The Babesia bovis hap2 gene is not required for blood stage replication, but expressed upon in vitro sexual stage induction

Abstract

Babesia bovis, is a tick borne apicomplexan parasite responsible for important cattle losses globally. Babesia parasites have a complex life cycle including asexual replication in the mammalian host and sexual reproduction in the tick vector. Novel control strategies aimed at limiting transmission of the parasite are needed, but transmission blocking vaccine candidates remain undefined. Expression of HAP2 has been recognized as critical for the fertilization of parasites in the Babesia-related Plasmodium, and is a leading candidate for a transmission blocking vaccine against malaria. Hereby we identified the B. bovis hap2 gene and demonstrated that it is widely conserved and differentially transcribed during development within the tick midgut, but not by blood stage parasites. The hap2 gene was disrupted by transfecting B. bovis with a plasmid containing the flanking regions of the hap2 gene and the GPF-BSD gene under the control of the ef-1α-B promoter. Comparison of in vitro growth between a hap2-KO B. bovis clonal line and its parental wild type strain showed that HAP2 is not required for the development of B. bovis in erythrocytes. However, xanthurenic acid-in vitro induction experiments of sexual stages of parasites recovered after tick transmission resulted in surface expression of HAP2 exclusively in sexual stage induced parasites. In addition, hap2-KO parasites were not able to develop such sexual stages as defined both by morphology and by expression of the B. bovis sexual marker genes 6-Cys A and B. Together, the data strongly suggests that tick midgut stage differential expression of hap2 is associated with the development of B. bovis sexual forms. Overall these studies are consistent with a role of HAP2 in tick stages of the parasite and suggest that HAP2 is a potential candidate for a transmission blocking vaccine against bovine babesiosis.

Fig 1. RT-PCR analysis of B. bovis hap2.

Analysis was performed in B. bovis from blood from an acutely infected animal, and in dissected tick midguts of female ticks that fed on animals acutely infected with B. bovis. Tick samples were obtained at 0–6 days post-repletion. Amplifications were performed on samples with (+) or without (-) the addition of reverse transcriptase. RBC: red blood cells. rap1: positive control. Molecular size markers in base pairs are indicated on the left side.

Fig 2. Schematic diagrams showing.

A. Structure of the targeted hap2 gene locus; B. Organization of the plasmid phap2-lucgfpbsd used to disrupt the B. bovis hap2 gene; C. Deduced structure of the disrupted hap2 gene locus in the transfected clonal lineTf-hap2KO-gfp-bsd.

Fig 3. Disruption of the B. bovis hap2 gene and cloning and phenotypic characterization of a hap2 KO strain.

A. Growth curve describing the kinetics of emergence of the transfected parasite line Tf-hap2KO-gfp-bsd upon blasticidin selection. B. Detection of GFP expression by the transfected B. bovis clonal line Tf-hap2KO-gfp-bsd-cln derived from the parasite line Tf-hap2KO-gfp-bsd, left panel is Tf-hap2KO-gfp-bsd-cln and right panel is the non-transfected B. bovis wild type strain T3B. Scale bar: 5 μm. C. Growth curves of the B. bovis Tf-hap2KO-gfp-bsd-cln clonal line and non-transfected B. bovis strain T3B parasite line (wild type) showing similar replication kinetics in vitro without blasticidin selection. The “X” axis represents the percentage of infected erythrocytes in in vitro cultures and the “Y” axis the days after the initiation of the in vitro cultures.

Fig 4. Photomicrograph of Giemsa-stained B. bovis smears.

A. Morphological changes of B. bovis wild type parasites developing in induced in vitro cultures, (1–4) lntraerythrocytic parasites, develop ray bodies (Strahlenkorper) whilst still intracellular. (5–7) Strahlenkorper egressing from erythrocytes. (8–11) Polymorphic multi-nucleated population of Strahlenkorper with long and short spikes. (12, 13) Two strahlenkorper adhering to each other along cell membranes. (14–16) Large aggregates of adhering multi-nucleated Strahlenkorper, occurring 20 h after induction. B. B. bovis Tf-hap2KO-gfp-bsd-cln parasites with induction medium (XA) at 26°C (1–4). Bars, 5 μm.

Fig 5. Transcription analysis of B. bovis blood stages and induced sexual stages.

A. RT-PCR analysis for the detection of 6-Cys A, 6-Cys B and hap2 transcripts in wild type B. bovis parasites. The RT-PCR analysis was performed using non-induced (NIC) and induced (IC) in vitro cultures. B. 6-Cys A, 6-Cys B RT-PCR products were not detectable in Tf-hap2KO-gfp-bsd-cln induced parasites. Amplifications were performed on samples with (+) or without (-) the addition of reverse transcriptase. RT-PCR amplification of rap1 was used as a positive control. M represents the sizes of molecular markers in base pairs. NIC: non-induced culture, IC: induced culture.

Fig 6. Western blot analysis using antibodies against 6-Cys A, and HAP2, performed on non-induced (NIC) and induced (IC) B. bovis parasites developed in in vitro cultures.

Monoclonal RAP-1 antibodies were used to detect RAP-1 protein during in vitro cultured B. bovis as a positive control, and pre-immune rabbit serum as a negative control PI rabbit serum. Size markers (M) in kDa are indicated at the left side.

Fig 7. Live immunofluorescence analysis for the expression of 6-Cys A and HAP2 in the surface of induced B. bovis extracellular parasites.

A. B. bovis induced cells incubated with anti-6-Cys A and goat anti-rabbit tagged with Alexa Fluor 555 and stained with DAPI; B. B. bovis induced cells incubated anti-HAP2 and goat anti-rabbit tagged with Alexa Fluor 555 and stained with DAPI; C. Negative control preimmune (PI) rabbit serum as primary antibody, and stained with DAPI; D. non-induced B. bovis cells Incubated with Anti 6-Cys A and Anti-HAP2 and goat anti-rabbit tagged with Alexa Fluor 555 and stained with DAP. NIC: non-induced culture, IC: induced culture, Bars, 5 μm.

Fig 8. HAP2 expression in the surface of B. bovis induced parasites.

A. B. bovis induced cells incubated with HAP2 and goat anti-rabbit tagged with Alexa Fluor 555 and stained with 6-CFDA and nucleic acid stain Hoechst 33342; B. B. bovis induced cells treated with trypsin and incubated with anti-HAP2 and goat anti-rabbit tagged with Alexa Fluor 555 and stained with 6-CFDA and nucleic acid stain Hoechst 33342; IC: induced culture, Bars, 5 μm.