Fig 1.
Microscopical description of isolated B. besnoiti tissue cysts.
(A) Binocular view of cysts freshly isolated from skin. (B) Conventional light microscopy. CW = cyst wall. (C) Light microscopy of semi-thin sections of epoxy-resin embedded cysts stained with methylene blue and basic fuchsin. (D-G) Scanning transmission electron microscopy of the tissue cyst wall. (H, I) Ruptured cyst releasing bradyzoites and cyst matrix. Bars in A = 500 μm, B = 140 μm, C = 110 μm, D = 170 μm, E and F = 40 μm, G = 1.6 μm, H and I = 140 μm.
Fig 2.
Transmission electron microscopy of tissue cysts.
(A-C) Compartments of the tissue cyst, with the fibrous outer cyst wall (OCW), host cell cytoplasm (HC), inner cyst wall (ICW) and the inner lumen of the tissue cyst (IL). Red arrows indicate peripheral cytoplasmic host cell-extensions into the OCW. The boxed area in (A) is magnified in (B), showing that the OCW is composed largely of fibrous material, and in (C) an even higher magnification depicts the collagen-like filament bundles (red arrows). (D) Host cell cytoplasm with cytoplasmic extensions (vertical red arrows) and inner cyst wall. The boxed area in (D) is enlarged in (E). (E-F) Larger magnification of the host cell cytoplasm adjacent to the inner cyst wall, with the presence of numerous electron-dense mitochondria (m) and many cytoskeletal filament bundles (horizontal red arrows). (G) Higher magnification view of the parasite-host cell interface, showing the ICW and its electron-dense granular substance, the parasitophorous vacuole membrane (pvm, yellow arrows), distinct vesicular structures adjacent to the ICW interior. Two bradyzoites are embedded in a parasitophorous vacuole network (PVN), exhibiting their apical parts with micronemes (mic), rhoptries (rop) and dense granules (dg).Bars in A = 2.8 μm, B = 0.55 μm, C = 0.25 μm, D = 0.55 μm, E = 0.25 μm, F = 0.35 μm, G = 0.15 μm.
Fig 3.
Transmission electron microscopy of the cyst interior and bradyzoites.
(A) Bradyzoites embedded in the tissue cyst matrix in the peripheral area of the cyst. Non-viable bradyzoites are marked with yellow arrows. (B-E) Higher magnification view of bradyzoites with typical zoite organelles. (D) enlarged inset from (C) showing details of the conoid (con) and the inner membrane complex, IMC (arrows). (F) Bradyzoites in the centrally located area of the tissue cyst. apg: amylopectin granules, con: conoid, dg: dense granules, ICW: inner cyst wall, mic: micronemes, nuc: nuclei, pvn: parasitophorous vacuole network, rop: rhoptries, v: vesiculated structures. Bars in A = 1.2 μm, B = 0.5 μm, C and E = 0.6 μm, D = 0.15 μm, F = 1.4 μm.
Fig 4.
Scanning and transmission electron microscopy of tachyzoites.
(A) Invasion of a single host fibroblast in vitro by multiple tachyzoites derived from tissue cysts (thin arrows). (B) Tachyzoite dividing by endodyogeny. Thicker arrows point towards the conoids. (C) Tachyzoites undergoing proliferation inside the parasitophorous vacuole forming a rosette (insert provides an overview at lower magnification). (D) Tightly packed tachyzoites within a parasitophorous vacuole (insert shows lower magnification view). apg: amylopectin granules, con: conoid, dg: dense granules, mic: micronemes, nuc: nuclei, rop: rhoptries. Bars in A = 2 μm, B = 0.3 μm, C and D = 0.6 μm.
Table 1.
Comparison of genome parameters from different Coccidia.
Fig 5.
Synteny and orthology between Besnoitia besnoiti, Toxoplasma gondii and Neospora caninum.
(A) Synteny mapping of the 19 contigs of B. besnoiti that are larger than 100 kb shows the genomic organization of the B. besnoiti genome to T. gondii and N. caninum. Syntenic regions are highlighted with coloured lines within the circle. Labelling of the B. besnoiti chromosomes is according to GenBank (B. besnoiti Ger1 reference strain). (B) Synteny mapping as in (A), but exclusively of T. gondii and N. caninum contigs. (C) Phylogenetic organization of B. besnoiti, T. gondii and N. caninum. Branch length represents substitution rate (substitutions per site). Chr: Chromosome; Unp: Unplaced contig.
Fig 6.
Orthology between species and unique B. besnoiti proteins.
Venn diagram with orthologous groups in B. besnoiti and four other apicomplexan species.
Table 2.
Number of genes in orthogroups.
Fig 7.
Differential expression of mRNA between B. besnoiti tachyzoites and tissue cysts and comparison to T. gondii.
