and comparative analysis of

527 apicomplexan hemoprotozoa. PLoS Pathog 3, 1401-1413.

528 Brobey, R.K., Sano, G.-i., Itoh, F., Aso, K., Kimura, M., Mitamura, T., Horii, T., 1996. Recombinant

529 Plasmodium falciparum dihydrofolate reductase-based in vitro screen for antifolate

530 antimalarials. Mol Biochem Parasitol 81, 225-237.

531 Burkard, G.S., Jutzi, P., Roditi, I., 2011. Genome-wide RNAi screens in bloodstream form

532 trypanosomes identify drug transporters. Mol Biochem Parasitol 175, 91-94.

533 Chan, J.N.Y., Nislow, C., Emili, A., 2010. Recent advances and method development for drug target

534 identification. Trends Pharmacol Sci 31, 82-88.

535 Chauvin, A., Moreau, E., Bonnet, S., Plantard, O., Malandrin, L., 2009. Babesia and its hosts:

536 adaptation to long-lasting interactions as a way to achieve efficient transmission. Vet Res

537 40, 37.

538 Chow, E.D., Lim, L., Fidock, D.A., Diagana, T.T., Winzeler, E.A., Bifani, P., 2016. UDP-galactose and

539 acetyl-CoA transporters as Plasmodium multidrug resistance genes. Nat Microbiol 1,

540 16166.

541 Coldham, N.G., Moore, A.S., Dave, M., Graham, P.J., Sivapathasundaram, S., Lake, B.G., Sauer,

542 M.J., 1995. Imidocarb residues in edible bovine tissues and in vitro assessment of

543 imidocarb metabolism and cytotoxicity. Drug Metab Dispos 23, 501-505.

544 Collins, C.R., Das, S., Wong, E.H., Andenmatten, N., Stallmach, R., Hackett, F., Herman, J.P., Müller,

545 S., Meissner, M., Blackman, M.J., 2013. Robust inducible Cre recombinase activity in the

546 human malaria parasite Plasmodium falciparum enables efficient gene deletion within a

547 single asexual erythrocytic growth cycle. Mol Microbiol 88, 687-701.

548 Cong, F., Cheung, A.K., Huang, S.M., 2012. Chemical genetics-based target identification in drug

549 discovery. Annu Rev Pharmacol Toxicol 52, 57-78.

550 Conrad, P.A., Kjemtrup, A.M., Carreno, R.A., Thomford, J., Wainwright, K., Eberhard, M., Quick, R.,

551 Telford, S.R., 3rd, Herwaldt, B.L., 2006. Description of Babesia duncani n.sp. (Apicomplexa:

552 Babesiidae) from humans and its differentiation from other piroplasms. Int J Parasitol 36,

553 779-789.

554 Corey, V.C., Lukens, A.K., Istvan, E.S., Lee, M.C., Franco, V., Magistrado, P., Coburn-Flynn, O.,

555 Sakata-Kato, T., Fuchs, O., Gnadig, N.F., Goldgof, G., Linares, M., Gomez-Lorenzo, M.G., De

556 Cozar, C., Lafuente-Monasterio, M.J., Prats, S., Meister, S., Tanaseichuk, O., Wree, M.,

557 Zhou, Y., Willis, P.A., Gamo, F.J., Goldberg, D.E., Fidock, D.A., Wirth, D.F., Winzeler, E.A.,
558 2016. A broad analysis of resistance development in the malaria parasite. Nat Commun 7,

559 11901.

560 Cornillot, E., Dassouli, A., Garg, A., Pachikara, N., Randazzo, S., Depoix, D., Carcy, B., Delbecq, S.,

561 Frutos, R., Silva, J.C., 2013. Whole genome mapping and re-organization of the nuclear and

562 mitochondrial genomes of Babesia microti isolates. PloS one 8, e72657.

563 Cornillot, E., Dassouli, A., Pachikara, N., Lawres, L., Renard, I., Francois, C., Randazzo, S., Bres, V.,

564 Garg, A., Brancato, J., Pazzi, J.E., Pablo, J., Hung, C., Teng, A., Shandling, A.D., Huynh, V.T.,

565 Krause, P.J., Lepore, T., Delbecq, S., Hermanson, G., Liang, X., Williams, S., Molina, D.M.,

566 Ben Mamoun, C., 2016. A targeted immunomic approach identifies diagnostic antigens in

567 the human pathogen Babesia microti. Transfusion 56, 2085-2099.

568 Cornillot, E., Hadj-Kaddour, K., Dassouli, A., Noel, B., Ranwez, V., Vacherie, B., Augagneur, Y., Bres,

569 V., Duclos, A., Randazzo, S., 2012a. Sequencing of the smallest Apicomplexan genome from

570 the human pathogen Babesia microti. Nucleic Acids Res 40, 9102-9114.

571 Cornillot, E., Hadj-Kaddour, K., Dassouli, A., Noel, B., Ranwez, V., Vacherie, B., Augagneur, Y., Bres,

572 V., Duclos, A., Randazzo, S., Carcy, B., Debierre-Grockiego, F., Delbecq, S., Moubri-Menage,

573 K., Shams-Eldin, H., Usmani-Brown, S., Bringaud, F., Wincker, P., Vivares, C.P., Schwarz,

574 R.T., Schetters, T.P., Krause, P.J., Gorenflot, A., Berry, V., Barbe, V., Ben Mamoun, C.,

575 2012b. Sequencing of the smallest Apicomplexan genome from the human pathogen

576 Babesia microti. Nucleic Acids Res 40, 9102-9114.

577 Cowell, A.N., Istvan, E.S., Lukens, A.K., Gomez-Lorenzo, M.G., Vanaerschot, M., Sakata-Kato, T.,

578 Flannery, E.L., Magistrado, P., Owen, E., Abraham, M., LaMonte, G., Painter, H.J., Williams,

579 R.M., Franco, V., Linares, M., Arriaga, I., Bopp, S., Corey, V.C., Gnadig, N.F., Coburn-Flynn,

580 O., Reimer, C., Gupta, P., Murithi, J.M., Moura, P.A., Fuchs, O., Sasaki, E., Kim, S.W., Teng,

581 C.H., Wang, L.T., Akidil, A., Adjalley, S., Willis, P.A., Siegel, D., Tanaseichuk, O., Zhong, Y.,
582 Zhou, Y., Llinas, M., Ottilie, S., Gamo, F.J., Lee, M.C.S., Goldberg, D.E., Fidock, D.A., Wirth,

583 D.F., Winzeler, E.A., 2018. Mapping the malaria parasite druggable genome by using in

584 vitro evolution and chemogenomics. Science 359, 191-199.

585 Cox, D.B.T., Gootenberg, J.S., Abudayyeh, O.O., Franklin, B., Kellner, M.J., Joung, J., Zhang, F., 2017.

586 RNA editing with CRISPR-Cas13. Science 358, 1019-1027.

587 Crawford, E.D., Quan, J., Horst, J.A., Ebert, D., Wu, W., DeRisi, J.L., 2017. Plasmid-free CRISPR/Cas9

588 genome editing in Plasmodium falciparum confirms mutations conferring resistance to the

589 dihydroisoquinolone clinical candidate SJ733. PloS one 12, e0178163.

590 Creek, D.J., Chua, H.H., Cobbold, S.A., Nijagal, B., MacRae, J.I., Dickerman, B.K., Gilson, P.R., Ralph,

591 S.A., McConville, M.J., 2016. Metabolomics-based screening of the Malaria Box reveals

592 both novel and established mechanisms of action. Antimicrob Agents Chemother 60, 6650-

593 6663.

594 Cuesta, I., Gonzalez, L.M., Estrada, K., Grande, R., Zaballos, A., Lobo, C.A., Barrera, J., Sanchez-

595 Flores, A., Montero, E., 2014a. High-Quality Draft Genome Sequence of Babesia divergens,

596 the Etiological Agent of Cattle and Human Babesiosis. Genome Announc 2.

597 Cuesta, I., González, L.M., Estrada, K., Grande, R., Zaballos, Á., Lobo, C.A., Barrera, J., Sanchez-

598 Flores, A., Montero, E., 2014b. High-quality draft genome sequence of Babesia divergens,

599 the etiological agent of cattle and human babesiosis. Genome Announc 2, e01194-01114.

