Figure 1.
EtMIC3 is a microneme protein.
(A) TEM/immunogold localization of EtMIC3 in an E. tenella sporozoite shows it to be located exclusively to the micronemes. (R) rhoptry, (M) microneme (RB) refractile body. The bar represents 1 µm and 0.1 µm in insert. (B) Immunofluorescence localization of EtMIC3 in fixed and permeabilized developing schizonts of E. tenella within a section of infected caecum shows it to be located at the apical tips of newly formed merozoites in a crescent shaped distribution. Blue counterstain is DAPI. Arrowheads indicate merozoites emerging from mature schizonts. (ES) early schizonts. The bar represents 10 µm. (C) Immunofluorescence localization (left panel) and corresponding differential interference contrast (DIC, right panel) of EtMIC3 (green) and beta tubulin (red) in fixed and permeabilized invading E. tenella sporozoites on MDBK monolayers. Blue counterstain is DAPI. Sporozoite attaches to the host cell (top panel) causing invagination of the host cell membrane (middle panel) and the sporozoite becomes committed to invasion with an extruded conoid (bottom panel). Note merged labelling of EtMIC3 and host cell tubulin at the moving junction (yellow) in the bottom panel. EtMIC3 is present at the apical end of the sporozoite throughout these early invasion stages. The bar represents 1 µm.
Figure 2.
Details of localization of EtMIC3 and EtAMA1 in invading parasites.
(a) Immunofluorescence localisation (left, top and bottom panels) and DIC (right, top and bottom panels) of EtMIC3 (green) and EtAMA1 (red) in fixed and permeabilised invading E. tenella sporozoites on MDBK monolayers. Blue counterstain is DAPI. Both EtMIC3 and EtAMA1 label necklace- like structures present at the junction between the invading parasite and the host cell; the labelling is closely associated but does not co-localize. The top panels indicate the position of the parasite-host junction and the bottom panels give a clear cross sectional image of the non-overlapping staining patterns. The bar represents 2 µm in top panels, 1 µm in bottom panels. (b) Immunofluorescence localisation (left panel) of EtMIC3 (red) and EtAMA1 (green) in fixed, unpermeabilized invading E. tenella sporozoite (left image) and combined with DIC (right panel). The parasite is invading from left to right and EtMIC3 is found on the surface at the region of the moving junction (arrowheads) and as a trail on the host cell surface. EtAMA1 was not detected. Blue counterstain is DAPI, N indicates parasite nucleus. The bar represents 2 µm. (c) Immunofluorescent localization of EtMIC3 (green) and EtMIC5 (red) in permeabilised E. tenella sporozoite attached to MDBK cell in culture. EtMIC3 is concentrated at the extended conoid and the apical surface of the sporozoite whereas EtMIC5 is slightly posterior to this region. The bar represents 3 µm.
Figure 3.
EtMIC3 binds cell surface sialylated carbohydrates.
(a) Western blot analysis of a sporozoite lysate (lys) and the cell-bound (CB) fraction of the lysate after incubation on MDBK cells. Proteins were detected using specific antisera to EtMIC1, EtMIC2, EtMIC3 and EtMIC4. All four proteins were detected in the sporozoite lysate, but only EtMIC3 was detected in the cell bound protein fraction of this assay this. It is useful to note that cell binding activity has been reported for the EtMIC4/5 complex [11], but the assay protocol used in this work is less sensitive and unable to detect it. The faint band below EtMIC3 is likely to be a break down product. (b) Dose-dependent bindg of a sporozoite lysate containing EtMIC3 to fixed MDBK cell monolayers determined by cell based ELISA. Sporozoite lysate was diluted in PBS at a range of concentrations from 2 mg/ml - 0.001 mg/ml. The lysate was incubated with gluteraldehyde fixed MDBK cell monolayers. Cells were washed to remove unbound protein and binding of EtMIC3 was determined by ELISA. Error bars indicate standard deviations. (c) Western blot analysis of EtMIC3 from a sporozoite lysate (20×106/ml) within cell bound (CB) or unbound (UB) fractions of a sporozoite lysate in the presence of neuraminidase (c), (d) fetuin or asialofetuin.