(A) Heatmap depicting log2 fold changes (LFC) of the top 10% of differentially expressed genes. N = 1172 (B) Heatmap showing the differences to the common mean (Z-score) of B. besnoiti orthologues of typical stage marker genes known in T. gondii. Tachyzoites (Z < 0, red) and tissue cysts (Z > 0, purple). (C) Heatmap showing the differences to the common mean (Z-score) of T. gondii tissue cyst-specific genes. (D) Scatter plot of TPM (Transcripts Per Kilobase Million, ToxoDB release 52) values of expressed genes in B. besnoiti. Values for tachyzoites are plotted against TPMs from tissue cysts. (E) Scatter plot of TPMs (ToxoDB release 52) values of protein coding genes in T. gondii. (F) Scatter plot with TPMs depicting genes encoding expressed dense granule proteins in B. besnoiti. (G) Scatter plot with TPMs depicting syntenic T. gondii genes from figure (F). BAG1: bradyzoite antigen 1; BPK1: bradyzoite pseudokinase 1; BRP1: bradyzoite rhoptry protein 1; CST: cyst wall protein; ENO: enolase; LDH: lactate dehydrogenase; MAG: matrix antigen; MIC: micronemal protein; SAG: surface antigen; SRS: SAG-related sequence; SUB: subtilisin.
Fig 8.
Metabolic pathways of B. besnoiti.
(A) Overview and classification of metabolic genes: number of annotated genes, pathways, and isoenzymes in B. besnoiti. Up- and downregulated genes have a relative fold-change >4 (log2 > |2|). Stage-specific isoenzymes are pairs that display upregulation only in one stage of the parasite (bradyzoites or tachyzoites; see also Tab E in S8 Table). (B) E.C. classification of all enzymes found in the B. besnoiti genome. Transferases are the most common class of enzymes, followed by transporters and enzymes with an unknown function (see also Tab F in S8 Table). (C) List of up-regulated genes encoding biosynthesis enzymes by the cyst-forming B. besnoiti bradyzoites. These enzymes are part of diverse metabolic pathways, including several tRNA-synthetases. (D) The 59 curated metabolic pathways identified in B. besnoiti, ordered by percentage of genes upregulated in the bradyzoite stage. Bubble size represents the number of genes (in brackets) in each pathway. Eighteen pathways have more than 25 percent of their genes upregulated in the bradyzoite stage.
Fig 9.
Upregulated pathways and correlation of gene expression levels the T. gondii transcriptomic profile in bradyzoites.
(A) The starch and galactose metabolic pathway with their gene expression in bradyzoites highlighted from red (downregulated) to blue (upregulated). Bubbles in shades of red or blue mark the level of regulation. Note that hexokinase belongs to the glycolysis pathway but is highlighted because its activity initiates the starch and galactose pathway. (B) Schematic representation of the polyamine salvage pathway and their high expression level in the latent stage of B. besnoiti. Bubbles in shades of blue highlight the high level of upregulation in B. besnoiti bradyzoites. (C) Correlation of the ratio of gene expression between bradyzoites and tachyzoites in B. besnoiti compared to T. gondii of all known metabolic genes in these coccidan species. Genes upregulated only in B. besnoiti but not in T. gondii are highlighted in blue and highlighted in red are genes upregulated only in T. gondii bradyzoites. 2,4-DECR: 2,4-dienoyl CoA reductase 2; 3-HACD: 3-hydroxyacyl-CoA dehydrogenase; 3-HBD: 3-hydroxyisobutyrate dehydrogenase; ACAAT: acetyl-CoA C-acyltransferase, ACDH: Acyl-CoA dehydrogenase domain-containing protein; AK: adenylate kinase; ALDH: alanine dehydrogenase; APT: aminopeptidase n; BCD: Butyryl-CoA dehydrogenase; CS: citrate (si)-synthase; DOXPS: 1-deoxy-D-xylulose 5-phosphate synthase; ENO1: enolase 1; ENO2: enolase 2; KDC: lysine decarboxylase family protein; LDH2: lactate dehydrogenase 2; NT1: nitrite transporter 1; NT2: nitrite transporter 2, PDH-E1b: pyruvate dehydrogenase E1 beta subunit; PEPCK: Phosphoenoylpyruvat (PEP) carboxykinase; PIG-T: GPI-anchor transamidase; PIG-V: mannosyltransferase; SPT1: serine C-palmitoyltransferase 1; TAG lipase: triacylglycerol lipase.
Fig 10.
B. besnoiti orthologues involved in sexual reproduction and oocyst formation.
(A) Bar chart showing the percentage of B. besnoiti orthologues identified for all T. gondii genes with expression maxima EES1-5 or oocysts (oocyst genes listed as in [38]). (B) Percentage of B. besnoiti genes (LFC > |2|) identified that are orthologous to upregulated genes in T. gondii tissue cysts (n = 814) (left graph) [38] This results in 483 unique B. besnoiti genes of which approximately 75% are not differentially regulated between tachyzoites and tissue cysts (right graph). (C) Percentage of T. gondii genes having expression maxima in a distinct coccidian stage. (D) As in (C), but for B. besnoiti orthologues.