600 Cursino-Santos, J.R., Singh, M., Pham, P., Lobo, C.A., 2017. A novel flow cytometric application

601 discriminates among the effects of chemical inhibitors on various phases of Babesia

602 divergens intraerythrocytic cycle. Cytometry A 91, 216-231.

603 Cursino-Santos, J.R., Singh, M., Pham, P., Rodriguez, M., Lobo, C.A., 2016. Babesia divergens builds

604 a complex population structure composed of specific ratios of infected cells to ensure a

605 prompt response to changing environmental conditions. Cell Microbiol 18, 859-874.
606 de Koning-Ward, T.F., Gilson, P.R., Crabb, B.S., 2015. Advances in molecular genetic systems in

607 malaria. Nature Rev Microbiol 13, 373-387.

608 de Rezende, J., Rangel, C.P., McIntosh, D., Silveira, J.A., Cunha, N.C., Ramos, C.A., Fonseca, A.H.,

609 2015. In vitro cultivation and cryopreservation of Babesia bigemina sporokinetes in

610 hemocytes of Rhipicephalus microplus. Vet Parasitol 212, 400-403.

611 Dhingra, S.K., Redhi, D., Combrinck, J.M., Yeo, T., Okombo, J., Henrich, P.P., Cowell, A.N., Gupta, P.,

612 Stegman, M.L., Hoke, J.M., 2017. A variant PfCRT isoform can contribute to Plasmodium

613 falciparum resistance to the first-line partner drug piperaquine. MBio 8, e00303-00317.

614 Dickerman, B.K., Elsworth, B., Cobbold, S.A., Nie, C.Q., McConville, M.J., Crabb, B.S., Gilson, P.R.,

615 2016. Identification of inhibitors that dually target the new permeability pathway and

616 dihydroorotate dehydrogenase in the blood stage of Plasmodium falciparum. Sci Rep 6,

617 37502.

618 Donald, R.G., Zhong, T., Wiersma, H., Nare, B., Yao, D., Lee, A., Allocco, J., Liberator, P.A., 2006.

619 Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-

620 dependent protein kinase. Mol Biochem Parasitol 149, 86-98.

621 Eichenberger, R.M., Ramakrishnan, C., Russo, G., Deplazes, P., Hehl, A.B., 2017. Genome-wide

622 analysis of gene expression and protein secretion of Babesia canis during virulent infection

623 identifies potential pathogenicity factors. Sci Rep 7, 3357.

624 Eisen, R.J., Gage, K.L., 2009. Adaptive strategies of Yersinia pestis to persist during inter-epizootic

625 and epizootic periods. Vet Res 40, 1.

626 Fernandez-Prada, C., Sharma, M., Plourde, M., Bresson, E., Roy, G., Leprohon, P., Ouellette, M.,

627 2018. High-throughput Cos-Seq screen with intracellular Leishmania infantum for the

628 discovery of novel drug-resistance mechanisms. Int J Parasitol Drugs Drug Resist 8, 165-

629 173.
630 Ganesan, S.M., Falla, A., Goldfless, S.J., Nasamu, A.S., Niles, J.C., 2016. Synthetic RNA–protein

631 modules integrated with native translation mechanisms to control gene expression in

632 malaria parasites. Nature Commun 7, 10727.

633 Ganesan, S.M., Morrisey, J.M., Ke, H., Painter, H.J., Laroiya, K., Phillips, M.A., Rathod, P.K., Mather,

634 M.W., Vaidya, A.B., 2011. Yeast dihydroorotate dehydrogenase as a new selectable marker

635 for Plasmodium falciparum transfection. Mol Biochem Parasitol 177, 29-34.

636 Gardiner, D.L., Trenholme, K.R., Skinner-Adams, T.S., Stack, C.M., Dalton, J.P., 2006.

637 Overexpression of Leucyl Aminopeptidase in Plasmodium falciparum Parasites target for

638 the antimalarial activity of bestatin. J Biol Chem 281, 1741-1745.

639 Gardner, M.J., Bishop, R., Shah, T., de Villiers, E.P., Carlton, J.M., Hall, N., Ren, Q., Paulsen, I.T.,

640 Pain, A., Berriman, M., Wilson, R.J., Sato, S., Ralph, S.A., Mann, D.J., Xiong, Z., Shallom, S.J.,

641 Weidman, J., Jiang, L., Lynn, J., Weaver, B., Shoaibi, A., Domingo, A.R., Wasawo, D.,

642 Crabtree, J., Wortman, J.R., Haas, B., Angiuoli, S.V., Creasy, T.H., Lu, C., Suh, B., Silva, J.C.,

643 Utterback, T.R., Feldblyum, T.V., Pertea, M., Allen, J., Nierman, W.C., Taracha, E.L.,

644 Salzberg, S.L., White, O.R., Fitzhugh, H.A., Morzaria, S., Venter, J.C., Fraser, C.M., Nene, V.,

645 2005. Genome sequence of Theileria parva, a bovine pathogen that transforms

646 lymphocytes. Science 309, 134-137.

647 Gardner, R.A., Molyneux, D.H., 1987. Babesia vesperuginis: natural and experimental infections in

648 British bats (Microchiroptera). Parasitology 95 ( Pt 3), 461-469.

649 Gazanion, É., Fernández-Prada, C., Papadopoulou, B., Leprohon, P., Ouellette, M., 2016. Cos-Seq

650 for high-throughput identification of drug target and resistance mechanisms in the

651 protozoan parasite Leishmania. Proc Nat Acad Sci 113, E3012-E3021.

652 Gharbi, M., Latrach, R., Sassi, L., Darghouth, M.A., 2012. Evaluation of a simple Theileria annulata

653 culture protocol from experimentally infected bovine whole blood. Parasite 19, 281-283.
654 Gilbert, L.A., Horlbeck, M.A., Adamson, B., Villalta, J.E., Chen, Y., Whitehead, E.H., Guimaraes, C.,

655 Panning, B., Ploegh, H.L., Bassik, M.C., Qi, L.S., Kampmann, M., Weissman, J.S., 2014.

656 Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell 159, 647-

657 661.

658 Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E., Stern-Ginossar, N., Brandman,

659 O., Whitehead, E.H., Doudna, J.A., Lim, W.A., Weissman, J.S., Qi, L.S., 2013. CRISPR-

660 mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442-451.

661 Goff, W.L., Yunker, C.E., 1986. Babesia bovis: increased percentage parasitized erythrocytes in

662 cultures and assessment of growth by incorporation of [3H]hypoxanthine. Exp Parasitol 62,

663 202-210.

664 Goff, W.L., Yunker, C.E., 1988. Effects of PH, buffers and medium-storage on the growth of Babesia

665 bovis in vitro. Int J Parasitol 18, 775-778.

666 Gohil, S., Kats, L.M., Sturm, A., Cooke, B.M., 2010. Recent insights into alteration of red blood cells

667 by Babesia bovis: mooving forward. Trends Parasitol 26, 591-599.

668 Goldfless, S.J., Wagner, J.C., Niles, J.C., 2014. Versatile control of Plasmodium falciparum gene

669 expression with an inducible protein–RNA interaction. Nature Commun 5, 5329.

670 Goo, Y.K., Xuan, X., 2014. New Molecules in Babesia gibsoni and their application for diagnosis,

671 vaccine development, and drug discovery. Korean J Parasitol 52, 345-353.

672 Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., Bujard, H., 1995. Transcriptional

673 activation by tetracyclines in mammalian cells. Science 268, 1766-1769.

674 Grande, N., Precigout, E., Ancelin, M.L., Moubri, K., Carcy, B., Lemesre, J.L., Vial, H., Gorenflot, A.,

675 1997. Continuous in vitro culture of Babesia divergens in a serum-free medium.

676 Parasitology 115 ( Pt 1), 81-89.

677 Guswanto, A., Sivakumar, T., Rizk, M.A., Elsayed, S.A., Youssef, M.A., ElSaid Eel, S., Yokoyama, N.,

678 Igarashi, I., 2014. Evaluation of a fluorescence-based method for antibabesial drug

679 screening. Antimicrob Agents Chemother 58, 4713-4717.

680 Hakimi, H., Yamagishi, J., Kegawa, Y., Kaneko, O., Kawazu, S.-i., Asada, M., 2016. Establishment of

681 transient and stable transfection systems for Babesia ovata. Parasites Vectors 9, 171.

