Fig 1.
Panning of B. bovis for binding to BBECs.
A) Selection of B. bovis for binding to BBECs. The number of bound iRBCs per 100 BBECs was counted. All data are expressed as mean ± SD. The assay was done in duplicate in 3 biological replicates. B) Giemsa-stained smear of cytoadherent B. bovis to BBECs. Small cells are iRBCs having single or paired forms of B. bovis. The larger cells are BBECs. Scale bar = 100 μm. C) Transmission electron micrograph of B. bovis-iRBC bound to BBEC through ridges (indicated by red arrows). Scale bar = 1 μm.
Fig 2.
Quantitative reverse transcription PCR (qRT-PCR) of ves1α and ves1β.
Absolute transcript levels of ves1α and ves1β in each cytoadherent parasite clone were quantified using qRT-PCR. All data are shown as mean ± SD. Transcript levels were normalized against methionyl-tRNA synthetase (Gene ID: BBOV_I001970).
Fig 3.
ves1α is the major determinant for binding of iRBCs to BBECs.
A) Schematic diagram of ves1α-gfp-hdhfr-expressing plasmid and diagnostic PCRs to confirm integration of ves1α-gfp-hdhfr-expressing plasmid into ef-1α locus. Stop codon is shown with a red asterisk. ef-1αIG-B, elongation factor-1α intergenic region B; ef-1α-3’NR, elongation factor-1α 3’ noncoding region; hdhfr, human dihydrofolate reductase. B) Western blot analysis of C2 clone and 2 transgenic parasite clones expressing C3 (α3) or C5 ves1α (α5) on C2 clone parasite. Anti-GFP shows the chimeric VESA1-a-GFP expression in the transgenic parasites. Anti-VESA1 and anti-SBP4 antibodies were used to detect VESA1, and SBP4 protein as a loading control. C) Cytoadhesion assay of ves1α-gfp-hdhfr-expressing parasite clones and WT parasites. C3, C2, C5: original clones before transfection; α3-Tg1 and Tg2: transfectants expressing C3 ves1α on C2 clone parasite; α5-Tg1 and Tg2: transfectants expressing C5 ves1α on C2 clone parasite. All data are expressed as mean ± SD of three independent experiments with two technical replicates per sample (*, P < 0.05; ns, not significant; determined by Tukey’s multiple comparison test).
Fig 4.
ves1β does not increase binding of iRBCs to BBECs.
A) Schematic diagram of ves1β-mCherry-bsd-expressing plasmid. Stop codons are shown with red asterisks. actin 5’NR, actin 5’ noncoding region/promoter; tpx-1 3’NR, thioredoxin peroxidase-1 3’ noncoding region; ef-1αIG-B, elongation factor-1α intergenic region B; bsd, basticidin S deaminase; rap-1 3’ NR; rhoptry associated protein-1 3’ noncoding region. B) Western blot analysis of C2 clone and 2 transgenic parasites episomally expressing C3 (β3) or C5 ves1β (β5) on C2 clone parasite. Anti-mCherry shows the chimeric VESA1b-mCherry expression in the transgenic parasites. Anti-VESA1 antibody was used to detect VESA1 protein as a loading control. C) Cytoadhesion assay of ves1β-mCherry-bsd-expressing parasites and WT uncloned cytoadherent line. β3-Tg1: transfectant episomally expressing C3 ves1β on C2 clone parasite; β5-Tg1: transfectant episomally expressing C5 ves1β on C2 clone parasite. All data are expressed as mean ± SD of three independent experiments (*, P < 0.05; ns, not significant; determined by Tukey’s multiple comparison test).
Fig 5.
Genome synteny between B. bovis T2Bo and cytoadherent clones.
A) The dot plot analyses among reference T2Bo, C1, C2, C3, and C5 identified genomic recombination. Dot plots were generated by D-Genies. The diagonal line shows the alignment between two parasite genomes. Short off-diagonal lines between C1 and T2Bo show structural variations in chromosomes 1 and 2. B) The synteny blocks of T2Bo were aligned with those of cytoadherent clones. Chromosome 4 of T2Bo has a gap and consists of two contigs (Ch4-1 and Ch4-2). The primary and alternative LATs are indicated with red and blue triangles, respectively. C) Integrative genomics viewer (IGV) of candidate LATs in cytoadherent clones. LATs were identified by aligning the RNA-seq reads on the genome of corresponding cytoadherent clones sequencing using MinION. The primary and alternative LATs are indicated with red and blue triangles, respectively. The aligned ves1 reads (black triangles) on the C5 genome did not reveal a major LAT.
Fig 6.
Pie chart of expressing ves genes in the cytoadherent clones.
Pie charts display ves genes expression profile of each cytoadherent clone population with each slice of the pie representing the expression level of a single ves gene. The expressing dominant ves1α and ves1β from a single LAT are labeled in each clone. Gene IDs are from T2Bo reference strain and are assigned based on ves1 sequence similarity.