Use of fluorescent nanoparticles to investigate nutrient acquisition by developing Eimeria maxima macrogametocytes

Abstract

The enteric disease coccidiosis, caused by the unicellular parasite Eimeria, is a major and reoccurring problem for the poultry industry. While the molecular machinery driving host cell invasion and oocyst wall formation has been well documented in Eimeria, relatively little is known about the host cell modifications which lead to acquisition of nutrients and parasite growth. In order to understand the mechanism(s) by which nutrients are acquired by developing intracellular gametocytes and oocysts, we have performed uptake experiments using polystyrene nanoparticles (NPs) of 40 nm and 100 nm in size, as model NPs typical of organic macromolecules. Cytochalasin D and nocodazole were used to inhibit, respectively, the polymerization of the actin and microtubules. The results indicated that NPs entered the parasite at all stages of macrogametocyte development and early oocyst maturation via an active energy dependent process. Interestingly, the smaller NPs were found throughout the parasite cytoplasm, while the larger NPs were mainly localised to the lumen of large type 1 wall forming body organelles. NP uptake was reduced after microfilament disruption and treatment with nocodazole. These observations suggest that E. maxima parasites utilize at least 2 or more uptake pathways to internalize exogenous material during the sexual stages of development.

Figure 1. Live cell filming of freshly harvested gametocytes.

(a) Micrographs of three time-points from a 3 hour time-lapse sequence of freshly extracted sexual stages that were acquired every 10 minutes using a combination of differential interference contrast DIC (top) and fluorescence microscopy (bottom). The cells were exposed to UV light (ex: 385 nm) to reveal the distribution of autofluorescent proteins in the macrogametocytes at early-, mid-, and late-stages of development, and in forming tissue oocysts. Note the presence of autofluorescing material in the wall of tissue oocyst at t0. As the sequence progresses, the macrogametocytes begin to exhibit blue autofluorescence (t1), at a different rate, which initially appears concentrated in the central cytoplasm, and spreads outwards to cover the entire macrogametocyte (t2). (b) Increase in blue autofluorescence intensity over time in live sexual stages. Autofluoresence intensities were measured by a monochromatic detector, CCD camera and ex: 385 nm. High resolution temporal information and relative intensity were recorded and time points 0, 20, 40, 80, 120, 160 and 180 minutes plotted. Scale bar = 5 μm. See also Movie 1 for a complete time-lapse experiment.

Figure 2. Nanoparticle uptake by freshly harvested gametocytes.

Freshly harvested sexual stages incubated with 40 nm (a) or 100 nm (b) particles for 1 h at 37 °C. LSM micrographs demonstrate beads of 100 nm, but not of 40 nm localise to the type 1 wall forming body vesicles. Inserts represent close up intracellular events of 40 nm and 100 nm particles in macrogametocytes. (c) Extracted sexual stages exposed to nanoparticles for 1 h at 4  °C. Note the absence of intracellular events and accumulation of the particles (green) at the surface of the parasite surrounded by the host cell. Scale bars correspond to 5 μm.

Figure 3. Effect of cytoskeletal inhibitors on uptake of nanoparticles.

(a) Fluorescent confocal 2D micrographs of fresh macrogametocytes incubated in the presence of fluorescent 40 nm nanoparticles prior to and after the addition of cytoskeletal inhibitors, cytochalsin D and nocodaloze. Intracellular 40 nm NPs are shown in green. Scale bar: 5 μm. (b) Quantification of foci containing intracellular 40 nm particles in cells with and without cytochalasin D and nocodazole (mean ± range, n = 3, P = two-tailed t-test). (c) Confocal micrographs showing uptake of 100 nm particles (particles: green) in untreated and drug-treated macrogametocytes. Scale bar: 5 μm. (d) Quantification of foci containing intracellular 100 nm particles in cells with and without cytochalasin D and nocodazole. Mean ± range, n = 3, P = two-tailed t-test.

Figure 4. Laser scanning microscopy imaging revealed colocalisation of 100 nm particles with large type 1 wall forming bodies.

(a) Distribution of internalized 100 nm NPs (green) and the anti-APGA antibodies (red) using conventional 2D confocal microscopy. Scale bar: 5 μm. Enlarged panels depict localisation of 100 nm beads (green) to anti-APGA positive type 1 wall forming bodies. Bottom: 3D computer reconstruction and isosurface-rendered images were generated from 5–20 confocal z-stacks of 0.5 μm thick optical cross sections. Quadrants = 5 μm. (b) Orthogonal cross-sections along the x axis highlight the spatial distribution of internalized 100 nm particles. The lumen of type 1 wall forming body vesicles (demarked by red antibody labelling) appears filled with numerous green 100 nm particles. Scare bar: 5 μm.

Figure 5. High resolution spatial imaging of green fluorescent 40 nm particles inside the mature macrogametocyte and forming tissue cysts counter-stained with Evans blue.

(a) 3D projection and corresponding isosurface-rendered images of the mature microgametocytes were generated from confocal z-stacks of 0.5 μm thick optical cross sections. The type 1 wall forming body vesicles are highlighted by Evans blue stain (red), as described in Materials and Methods. Cellular distribution of 40 nm nanoparticles is shown in green. Enlarged box shows magnified cellular compartment illustrating intracellular distribution of 40 nm particles. Quadrants = 10 μm. (b) Three-dimensional distribution of 40 nm (green) beads in an early-stage oocyst labelled with Evans blue (red). Cellular distribution of 40 nm nanoparticles is shown in green. Close up top view of the region depicted in (b) showing 40 nm particles (green) localised to the cell surface and within the cytoplasm, with nanoparticles accumulated in small aggregates. Orthogonal view of an image in (b) showing the luminal to basal surface along the Z axis (z slicing 0.5 μm). Micrographs represent the intracellular distribution of 40 nm particles. Quadrants = 10 μm. See Movies 2 and 3 for 3D spatial distribution of 40 nm NPs in mature macrogametocytes.