682 Hanson, K.K., Ressurreicao, A.S., Buchholz, K., Prudencio, M., Herman-Ornelas, J.D., Rebelo, M.,

683 Beatty, W.L., Wirth, D.F., Hanscheid, T., Moreira, R., Marti, M., Mota, M.M., 2013. Torins

684 are potent antimalarials that block replenishment of Plasmodium liver stage

685 parasitophorous vacuole membrane proteins. Proc Natl Acad Sci U S A 110, E2838-2847.

686 Hayashida, K., Hara, Y., Abe, T., Yamasaki, C., Toyoda, A., Kosuge, T., Suzuki, Y., Sato, Y.,

687 Kawashima, S., Katayama, T., Wakaguri, H., Inoue, N., Homma, K., Tada-Umezaki, M., Yagi,

688 Y., Fujii, Y., Habara, T., Kanehisa, M., Watanabe, H., Ito, K., Gojobori, T., Sugawara, H.,

689 Imanishi, T., Weir, W., Gardner, M., Pain, A., Shiels, B., Hattori, M., Nene, V., Sugimoto, C.,

690 2012. Comparative genome analysis of three eukaryotic parasites with differing abilities to

691 transform leukocytes reveals key mediators of Theileria-induced leukocyte transformation.

692 MBio 3, e00204-00212.

693 Herwaldt, B., Persing, D.H., Precigout, E.A., Goff, W.L., Mathiesen, D.A., Taylor, P.W., Eberhard,

694 M.L., Gorenflot, A.F., 1996. A fatal case of babesiosis in Missouri: identification of another

695 piroplasm that infects humans. Ann Intern Med 124, 643-650.

696 Herwaldt, B.L., Caccio, S., Gherlinzoni, F., Aspock, H., Slemenda, S.B., Piccaluga, P., Martinelli, G.,

697 Edelhofer, R., Hollenstein, U., Poletti, G., Pampiglione, S., Loschenberger, K., Tura, S.,

698 Pieniazek, N.J., 2003. Molecular characterization of a non-Babesia divergens organism

699 causing zoonotic babesiosis in Europe. Emerg Infect Dis 9, 942-948.

700 Hoepfner, D., McNamara, C.W., Lim, C.S., Studer, C., Riedl, R., Aust, T., McCormack, S.L., Plouffe,

701 D.M., Meister, S., Schuierer, S., 2012. Selective and specific inhibition of the Plasmodium

702 falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin. Cell host

703 Microbe 11, 654-663.

704 Holman, P.J., Chieves, L., Frerichs, W.M., Olson, D., Wagner, G.G., 1994a. Babesia equi erythrocytic

705 stage continuously cultured in an enriched medium. J Parasitol 80, 232-236.

706 Holman, P.J., Craig, T.M., Crider, D.L., Petrini, K.R., Rhyan, J., Wagner, G.G., 1994b. Culture

707 isolation and partial characterization of a Babesia sp. from a North American elk (Cervus

708 elaphus). J Wildl Dis 30, 460-465.

709 Holman, P.J., Petrini, K., Rhyan, J., Wagner, G.G., 1994c. In vitro isolation and cultivation of a

710 Babesia from an American woodland caribou (Rangifer tarandus caribou). J Wildl Dis 30,

711 195-200.

712 Holman, P.J., Spencer, A.M., Droleskey, R.E., Goethert, H.K., Telford, S.R., 3rd, 2005. In vitro

713 cultivation of a zoonotic Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus

714 floridanus) on Nantucket Island, Massachusetts. J Clin Microbiol 43, 3995-4001.

715 Holman, P.J., Waldrup, K.A., Wagner, G.C., 1988. In vitro cultivation of a Babesia isolated from a

716 white-tailed deer (Odocoileus virginianus). J Parasitol 74, 111-115.

717 Hostettler, I., Muller, J., Hemphill, A., 2016. In Vitro Screening of the Open-Source Medicines for

718 Malaria Venture Malaria Box Reveals Novel Compounds with Profound Activities against

719 Theileria annulata Schizonts. Antimicrob Agents Chemother 60, 3301-3308.

720 Hunfeld, K.P., Hildebrandt, A., Gray, J.S., 2008. Babesiosis: recent insights into an ancient disease.

721 Int J Parasitol 38, 1219-1237.

722 Hwang, S.J., Yamasaki, M., Nakamura, K., Sasaki, N., Murakami, M., Wickramasekara

723 Rajapakshage, B.K., Ohta, H., Maede, Y., Takiguchi, M., 2010. Development and
724 characterization of a strain of Babesia gibsoni resistant to diminazene aceturate in vitro. J

725 Vet Med Sci 72, 765-771.

726 Igarashi, I., Avarzed, A., Tanaka, T., Inoue, N., Ito, M., Omata, Y., Saito, A., Suzuki, N., 1994.

727 Continuous in vitro cultivation of Babesia ovata. J Protozool.

728 Irvin, A.D., Young, E.R., Adams, P.J., 1979. The effects of irradiation on Babesia maintained in vitro.

729 Res Vet Sci 27, 200-204.

730 Iwamoto, M., Björklund, T., Lundberg, C., Kirik, D., Wandless, T.J., 2010. A general chemical

731 method to regulate protein stability in the mammalian central nervous system. Chem Biol

732 17, 981-988.

733 Jackson, A.P., Otto, T.D., Darby, A., Ramaprasad, A., Xia, D., Echaide, I.E., Farber, M., Gahlot, S.,

734 Gamble, J., Gupta, D., 2014a. The evolutionary dynamics of variant antigen genes in

735 Babesia reveal a history of genomic innovation underlying host–parasite interaction.

736 Nucleic Acids Res 42, 7113-7131.

737 Jackson, A.P., Otto, T.D., Darby, A., Ramaprasad, A., Xia, D., Echaide, I.E., Farber, M., Gahlot, S.,

738 Gamble, J., Gupta, D., Gupta, Y., Jackson, L., Malandrin, L., Malas, T.B., Moussa, E., Nair, M.,

739 Reid, A.J., Sanders, M., Sharma, J., Tracey, A., Quail, M.A., Weir, W., Wastling, J.M., Hall, N.,

740 Willadsen, P., Lingelbach, K., Shiels, B., Tait, A., Berriman, M., Allred, D.R., Pain, A., 2014b.

741 The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic

742 innovation underlying host–parasite interaction. Nucleic Acids Res 42, 7113-7131.

743 Jia, N., Zheng, Y.C., Jiang, J.F., Jiang, R.R., Jiang, B.G., Wei, R., Liu, H.B., Huo, Q.B., Sun, Y., Chu, Y.L.,

744 Fan, H., Chang, Q.C., Yao, N.N., Zhang, W.H., Wang, H., Guo, D.H., Fu, X., Wang, Y.W.,

745 Krause, P.J., Song, J.L., Cao, W.C., 2018. Human Babesiosis Caused by a Babesia crassa-like

746 Pathogen: A Case Series. Clin Infect Dis. 67 (7), 1110-1119.

747 Johnson, W.C., Taus, N.S., Reif, K.E., Bohaliga, G.A., Kappmeyer, L.S., Ueti, M.W., 2017. Analysis of

748 Stage-Specific Protein Expression during Babesia Bovis Development within Female

749 Rhipicephalus Microplus. J Proteome Res 16, 1327-1338.

750 Joung, J., Konermann, S., Gootenberg, J.S., Abudayyeh, O.O., Platt, R.J., Brigham, M.D., Sanjana,

751 N.E., Zhang, F., 2017. Genome-scale CRISPR-Cas9 knockout and transcriptional activation

752 screening. Nature Protocols 12, 828.

753 Kamyingkird, K., Cao, S., Masatani, T., Moumouni, P.F.A., Vudriko, P., Mousa, A.A.E.M., Terkawi,

754 M.A., Nishikawa, Y., Igarashi, I., Xuan, X., 2014. Babesia bovis Dihydroorotate

755 Dehydrogenase (BboDHODH) is a Novel Molecular Target of Drug for Bovine Babesiosis. J

756 Vet Med Sci 76, 323-330.

757 Kappmeyer, L.S., Thiagarajan, M., Herndon, D.R., Ramsay, J.D., Caler, E., Djikeng, A., Gillespie, J.J.,

758 Lau, A.O., Roalson, E.H., Silva, J.C., Silva, M.G., Suarez, C.E., Ueti, M.W., Nene, V.M.,

759 Mealey, R.H., Knowles, D.P., Brayton, K.A., 2012. Comparative genomic analysis and

760 phylogenetic position of Theileria equi. BMC Genomics 13, 603.

