Expression of 6-Cys Gene Superfamily Defines Babesia bovis Sexual Stage Development within Rhipicephalus microplus

Abstract

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector.

Fig 1. Bioinformatic analysis of Bbo 6-Cys family genes.

Schematic representation of the locations and relative orientations of the Bbo 6-Cys genes family members into the four B. bovis nuclear chromosomes. The lengths of the ORFs, the intergenic regions among them, and their distance from the chromosomes ends are indicated in bp. The size of the genes is not represented in a proportional scale.

Fig 2. Characterization of Bbo 6-Cys proteins and 6-Cys domain.

A. Schematic representation of 6-Cys family proteins. The number and relative localization of the 6-Cys domains, represented as blue boxes, the predicted signal peptides (SP) and transmembrane domains (Tm), and the number of amino acids are indicated. B. Schematic representation of the arrangement and number of the 6-Cys domains in each of the B. bovis 6-Cys proteins. The number of residues found in each domain is represented inside each box. The lengths of the inter-domain regions (IDR) in the proteins containing two 6-Cys domains are relatively similar at 163,161,159,162, 164, in the A, B, C, D and E proteins, whereas it contains 227 and 521 aa in H and G’s 6-Cys domain, respectively. Since protein F has three 6-Cys domains so it has two IDRs with lengths 164 aa and 214 aa for IDR1 and IDR2 respectively. C. Schematic representation of the predicted disulfide bonds in the B. bovis 6-Cys proteins using the Prosite program (top). Description of the three conserved domains identified in the 6-Cys domains among the ten 6-Cys B. bovis proteins using MEME analysis. Amino acids are represented using letter symbols. The highest letters represent strictly conserved residues. Font sizes are proportional to the relative frequency of each residue for each position.

Fig 3. Transcriptional analysis of the Bbo 6-Cys genes in cultured blood stages.

A. and B. RNA seq analysis performed on the virulent (red) and attenuated (blue) parasites derived from the L17 strain of B. bovis. The Y axis indicates relative transcriptional levels. The X axis represents the name of the 6-Cys and rap-1 control genes. Transcripts for the B. bovis 6-Cys genes C, D, G, H, I and J were not detected using RNA seq. C. and D. Microarray analysis of the virulent (red) and attenuated (blue) parasites derived from the T2Bo strain of B. bovis. The Y axis indicates relative transcriptional levels. The X axis represents the name of the 6-Cys and rap-1 control genes.

Fig 4. Differential transcription of the Bbo 6-Cys family gene members among three distinct developmental stages of the life cycle of B. bovis.

First panel: PCR amplifications of the full ORF of each Bbo 6-Cys family gene members (A-J) using gDNA extracted from cultured B. bovis T3bo strain parasites. Amplifications were performed on samples with (+) or without (-) DNA template. PCR amplification of the B. bovis RAP-1 gene was used as a control (left panel). Second panel: Transcription analysis of the Bbo 6-Cys family gene members(A-J) using cDNA generated from total RNA extracted from in vitro blood culture T3bo strain parasites. Identical results were obtained using RNA extracted from in vitro cultured Mo7 clonal line, L17 strain; in vivo T3Bo strain and T strain pair virulent and attenuated (data not shown). Identical amplifications using RAP-1 primers generated a 300 bp PCR product (indicated in the left panel). Third panel: Transcription analysis of the Bbo 6-Cys family gene members (A-J) performed on cDNA generated from total RNA extracted from B. bovis-infected R. microplus midgut. Total RNA was extracted from midguts at different days upon incubation (2, 3 and 4 day) at 28°C. Asterisk (*) indicate presence of faint band for 6-Cys I gene amplification. Amplification of the control rap-1 gene is shown in the left panel. Fourth panel: Transcription analysis of the Bbo 6-Cys family gene members (A-J) using cDNA generated from total RNA extracted from B. bovis infected R. microplus hemolymph. Control gene RAP-1 was not amplified from infected hemolymph. Size markers (SM) are indicated on the left of the large panels.

Fig 5

Immunoblot analysis for expression of the B. bovis 6-Cys A and B proteins in blood, midgut, and hemolymph stages. A. Immunoblot analysis of uninfected RBC and cultured B. bovis infected RBC with rap-1 mAb BABB75, rabbit polyclonal sera against 6-Cys A-specific synthetic peptide and 6-Cys B-specific synthetic peptide. Synthetic A and B peptides were used as positive controls indicated by the black arrow. The size of peptide A is 21.6 D and peptide B is 24 D. B. Immunoblot analysis of non-infected and B. bovis infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys A-specific synthetic peptide, and pre-immune rabbit sera (PI). C. Immunoblot analysis of non-infected and B. bovis-infected R. microplus midgut protein extract and hemolymph with rabbit polyclonal sera against 6-Cys B-specific synthetic peptide, and pre-immune rabbit sera. Size markers (M) in kDa are indicated at the left of each panel.