Genome sequence of Babesia bovis and comparative analysis of apicomplexan hemoprotozoa

Abstract

Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The approximately 150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development.

Figure 1. Representation of B. bovis Centromeres, ves1, and smorf Genes

Each chromosome is represented by a black line, with the chromosome number shown on the left. Centromeres are depicted as black dots. ves1 loci are depicted as colored boxes, and the number of ves1 and smorf genes in each locus is shown above or below the colored boxes, respectively. Red and blue boxes indicate the presence of at least one ves1α/ves1β or ves1α/ves1α pair, respectively, within the cluster.

Figure 2. Comparison of Major Metabolic Pathways in B. bovis, T. parva, and P. falciparum

Solid arrows indicate single step enzymatic reactions, dashed lines indicate multi-step reactions, and dotted lines indicate incomplete or unknown pathways. Inhibitory drugs are indicated with red arrows. Glucose is assumed to be the major carbon and energy source. Non-functional pathways in P. falciparum, T. parva, and B. bovis are shown in boxes with a red X. Pathways denoted with blue text are present in P. falciparum, T. parva, and B. bovis. Pathways denoted with green text are present in P. falciparum and T. parva but not in B. bovis, and pathways denoted with red text are exclusively found in P. falciparum.

Figure 3. Diagram of a Locus Containing the ves1α, ves1β, and smorf Genes

The genome backbone is a gray line, ves1α exons are blue, ves1β exons are red, and the SmORF exon is yellow. Introns are shown as blank boxes through which the genome backbone is seen. The systematic gene name for each gene is shown. Transmembrane helices (black bars), coiled-coil domains (green boxes), variant domains with conserved sequences 1 and 2 (pink boxes), and the SmORF signal peptide (orange box) are indicated. Arrows represent the positions of the primers for each of the cDNA experiments. The experiment number is indicated to the right of the primer sets, with experiment 1 targeting specific genes, experiment 2 sets of genes, and experiment 3 the published LAT.

Figure 4. Diagram of Chromosomal Synteny between B. bovis and T. parva

(A) Synteny at the chromosomal level. B. bovis chromosome number is indicated at the top. Bars on the right side of each chromosome diagram designate B. bovis genes, with black bars indicating B. bovis ves1 genes and gray bars indicating other genes. The colors on the left of each chromosome diagram indicate to which T. parva chromosome an ortholog belongs as follows: Tp1 = red, Tp2 = green, Tp3 = blue, Tp4 = orange. (B) Comparison of telomeric arrangement of genes for B. bovis and T. parva chromosome 2. The gray line indicates the chromosomal backbone, with black dots indicating the telomere. Large genes are depicted as arrows with coding direction indicated, while small genes have an arrowhead beneath the gene to indicate the direction of transcription. Gray double arrowheads indicate that the chromosome continues. Colors indicate gene content as follows: blue = B. bovis ves1α, red = B. bovis ves1β, yellow = B. bovis SmORF, pale green = putative membrane protein, pale blue = annotated genes with predicted function, black = hypothetical, orange = T. parva family 3 hypothetical, purple = T. parva family 1 hypothetical, green = ABC transporter.