761 Kessl, J.J., Lange, B.B., Merbitz-Zahradnik, T., Zwicker, K., Hill, P., Meunier, B., Palsdottir, H., Hunte,

762 C., Meshnick, S., Trumpower, B.L., 2003. Molecular basis for atovaquone binding to the

763 cytochrome bc1 complex. J Biol Chem 278, 31312-31318.

764 Khare, S., Roach, S.L., Barnes, S.W., Hoepfner, D., Walker, J.R., Chatterjee, A.K., Neitz, R.J., Arkin,

765 M.R., McNamara, C.W., Ballard, J., Lai, Y., Fu, Y., Molteni, V., Yeh, V., McKerrow, J.H.,

766 Glynne, R.J., Supek, F., 2015. Utilizing Chemical Genomics to Identify Cytochrome b as a

767 Novel Drug Target for Chagas Disease. PLOS Path 11, e1005058.

768 Konermann, S., Brigham, M.D., Trevino, A.E., Joung, J., Abudayyeh, O.O., Barcena, C., Hsu, P.D.,

769 Habib, N., Gootenberg, J.S., Nishimasu, H., Nureki, O., Zhang, F., 2015. Genome-scale

770 transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583-588.

771 Konermann, S., Lotfy, P., Brideau, N.J., Oki, J., Shokhirev, M.N., Hsu, P.D., 2018. Transcriptome

772 Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell 173, 665-676.e614.

773 Koushik, A.B., Welter, B.H., Rock, M.L., Temesvari, L.A., 2014. A genomewide overexpression

774 screen identifies genes involved in the phosphatidylinositol 3-kinase pathway in the human

775 protozoan parasite Entamoeba histolytica. Eukaryotic cell 13, 401-411.

776 LaMonte, G., Lim, M.Y.-X., Wree, M., Reimer, C., Nachon, M., Corey, V., Gedeck, P., Plouffe, D., Du,

777 A., Figueroa, N., 2016. Mutations in the Plasmodium falciparum cyclic amine resistance

778 locus (PfCARL) confer multidrug resistance. MBio 7, e00696-00616.

779 Lau, A.O., 2009. An overview of the Babesia, Plasmodium and Theileria genomes: a comparative

780 perspective. Mol Biochem Parasitol 164, 1-8.

781 Laughery, J.M., Knowles, D.P., Schneider, D.A., Bastos, R.G., McElwain, T.F., Suarez, C.E., 2014.

782 Targeted surface expression of an exogenous antigen in stably transfected Babesia bovis.

783 PloS one 9, e97890.

784 Lawres, L.A., Garg, A., Kumar, V., Bruzual, I., Forquer, I.P., Renard, I., Virji, A.Z., Boulard, P.,

785 Rodriguez, E.X., Allen, A.J., Pou, S., Wegmann, K.W., Winter, R.W., Nilsen, A., Mao, J.,

786 Preston, D.A., Belperron, A.A., Bockenstedt, L.K., Hinrichs, D.J., Riscoe, M.K., Doggett, J.S.,

787 Ben Mamoun, C., 2016. Radical cure of experimental babesiosis in immunodeficient mice

788 using a combination of an endochin-like quinolone and atovaquone. J Exp Med 213, 1307-

789 1318.

790 Lemieux, J.E., Tran, A.D., Freimark, L., Schaffner, S.F., Goethert, H., Andersen, K.G., Bazner, S., Li,

791 A., McGrath, G., Sloan, L., Vannier, E., Milner, D., Pritt, B., Rosenberg, E., Telford, S., 3rd,

792 Bailey, J.A., Sabeti, P.C., 2016. A global map of genetic diversity in Babesia microti reveals

793 strong population structure and identifies variants associated with clinical relapse. Nat

794 Microbiol 1, 16079.

795 Levy, M.G., Ristic, M., 1980. Babesia bovis: continuous cultivation in a microaerophilous stationary

796 phase culture. Science 207, 1218-1220.

797 Liu, M., Asada, M., Cao, S., Moumouni, P.F.A., Vudriko, P., Efstratiou, A., Hakimi, H., Masatani, T.,

798 Sunaga, F., Kawazu, S.-i., 2017a. Transient transfection of intraerythrocytic Babesia gibsoni

799 using elongation factor-1 alpha promoter. Mol Biochem Parasitol.

800 Liu, M., Moumouni, P.F.A., Asada, M., Hakimi, H., Masatani, T., Vudriko, P., Lee, S.-H., Kawazu, S.-i.,

801 Yamagishi, J., Xuan, X., 2018. Establishment of a stable transfection system for genetic

802 manipulation of Babesia gibsoni. Parasites vectors 11, 260.

803 Liu, M., Moumouni, P.F.A., Cao, S., Asada, M., Wang, G., Gao, Y., Guo, H., Li, J., Vudriko, P.,

804 Efstratiou, A., 2017b. Identification and characterization of interchangeable cross-species

805 functional promoters between Babesia gibsoni and Babesia bovis. Ticks Tick-Borne Dis.

806 Lourido, S., Shuman, J., Zhang, C., Shokat, K.M., Hui, R., Sibley, L.D., 2010. Calcium-dependent

807 protein kinase 1 is an essential regulator of exocytosis in Toxoplasma. Nature 465, 359-362.

808 Luth, M.R., Gupta, P., Ottilie, S., Winzeler, E.A., 2018. Using in Vitro Evolution and Whole Genome

809 Analysis To Discover Next Generation Targets for Antimalarial Drug Discovery. ACS Infect

810 Dis 4, 301-314.

811 Mackenstedt, U., Gauer, M., Mehlhorn, H., Schein, E., Hauschild, S., 1990. Sexual cycle of Babesia

812 divergens confirmed by DNA measurements. Parasitol Res 76, 199-206.

813 McFadden, D.C., Camps, M., Boothroyd, J.C., 2001. Resistance as a tool in the study of old and new

814 drug targets in Toxoplasma. Drug Resist Updates 4, 79-84.

815 McHardy, N., Woollon, R.M., Clampitt, R.B., James, J.A., Crawley, R.J., 1986. Efficacy, toxicity and

816 metabolism of imidocarb dipropionate in the treatment of Babesia ovis infection in sheep.

817 Res Vet Sci 41, 14-20.

818 Meissner, M., Brecht, S., Bujard, H., Soldati, D., 2001. Modulation of myosin A expression by a

819 newly established tetracycline repressor-based inducible system in Toxoplasma gondii.

820 Nucleic Acids Res 29, e115-e115.

821 Meissner, M., Krejany, E., Gilson, P.R., de Koning-Ward, T.F., Soldati, D., Crabb, B.S., 2005.

822 Tetracycline analogue-regulated transgene expression in Plasmodium falciparum blood

823 stages using Toxoplasma gondii transactivators. Proc Nat Acad Sci USA 102, 2980-2985.

824 Molinar, E., James, M.A., Kakoma, I., Holland, C., Ristic, M., 1982. Antigenic and immunogenic

825 studies on cell culture-derived Babesia canis. Vet Parasitol 10, 29-40.

826 Mosqueda, J., Olvera-Ramirez, A., Aguilar-Tipacamu, G., J Canto, G., 2012. Current advances in

827 detection and treatment of babesiosis. Curr Med Chem 19, 1504-1518.

828 Muralidharan, V., Oksman, A., Iwamoto, M., Wandless, T.J., Goldberg, D.E., 2011. Asparagine

829 repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent

830 affinity tag. Proc Nat Acad Sci 108, 4411-4416.

831 Musa, N.B., Abdel Gawad, M.A., 2004. Adaptation of three Babesia divergens isolates to

832 continuous culture in rat erythrocytes. J Egypt Soc Parasitol 34, 333-344.

833 Nagai, A., Yokoyama, N., Matsuo, T., Bork, S., Hirata, H., Xuan, X., Zhu, Y., Claveria, F.G., Fujisaki, K.,

834 Igarashi, I., 2003. Growth-Inhibitory Effects of Artesunate, Pyrimethamine, and Pamaquine

835 against Babesia equi and Babesia caballi in In Vitro Cultures. Antimicrob Agents Chemother

836 47, 800-803.

