Identification of a putative methyltransferase gene of Babesia bigemina as a novel molecular biomarker uniquely expressed in parasite tick stages

Abstract

BackgroundBovine babesiosis is caused by apicomplexan pathogens of the genus Babesia such as B. bigemina and B. bovis. These tick-borne pathogens have a complex life-cycle involving asexual multiplication in vertebrate hosts and sexual reproduction in invertebrate vectors. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. Understanding the mechanisms that underlie formation of extracellular Babesia tick stages is an important step towards developing control strategies for preventing tick infection and subsequent parasite transmission.ResultsWe induced B. bigemina sexual stages in vitro by exposing parasites to Tris 2-carboxyethyl phosphine (TCEP). Subsequently, we identified a novel putative methyltransferase gene (BBBOND_0204030) that is expressed uniquely in all B. bigemina tick stages but not in blood stages. In vitro TCEP-exposed B. bigemina presented diverse morphology including parasites with long projections, round forms and clusters of round forms indicative of sexual stage induction. We confirmed the development of sexual stages by detecting upregulation of previously defined B. bigemina sexual stage marker genes, ccp2 and 3, and their respective protein expression in TCEP-induced B. bigemina cultures. Next, transcription analysis of in vitro TCEP-induced B. bigemina culture based on an in silico derived list of homologs of Plasmodium falciparum gamete-specific genes demonstrated differential expression of the gene BBBOND_0204030 in induced cells. Further examination of ex vivo infected ticks demonstrated that BBBOND_0204030 is transcribed by multiple stages of B. bigemina during parasite development in tick midgut, ovary and hemolymph. Interestingly, ex vivo results confirmed our in vitro observation that blood stages of B. bigemina do not express BBBOND_0204030 and validated the in vitro system of inducing sexual stages.ConclusionsHerein we describe the identification of a B. bigemina gene transcribed exclusively by parasites infecting ticks using a novel method of inducing B. bigemina sexual stages in vitro. We propose that this gene can be used as a marker for parasite development within the tick vector. Together, these tools will facilitate our understanding of parasite-tick interactions, the identification of novel vaccine targets and, consequently, the development of additional strategies to control bovine babesiosis.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-3052-9) contains supplementary material, which is available to authorized users.

Identification of proteins expressed by Babesia bigemina kinetes

“…Babesia bigemina sexual stages were produced as previously described [11]. In brief, B. bigemina infected erythrocytes were grown in HL-1 medium (Lonza, Walkersville, MD) supplemented with 40% normal bovine serum, 10 mM 3-[N-tris (hydroxymethyl) methylanino]-2-hydroxypropanesulfonic acid (Sigma-Aldrich, St. Louis, MO, USA), and antibiotic/antimycotic (Sigma-Aldrich, St. Louis, MO, USA), pH 7.2, with a 5% packed cell volume of bovine red blood cells.…”

“…To increase parasitemia, cultures were incubated at 37 °C in 5% CO 2 and expanded without addition of erythrocytes [18]. When the parasitemia exceeded 10%, B. bigemina sexual stages were induced with 20 mM tris(2-carboxyethyl)phosphine (Thermo Fisher scientific) and incubated for 1 h at 37 °C with 5% CO 2 as previously described [11]. Cultures were then washed once with fresh medium to remove excess reducing agent by centrifugation for 3 min at 2655× g .…”

“…In silico and experimental data demonstrated that CCp2 and CCp3 are well conserved among arthropod-borne apicomplexan parasites including Plasmodium , Theileria and Babesia and are consistently expressed in in vitro induced sexual stages [9, 10]. Transcription of tick stage-specific genes, and associated protein expression, of B. bigemina has recently been documented during the parasite’s development in the vector [11]. Bohaliga et al [11] identified BBBOND_0204030 as a gene uniquely transcribed by B. bigemina tick stages and by in vitro induced B. bigemina sexual stages.…”

“…Transcription of tick stage-specific genes, and associated protein expression, of B. bigemina has recently been documented during the parasite’s development in the vector [11]. Bohaliga et al [11] identified BBBOND_0204030 as a gene uniquely transcribed by B. bigemina tick stages and by in vitro induced B. bigemina sexual stages. The B. bigemina HAP2 protein, that is associated with the development of gametes in apicomplexan parasites [12], was found to be expressed by parasite stages developing in the midgut of infected ticks at day 3 post-repletion of female R .…”

“…microplus ticks. In addition, studies identified and demonstrated protein expression of B. bigemina CCp1-3, a gene family also related to the development of sexual stages in apicomplexans [10], by B. bigemina in vitro induced sexual stages but not blood stages [10, 11]. Finally, Johnson et al [13] identified kinete stage-specific genes that are highly transcribed by B. bovis and B. bigemina kinetes as compared to blood stages.…”