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

Sonja Frölich,Michael Wallach

doi:10.1038/srep29030

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.

Highlights

To acquire nutrients needed for development into an infectious cyst form, remains an open question
Autofluorescence increases over time in developing E. maxima macrogametocytes, which is indicative of enzyme-mediated formation of dityrosine bonds
We have investigated the viability of freshly extracted sexual stages by observing and measuring changes in blue autofluorescence over time using a Nikon Ti microscope equipped with the automatic focus correction system (PFS), a LED-based epi-fluorescence illuminator and a high sensitivity cooled (CCD) camera, which allows capture of clear, high contrast images of low light fluorescence signals

Summary

Introduction

To acquire nutrients needed for development into an infectious cyst form, remains an open question. Studies of the encapsulation process in E. maxima-infected enterocytes in situ suggested that surface membranes of developing macrogametocytes may contain micropores[18]. These membranous structures were proposed to play a role in nutrient intake. We investigated interactions of developing E. maxima sexual stages with negatively charged carboxylated-modified polystyrene nanoparticles (NPs) of 40 nm and 100 nm in size, as representatives of typical endocytotic cargoes widely used to study cell biology of uptake mechanisms[19,20,21,22,23]. The uptake of the model polystyrene NPs was investigated in the presence and absence of pharmacological inhibitors of different aspects of endocytosis. The results reported here indicate that there are at least two pathways by which nanoparticles can gain access to the intracellular parasite, and that nutrient acquisition requires an intact and active cytoskeleton

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