837 Ng, C.L., Siciliano, G., Lee, M., de Almeida, M.J., Corey, V.C., Bopp, S.E., Bertuccini, L., Wittlin, S.,

838 Kasdin, R.G., Le Bihan, A., 2016. CRISPR-Cas9-modified pfmdr1 protects Plasmodium

839 falciparum asexual blood stages and gametocytes against a class of piperazine-containing

840 compounds but potentiates artemisinin-based combination therapy partner drugs. Mol

841 Microbiol 101, 381-393.

842 Nkrumah, L.J., Riegelhaupt, P.M., Moura, P., Johnson, D.J., Patel, J., Hayton, K., Ferdig, M.T.,

843 Wellems, T.E., Akabas, M.H., Fidock, D.A., 2009. Probing the multifactorial basis of

844 Plasmodium falciparum quinine resistance: Evidence for a strain-specific contribution of

845 the sodium-proton exchanger PfNHE. Mol Biochem Parasitol 165, 122-131.

846 Okubo, K., Yokoyama, N., Govind, Y., Alhassan, A., Igarashi, I., 2007. Babesia bovis: effects of

847 cysteine protease inhibitors on in vitro growth. Exp Parasitol 117, 214-217.

848 Oldiges, D.P., Laughery, J.M., Tagliari, N.J., Leite Filho, R.V., Davis, W.C., da Silva Vaz Jr, I.,

849 Termignoni, C., Knowles, D.P., Suarez, C.E., 2016. Transfected Babesia bovis Expressing a

850 Tick GST as a Live Vector Vaccine. PLOS Negl Trop Dis 10, e0005152.

851 Omar, M.A., Salama, A., Elsify, A., Rizk, M.A., Al-Aboody, M.S., AbouLaila, M., El-Sayed, S.A.,

852 Igarashi, I., 2016. Evaluation of in vitro inhibitory effect of enoxacin on Babesia and

853 Theileria parasites. Exp Parasitol 161, 62-67.

854 Ord, R.L., Lobo, C.A., 2015. Human Babesiosis: Pathogens, Prevalence, Diagnosis and Treatment.

855 Curr Clin Microbiol Rep 2, 173-181.

856 Pain, A., Renauld, H., Berriman, M., Murphy, L., Yeats, C.A., Weir, W., Kerhornou, A., Aslett, M.,

857 Bishop, R., Bouchier, C., Cochet, M., Coulson, R.M., Cronin, A., de Villiers, E.P., Fraser, A.,

858 Fosker, N., Gardner, M., Goble, A., Griffiths-Jones, S., Harris, D.E., Katzer, F., Larke, N., Lord,

859 A., Maser, P., McKellar, S., Mooney, P., Morton, F., Nene, V., O'Neil, S., Price, C., Quail,

860 M.A., Rabbinowitsch, E., Rawlings, N.D., Rutter, S., Saunders, D., Seeger, K., Shah, T.,

861 Squares, R., Squares, S., Tivey, A., Walker, A.R., Woodward, J., Dobbelaere, D.A., Langsley,

862 G., Rajandream, M.A., McKeever, D., Shiels, B., Tait, A., Barrell, B., Hall, N., 2005. Genome

863 of the host-cell transforming parasite Theileria annulata compared with T. parva. Science

864 309, 131-133.

865 Painter, H.J., Morrisey, J.M., Mather, M.W., Vaidya, A.B., 2007. Specific role of mitochondrial

866 electron transport in blood-stage Plasmodium falciparum. Nature 446, 88.

867 Park, H., Hong, S.H., Kim, K., Cho, S.H., Lee, W.J., Kim, Y., Lee, S.E., Park, Y., 2015. Characterizations

868 of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic

869 microscopy. Sci Rep 5, 10827.

870 Paul, A.S., Moreira, C.K., Elsworth, B., Allred, D.R., Duraisingh, M.T., 2016. Extensive Shared

871 Chemosensitivity between Malaria and Babesiosis Blood-Stage Parasites. Antimicrob

872 Agents Chemother 60, 5059-5063.

873 Pedroni, M.J., Sondgeroth, K.S., Gallego-Lopez, G.M., Echaide, I., Lau, A.O., 2013. Comparative

874 transcriptome analysis of geographically distinct virulent and attenuated Babesia bovis

875 strains reveals similar gene expression changes through attenuation. BMC Genomics 14,

876 763.

877 Pedroni, M.J., Vidadala, R.S., Choi, R., Keyloun, K.R., Reid, M.C., Murphy, R.C., Barrett, L.K., Van

878 Voorhis, W.C., Maly, D.J., Ojo, K.K., Lau, A.O., 2016. Bumped kinase inhibitor prohibits

879 egression in i. Vet Parasitol 215, 22-28.

880 Pellé, K.G., Jiang, R.H., Mantel, P.Y., Xiao, Y.P., Hjelmqvist, D., Gallego-Lopez, G.M., OT Lau, A.,

881 Kang, B.H., Allred, D.R., Marti, M., 2015. Shared elements of host-targeting pathways

882 among apicomplexan parasites of differing lifestyles. Cell Microbiol 17, 1618-1639.

883 Penzhorn, B.L., Lewis, B.D., Lopez-Rebollar, L.M., Swan, G.E., 2000. Screening of five drugs for

884 efficacy against Babesia felis in experimentally infected cats. J South African Vet Assoc 71,

885 53-57.

886 Phillips, M.A., Lotharius, J., Marsh, K., White, J., Dayan, A., White, K.L., Njoroge, J.W., El Mazouni,

887 F., Lao, Y., Kokkonda, S., 2015. A long-duration dihydroorotate dehydrogenase inhibitor
888 (DSM265) for prevention and treatment of malaria. Sci Translat Med 7, 296ra111-

889 296ra111.

890 Phillips, M.A., White, K.L., Kokkonda, S., Deng, X., White, J., El Mazouni, F., Marsh, K., Tomchick,

891 D.R., Manjalanagara, K., Rudra, K.R., 2016. A Triazolopyrimidine-Based Dihydroorotate

892 Dehydrogenase Inhibitor with Improved Drug-like Properties for Treatment and Prevention

893 of Malaria. ACS Infect Dis 2, 945-957.

894 Pillai, A.D., Pain, M., Solomon, T., Bokhari, A.A., Desai, S.A., 2010. A cell-based high-throughput

895 screen validates the plasmodial surface anion channel as an antimalarial target. Mol

896 Pharmacol 77, 724-733.

897 Pino, P., Sebastian, S., Kim, E.A., Bush, E., Brochet, M., Volkmann, K., Kozlowski, E., Llinás, M.,

898 Billker, O., Soldati-Favre, D., 2012. A tetracycline-repressible transactivator system to study

899 essential genes in malaria parasites. Cell Host Microbe 12, 824-834.

900 Posnett, E.S., Metaferia, E., Wiliamson, S., Brown, C.G., Canning, E.U., 1998. In vitro cultivation of

901 an African strain of Babesia bigemina, its characterisation and infectivity in cattle. Parasitol

902 Res 84, 302-309.

903 Pradhan, A., Siwo, G.H., Singh, N., Martens, B., Balu, B., Button-Simons, K.A., Tan, A., Zhang, M.,

904 Udenze, K.O., Jiang, R.H.Y., Ferdig, M.T., Adams, J.H., Kyle, D.E., 2015. Chemogenomic

905 profiling of Plasmodium falciparum as a tool to aid antimalarial drug discovery. Sci Rep 5,

906 15930.

907 Prommana, P., Uthaipibull, C., Wongsombat, C., Kamchonwongpaisan, S., Yuthavong, Y., Knuepfer,

908 E., Holder, A.A., Shaw, P.J., 2013. Inducible knockdown of Plasmodium gene expression

909 using the glmS ribozyme. PloS one 8, e73783.

910 Qian, X., Liang, G.-B., Feng, D., Fisher, M., Crumley, T., Rattray, S., Dulski, P.M., Gurnett, A., Leavitt,

911 P.S., Liberator, P.A., Misura, A.S., Samaras, S., Tamas, T., Schmatz, D.M., Wyvratt, M., Biftu,
912 T., 2006. Synthesis and SAR Studies of diarylpyrrole anticoccidial agents. Bioorgan Med

913 Chem Lett 16, 2817-2821.

914 Rajkumari, N., 2015. Epidemiological profile of “Babesia venatorum”. Lancet Infect Dis 15, 877-

915 878.