Figure 4.
EtMIC3 targets sialic acid bearing glycans on host cells.
(a) Western blot analysis of a cell bound fraction of EtMIC3 from a sporozoite lysate (20×106/ml) in presence of increasing amounts of either α2–3 sialyllactose or α2–6 sialyllactose. (b) Western blot analysis of a cell bound fraction of EtMIC3 from a sporozoite lysate (20×106/ml) in presence of increasing amounts of lectins from Sambucus nigra (SNA) or Maackia amurensis (MAA). (c) Histology analysis of carbohydrate presentation within chicken intestine. Alkaline phosphatase staining of histological sections of chicken intestinal tissue derived from the upper, mid and lower intestine and the caecum following incubation with plant lectins SNA or MAAII, with recombinant EtMIC3-MAR5 protein or with thioredoxin as a control protein (C). EtMIC3 and MAAII bind abundantly to epithelial cells of the caecum indicating that the preferred binding site of EtMIC3 is in the region of the intestine that expresses a high level of α2–3 sialylated glycans.
Figure 5.
Carbohydrate microarray analyses of MAR domains.
Carbohydrate microarray analyses of recombinant EtMIC3-MAR1b (a), EtMIC3-MAR5 (b) and TgMIC1-MARR (c) using microarrays of 115 lipid-linked oligosaccharide probes. Numerical scores of the binding signals are means of duplicate spots at 5 fmol/spot (with error bars). The various types of terminal sialic acid linkage are indicated by the coloured panels as defined at the bottom of the figure. The list of probes and their sequences and binding scores are in Table 1.
Table 1.
Comparison of the binding intensities elicited by selected glycan probes in carbohydrate microarrays with EtMIC3-MAR5 and TgMIC1-MARR.
Figure 6.
Three-dimensional structure of the MAR1b domain from EtMIC3.
(a) Superposition of EtMIC3-MAR1b (red; PDB code 2LBO) on the MAR2 domain from TgMIC1 (PDB code 2JH1; cyan) showing the position of the disulfide bonds and ‘MAR1 insertion’ [4], [5]. Left and right images represent views from two orientations (Figure S1 and S4). (b) Superposition of EtMIC3-MAR1b (red; PDB code 2LBO) on the MAR2 domain from TgMIC1 (PDB code 2JH1; cyan) in complex with Siaα2–3Gal. For clarity only the sialic acid units are shown for both structures. Additional side-chain contacts from the MAR1 domain are shown in red. Conserved binding site HxT side chains are shown in purple. Left and right images represent views from two orientations (Figure S4).
Table 2.
Structural statistics from the solution structure calculation for EtMIC3.
Figure 7.
Sporozoite invasion of MDBK cells can be inhibited by sialic acid competitors.
Freshly excysted sporozoites of E. tenella were incubated for 10 mins at room temperature with varying concentrations of sialylated molecules, and then allowed to invade semi-confluent monolayers of MDBK cells for 15 mins at 41°C. Cells were then fixed in methanol, stained in haematoxylin and eosin and the number of intracellular sporozoites enumerated.
Figure 8.
Vaccination trials with MARR from EtMIC3.
(a) Counts for individual birds for each antigen are shown as overlapping columns and the average shown as a separate column with error bar. Immunization with recombinantly-expressed EtMIC3-MAR1c fusion protein resulted in an overall reduction in oocyst output after challenge of 54% compared to treatment with the thioredoxin fusion protein alone (See Table S3). (b) Counts for individual birds for each antigen are shown as overlapping columns and the average shown as a separate column with error bar. DNA immunization with pcDNA3.1 vector containing EtMIC3-MAR5 or EtMIC3-MAR1c resulted in an overall reduction in oocyst outputs after challenge of 48 and 51% respectively compared to treatment with the pcDNA3.1 vector alone (See Table S3).