916 Reynolds, M.G., Oh, J., Roos, D.S., 2001. In vitro generation of novel pyrimethamine resistance

917 mutations in the Toxoplasma gondii dihydrofolate reductase. Antimicrob Agents

918 Chemother 45, 1271-1277.

919 Rizk, M.A., El-Sayed, S.A., AbouLaila, M., Tuvshintulga, B., Yokoyama, N., Igarashi, I., 2016. Large-

920 scale drug screening against Babesia divergens parasite using a fluorescence-based high-

921 throughput screening assay. Vet Parasitol 227, 93-97.

922 Rizk, M.A., El-Sayed, S.A., Terkawi, M.A., Youssef, M.A., El Said el Sel, S., Elsayed, G., El-Khodery, S.,

923 El-Ashker, M., Elsify, A., Omar, M., Salama, A., Yokoyama, N., Igarashi, I., 2015.

924 Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia

925 and Theileria Parasites. PLoS One 10, e0125276.

926 Rizk, M.A., El-Sayed, S.A.E., AbouLaila, M., Eltaysh, R., Yokoyama, N., Igarashi, I., 2017.

927 Performance and consistency of a fluorescence-based high-throughput screening assay for

928 use in Babesia drug screening in mice. Sci Rep 7, 12774.

929 Robinson, M.S., Sahlender, D.A., Foster, S.D., 2010. Rapid inactivation of proteins by rapamycin-

930 induced rerouting to mitochondria. Develop cell 18, 324-331.

931 Rodriguez, R.I., Trees, A.J., 1996. In vitro responsiveness of Babesia bovis to imidocarb

932 dipropionate and the selection of a drug-adapted line. Vet Parasitol 62, 35-41.

933 Rossouw, I., Maritz-Olivier, C., Niemand, J., van Biljon, R., Smit, A., Olivier, N.A., Birkholtz, L.M.,

934 2015. Morphological and Molecular Descriptors of the Developmental Cycle of Babesia

935 divergens Parasites in Human Erythrocytes. PLoS Negl Trop Dis 9, e0003711.
936 Saito-Ito, A., Kawai, A., Ohmori, S., Nagano-Fujii, M., 2016. Continuous in vivo culture and indirect

937 fluorescent antibody test for zoonotic protozoa of Babesia microti. Parasitol Int 65, 526-

938 531.

939 Sakuma, M., Setoguchi, A., Endo, Y., 2009. Possible emergence of drug-resistant variants of

940 Babesia gibsoni in clinical cases treated with atovaquone and azithromycin. J Vet Intern

941 Med 23, 493-498.

942 Salmon, B. L., Oksman A. and Goldberg D. E., 2001. Malaria parasite exit from the host

943 erythrocyte: a two-step process requiring extraerythrocytic proteolysis. Proc Nat Acad Sci

944 98(1): 271-276.

945 Schenone, M., Dancik, V., Wagner, B.K., Clemons, P.A., 2013. Target identification and mechanism

946 of action in chemical biology and drug discovery. Nat Chem Biol 9, 232-240.

947 Schreeg, M.E., Marr, H.S., Tarigo, J.L., Cohn, L.A., Bird, D.M., Scholl, E.H., Levy, M.G., Wiegmann,

948 B.M., Birkenheuer, A.J., 2016. Mitochondrial Genome Sequences and Structures Aid in the

949 Resolution of Piroplasmida phylogeny. PLoS One 11, e0165702.

950 Schuster, F.L., 2002. Cultivation of Babesia and Babesia-Like Blood Parasites: Agents of an

951 Emerging Zoonotic Disease. Clin Microbiol Rev 15, 365-373.

952 Shikano, S., Nakada, K., Hashiguchi, R., Shimada, T., Ono, K., 1995. A short term in vitro cultivation

953 of Babesia rodhaini and Babesia microti. J. Vet. Med. Sci. 57, 955-957.

954 Sidik, S.M., Hortua Triana, M.A., Paul, A.S., El Bakkouri, M., Hackett, C.G., Tran, F., Westwood, N.J.,

955 Hui, R., Zuercher, W.J., Duraisingh, M.T., Moreno, S.N.J., Lourido, S., 2016. Using a

956 Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling. J

957 Biol Chem 291, 9566-9580.

958 Silva, J.C., Cornillot, E., McCracken, C., Usmani-Brown, S., Dwivedi, A., Ifeonu, O.O., Crabtree, J.,

959 Gotia, H.T., Virji, A.Z., Reynes, C., Colinge, J., Kumar, V., Lawres, L., Pazzi, J.E., Pablo, J.V.,
960 Hung, C., Brancato, J., Kumari, P., Orvis, J., Tretina, K., Chibucos, M., Ott, S., Sadzewicz, L.,

961 Sengamalay, N., Shetty, A.C., Su, Q., Tallon, L., Fraser, C.M., Frutos, R., Molina, D.M.,

962 Krause, P.J., Ben Mamoun, C., 2016a. Genome-wide diversity and gene expression profiling

963 of Babesia microti isolates identify polymorphic genes that mediate host-pathogen

964 interactions. Sci Rep 6, 35284.

965 Silva, M.G., Domingos, A., Esteves, M.A., Cruz, M.E.M., Suarez, C.E., 2013. Evaluation of the

966 growth-inhibitory effect of trifluralin analogues on in vitro cultured Babesia bovis parasites.

967 Int J Parasitol Drugs Drug Resist 3, 59-68.

968 Silva, M.G., Knowles, D.P., Mazuz, M.L., Cooke, B.M., Suarez, C.E., 2018a. Stable transformation of

969 Babesia bigemina and Babesia bovis using a single transfection plasmid. Sci Rep 8, 6096.

970 Silva, M.G., Knowles, D.P., Suarez, C.E., 2016b. Identification of interchangeable cross-species

971 function of elongation factor-1 alpha promoters in Babesia bigemina and Babesia bovis.

972 Parasites vectors 9, 576.

973 Silva, M.G., Villarino, N.F., Knowles, D.P., Suarez, C.E., 2018b. Assessment of Draxxin®

974 (tulathromycin) as an inhibitor of in vitro growth of Babesia bovis, Babesia bigemina and

975 Theileria equi. Int J Parasitol Drugs Drug Resist 8, 265-270.

976 Simon, M.S., Westblade, L.F., Dziedziech, A., Visone, J.E., Furman, R.R., Jenkins, S.G., Schuetz, A.N.,

977 Kirkman, L.A., 2017. Clinical and Molecular Evidence of Atovaquone and Azithromycin

978 Resistance in Relapsed Babesia microti infection associated with Rituximab and Chronic

979 Lymphocytic Leukemia. Clin Infect Dis. 65(7), 1222-1225.

980 Skinner, T.S., Manning, L.S., Johnston, W.A., Davis, T.M.E., 1996. In vitro stage-specific sensitivity

981 of Plasmodium falciparum to quinine and artemisinin drugs. Int J Parasitol 26, 519-525.

982 Sleebs, B.E., Lopaticki, S., Marapana, D.S., O'Neill, M.T., Rajasekaran, P., Gazdik, M., Günther, S.,

983 Whitehead, L.W., Lowes, K.N., Barfod, L., 2014. Inhibition of Plasmepsin V activity
984 demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria

985 parasites. PLoS Biol 12, e1001897.

986 Solano-Gallego, L., Sainz, A., Roura, X., Estrada-Pena, A., Miro, G., 2016. A review of canine

987 babesiosis: the European perspective. Parasit Vectors 9, 336.

988 Sonoiki, E., Ng, C.L., Lee, M.C., Guo, D., Zhang, Y.-K., Zhou, Y., Alley, M., Ahyong, V., Sanz, L.M.,

989 Lafuente-Monasterio, M.J., 2017. A potent antimalarial benzoxaborole targets a

990 Plasmodium falciparum cleavage and polyadenylation specificity factor homologue. Nature

991 Commun 8, 14574.

992 Sriwilaijareon, N., Petmitr, S., Mutirangura, A., Ponglikitmongkol, M., Wilairat, P., 2002. Stage

993 specificity of Plasmodium falciparum telomerase and its inhibition by berberine. Parasitol

994 Int 51, 99-103.

995 Suarez, C.E., Johnson, W.C., Herndon, D.R., Laughery, J.M., Davis, W.C., 2015. Integration of a

996 transfected gene into the genome of Babesia bovis occurs by legitimate homologous

997 recombination mechanisms. Mol Biochem Parasitol 202, 23-28.

998 Suarez, C.E., Laughery, J.M., Schneider, D.A., Sondgeroth, K.S., McElwain, T.F., 2012. Acute and

999 persistent infection by a transfected Mo7 strain of Babesia bovis. Mol Biochem Parasitol

1000 185, 52-57.

1001 Suarez, C.E., McElwain, T.F., 2008. Transient transfection of purified Babesia bovis merozoites. Exp

1002 Parasitol 118, 498-504.

1003 Suarez, C.E., McElwain, T.F., 2009. Stable expression of a GFP-BSD fusion protein in Babesia bovis

1004 merozoites. Int J Parasitol 39, 289-297.

1005 Suarez, C.E., Norimine, J., Lacy, P., McElwain, T.F., 2006. Characterization and gene expression of

1006 Babesia bovis elongation factor-1α. Int J Parasitol 36, 965-973.

1007 Suarez, C.E., Palmer, G.H., LeRoith, T., Florin-Christensen, M., Crabb, B., McElwain, T.F., 2004.

1008 Intergenic regions in the rhoptry associated protein-1 (rap-1) locus promote exogenous

1009 gene expression in Babesia bovis. Int J Parasitol 34, 1177-1184.

1010 Sun, W., Tanaka, T.Q., Magle, C.T., Huang, W., Southall, N., Huang, R., Dehdashti, S.J., McKew, J.C.,

1011 Williamson, K.C., Zheng, W., 2014a. Chemical signatures and new drug targets for

1012 gametocytocidal drug development. Sci Rep 4, 3743.

1013 Sun, W., Tanaka, T.Q., Magle, C.T., Huang, W., Southall, N., Huang, R., Dehdashti, S.J., McKew, J.C.,

1014 Williamson, K.C., Zheng, W., 2014b. Chemical signatures and new drug targets for

1015 gametocytocidal drug development. Sci Rep 4, 3743.

1016 Tejera Nevado, P., Bifeld, E., Höhn, K., Clos, J., 2016. A Telomeric Cluster of Antimony Resistance

1017 Genes on Chromosome 34 of Leishmania infantum. Antimicrob Agents Chemother 60,

1018 5262-5275.

1019 Thomford, J.W., Conrad, P.A., Boyce, W.M., Holman, P.J., Jessup, D.A., 1993. Isolation and in vitro

1020 cultivation of Babesia parasites from free-ranging desert bighorn sheep (Ovis canadensis

1021 nelsoni) and mule deer (Odocoileus hemionus) in California. J Parasitol 79, 77-84.

1022 Thomford, J.W., Conrad, P.A., Telford, S.R., 3rd, Mathiesen, D., Bowman, B.H., Spielman, A.,

1023 Eberhard, M.L., Herwaldt, B.L., Quick, R.E., Persing, D.H., 1994. Cultivation and

1024 phylogenetic characterization of a newly recognized human pathogenic protozoan. J Infect

1025 Dis 169, 1050-1056.

1026 Thomson, J.G., Fantham, H.B., 1914. The successful cultivation of Babesia (Piroplasma) canis in

1027 vitro, following the method of Bass. Trans Royal Soc Trop Med Hyg 7, 119-125.

1028 Tuvshintulga, B., AbouLaila, M., Sivakumar, T., Tayebwa, D.S., Gantuya, S., Naranbaatar, K.,

1029 Ishiyama, A., Iwatsuki, M., Otoguro, K., Omura, S., Terkawi, M.A., Guswanto, A., Rizk, M.A.,

1030 Yokoyama, N., Igarashi, I., 2017. Chemotherapeutic efficacies of a clofazimine and
1031 diminazene aceturate combination against piroplasm parasites and their AT-rich DNA-

1032 binding activity on Babesia bovis. Sci Rep 7, 13888.

1033 Van Niekerk, C.J., Zweygarth, E., 1996. In vitro cultivation of Babesia occultans. Onderstepoort J

1034 Vet Res 63, 259-261.

1035 Van Voorhis, W.C., Adams, J.H., Adelfio, R., Ahyong, V., Akabas, M.H., Alano, P., Alday, A., Aleman

1036 Resto, Y., Alsibaee, A., Alzualde, A., Andrews, K.T., Avery, S.V., Avery, V.M., Ayong, L.,

1037 Baker, M., Baker, S., Ben Mamoun, C., Bhatia, S., Bickle, Q., Bounaadja, L., Bowling, T.,

1038 Bosch, J., Boucher, L.E., Boyom, F.F., Brea, J., Brennan, M., Burton, A., Caffrey, C.R.,

1039 Camarda, G., Carrasquilla, M., Carter, D., Belen Cassera, M., Chih-Chien Cheng, K.,

1040 Chindaudomsate, W., Chubb, A., Colon, B.L., Colon-Lopez, D.D., Corbett, Y., Crowther, G.J.,

1041 Cowan, N., D'Alessandro, S., Le Dang, N., Delves, M., DeRisi, J.L., Du, A.Y., Duffy, S., Abd El-

1042 Salam El-Sayed, S., Ferdig, M.T., Fernandez Robledo, J.A., Fidock, D.A., Florent, I., Fokou,

1043 P.V., Galstian, A., Gamo, F.J., Gokool, S., Gold, B., Golub, T., Goldgof, G.M., Guha, R.,

1044 Guiguemde, W.A., Gural, N., Guy, R.K., Hansen, M.A., Hanson, K.K., Hemphill, A., Hooft van

1045 Huijsduijnen, R., Horii, T., Horrocks, P., Hughes, T.B., Huston, C., Igarashi, I., Ingram-Sieber,

1046 K., Itoe, M.A., Jadhav, A., Naranuntarat Jensen, A., Jensen, L.T., Jiang, R.H., Kaiser, A.,

1047 Keiser, J., Ketas, T., Kicka, S., Kim, S., Kirk, K., Kumar, V.P., Kyle, D.E., Lafuente, M.J.,

1048 Landfear, S., Lee, N., Lee, S., Lehane, A.M., Li, F., Little, D., Liu, L., Llinas, M., Loza, M.I.,

1049 Lubar, A., Lucantoni, L., Lucet, I., Maes, L., Mancama, D., Mansour, N.R., March, S.,

1050 McGowan, S., Medina Vera, I., Meister, S., Mercer, L., Mestres, J., Mfopa, A.N., Misra, R.N.,

1051 Moon, S., Moore, J.P., Morais Rodrigues da Costa, F., Muller, J., Muriana, A., Nakazawa

1052 Hewitt, S., Nare, B., Nathan, C., Narraidoo, N., Nawaratna, S., Ojo, K.K., Ortiz, D., Panic, G.,

1053 Papadatos, G., Parapini, S., Patra, K., Pham, N., Prats, S., Plouffe, D.M., Poulsen, S.A.,

1054 Pradhan, A., Quevedo, C., Quinn, R.J., Rice, C.A., Abdo Rizk, M., Ruecker, A., St Onge, R.,
1055 Salgado Ferreira, R., Samra, J., Robinett, N.G., Schlecht, U., Schmitt, M., Silva Villela, F.,

1056 Silvestrini, F., Sinden, R., Smith, D.A., Soldati, T., Spitzmuller, A., Stamm, S.M., Sullivan, D.J.,

1057 Sullivan, W., Suresh, S., Suzuki, B.M., Suzuki, Y., Swamidass, S.J., Taramelli, D., Tchokouaha,

1058 L.R., Theron, A., Thomas, D., Tonissen, K.F., Townson, S., Tripathi, A.K., Trofimov, V.,

1059 Udenze, K.O., Ullah, I., Vallieres, C., Vigil, E., Vinetz, J.M., Voong Vinh, P., Vu, H., Watanabe,

1060 N.A., Weatherby, K., White, P.M., Wilks, A.F., Winzeler, E.A., Wojcik, E., Wree, M., Wu, W.,

1061 Yokoyama, N., Zollo, P.H., Abla, N., Blasco, B., Burrows, J., Laleu, B., Leroy, D., Spangenberg,

1062 T., Wells, T., Willis, P.A., 2016. Open Source Drug Discovery with the Malaria Box

1063 Compound Collection for Neglected Diseases and Beyond. PLoS Pathog 12, e1005763.

1064 Vannier, E., Gewurz, B.E., Krause, P.J., 2008. Human babesiosis. Infect Dis Clin North Am 22, 469-

1065 488.

1066 Vannier, E.G., Diuk-Wasser, M.A., Ben Mamoun, C., Krause, P.J., 2015. Babesiosis. Infect Dis Clin

1067 North Am 29, 357-370.

1068 Vayrynen, R., Tuomi, J., 1982. Continuous in vitro cultivation of Babesia divergens. Acta Vet Scand

1069 23, 471-472.

1070 Vega, C.A., Buening, G.M., Green, T.J., Carson, C.A., 1985a. In vitro cultivation of Babesia bigemina.

1071 Am J Vet Res 46, 416-420.

1072 Vega, C.A., Buening, G.M., Rodriguez, S.D., Carson, C.A., McLaughlin, K., 1985b. Cryopreservation

1073 of Babesia bigemina for in vitro cultivation. Am J Vet Res 46, 421-423.

1074 Viseras, J., Garcia-Fernandez, P., Adroher, F.J., 1997. Isolation and establishment in in vitro culture

1075 of a Theileria annulata-infected cell line from Spain. Parasitol Res 83, 394-396.

1076 Waller, K.L., Muhle, R.A., Ursos, L.M., Horrocks, P., Verdier-Pinard, D., Sidhu, A.B.S., Fujioka, H.,

1077 Roepe, P.D., Fidock, D.A., 2003. Chloroquine Resistance Modulated in Vitro by Expression
1078 Levels of the Plasmodium falciparum Chloroquine Resistance Transporter. J Biol Chem 278,

1079 33593-33601.

1080 Wang, X., Xiao, Y.-P., Bouchut, A., Al-Khedery, B., Wang, H., Allred, D.R., 2012. Characterization of

1081 the unusual bidirectional ves promoters driving VESA1 expression and associated with

1082 antigenic variation in Babesia bovis. Eukaryot Cell 11, 260-269.

1083 Wirtz, E., Clayton, C., 1995. Inducible gene expression in trypanosomes mediated by a prokaryotic

1084 repressor. Science 268, 1179-1183.

1085 Wormser, G.P., Prasad, A., Neuhaus, E., Joshi, S., Nowakowski, J., Nelson, J., Mittleman, A.,

1086 Aguero-Rosenfeld, M., Topal, J., Krause, P.J., 2010. Emergence of Resistance to

1087 Azithromycin-Atovaquone in Immunocompromised Patients with Babesia microti Infection.

1088 Clin Infect Dis 50, 381-386.

1089 Wu, W., Herrera, Z., Ebert, D., Baska, K., Cho, S.H., DeRisi, J.L., Yeh, E., 2015. A chemical rescue

1090 screen identifies a Plasmodium falciparum apicoplast inhibitor targeting MEP isoprenoid

1091 precursor biosynthesis. Antimicrob Agents Chemother 59, 356-364.

1092 Yabsley, M.J., Shock, B.C., 2013. Natural history of Zoonotic Babesia: Role of wildlife reservoirs. Int

1093 J Parasitol Parasites Wildl 2, 18-31.

1094 Yamagishi, J., Asada, M., Hakimi, H., Tanaka, T.Q., Sugimoto, C., Kawazu, S.-i., 2017. Whole-

1095 genome assembly of Babesia ovata and comparative genomics between closely related

1096 pathogens. BMC Genomics 18, 832.

1097 Yamasaki, M., Asano, H., Otsuka, Y., Yamato, O., Tajima, M., Maede, Y., 2000. Use of canine red

1098 blood cell with high concentrations of potassium, reduced glutathione, and free amino acid

1099 as host cells for in vitro cultivation of Babesia gibsoni. Am J Vet Res 61, 1520-1524.

1100 Yeruham, I., Pipano, E., Davidson, M., 1985. A field strain of Babesia bovis apparently resistant to

1101 amicarbalide isethionate. Trop Anim Health Prod 17, 29-30.

1102 Zhang, C., Ondeyka, J.G., Herath, K.B., Guan, Z., Collado, J., Pelaez, F., Leavitt, P.S., Gurnett, A.,

1103 Nare, B., Liberator, P., 2006. Highly substituted terphenyls as inhibitors of parasite cGMP-

1104 dependent protein kinase activity. J Nat Prod 69, 710-712.

1105 Zhang, M., Wang, C., Otto, T.D., Oberstaller, J., Liao, X., Adapa, S.R., Udenze, K., Bronner, I.F.,

1106 Casandra, D., Mayho, M., Brown, J., Li, S., Swanson, J., Rayner, J.C., Jiang, R.H.Y., Adams,

1107 J.H., 2018. Uncovering the essential genes of the human malaria parasite Plasmodium

1108 falciparum by saturation mutagenesis. Science 360, 6388.

1109 Zhao, J., Liu, Z., Yao, B., Ma, L., 2002. Culture-derived Babesia orientalis exoantigens used as a

1110 vaccine against buffalo babesiosis. Parasitol Res 88, S38-S40.

1111 Zweygarth, E., Just, M.C., de Waal, D.T., 1995. Continuous in vitro cultivation of erythrocytic stages

1112 of Babesia equi. Parasitol Res 81, 355-358.

1113 Zweygarth, E., Lopez-Rebollar, L.M., 2000. Continuous in vitro cultivation of Babesia gibsoni.

1114 Parasitol Res 86, 905-907.

1115 Zweygarth, E., van Niekerk, C.J., de Waal, D.T., 1999. Continuous in vitro cultivation of Babesia

1116 caballi in serum-free medium. Parasitol Res 85, 413-416.

1117

1118

1119 Figure legends

1120 Fig. 1. Presented is an illustrative phylogeny based loosely on available 18S rRNA sequence data

1121 and current phylogenetic studies of piroplasm species of veterinary and medical importance, as

1122 well as all species which can currently be cultured in vitro. Filled circles represent the existence of

1123 the denoted system (green: in vitro culture; blue: in vivo model; purple: genetic system

1124 established). Parasites of zoonotic importance are denoted by a red human silhouette. B., Babesia;

1125 T., Trypanosoma.

1126 Highlights

1127  The poorly understood biology and the large diversity of piroplasmids poses a challenge to

1128 drug development.

1129  The ability to culture many Babesia spp. in vitro and in vivo provides an opportunity to

1130 identify core, conserved biology.

1131  The availability of many genetic tools in related parasites will help accelerate genetic

1132 technology development in Babesia.

1133  The use of sophisticated genetic techniques will be essential for the identification and

1134 validation of drug targets.

1135
1136

1137
1138 Table 1. Genetic tools available for drug target identification. ‘Yes’ represents technologies that

1139 are currently available for that organism. ‘Possible’ represents technologies where there are no

1140 obvious biological features of the organism that would prevent its development. ‘Not possible’

1141 represents technologies where the biology of the organism is expected to prevent the successful

1142 use in that organism.

1143
Genetic tool Mammalian/ Trypanosoma Toxoplasma Plasmodium Babesia
Yeast brucei gondii falciparum spp.

Gene editing

Homologous Yes Yes Yes Yes Yes

recombination

CRISPR/Cas9 Yes Yes Yes Yes Possible

Overexpression

Stable episomal Yes Yes Yes Yes Yes

expression
Stable integration Yes Yes Yes Yes Yes

Genome-wide Yes Yes Possible Possible Possible

overexpression
library
CRISPR-a Yes Possible Possible Possible Possible

Reduced expression

Conditional Yes Yes Yes Yes Possible

transcription
Conditional mRNA Yes Yes Yes Yes Possible
stability / translation
Conditional protein Yes Possible Yes Yes Possible
stability / localization
Haploinsufficiency Yes Not possible Not Not Not
 possible possible possible

RNA interference Yes Yes Not Not Not

possible possible possible
CRISPR-i/Cas13 RNA Yes Possible Possible Possible Possible
targeting
Gene deletion

Homologous Yes Yes Yes Yes Yes

recombination
CRISPR/Cas9 -NHEJ Yes Not possible Yes Not Not
possible possible
Transposon Yes Yes Possible Yes Possible
mutagenesis
1144
1145
1146