Development of the oocyst wall in Eimeria maxima and biochemical analysis of gametocyte wall forming bodies
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Eimeria is a cyst-forming intracellular parasite that causes the economically important disease, coccidiosis, in intensely reared broiler chickens worldwide. The ability of the Eimeria parasite to replicate very rapidly and to synthesize an impenetrable, highly resistant oocyst wall, allows it to build up to very large numbers in the litter of broiler flocks. The molecular machinery involved in the assembly of the oocyst wall is housed in the two types of wall forming bodies (WFB1 and WFB2) of the sexual stage parasites (macrogametocytes). The current project aimed to expand our understanding of the fundamental mechanisms involved in oocyst wall formation by: (1) characterising the morphological changes involved in oocyst wall assembly during parasite development; (2) developing a method to isolate gametocyte WFBs in order to characterise their molecular composition; and (3) studying the nature and characterizing the mechanisms of nutrient acquisition in developing E. maxima gametocytes in vitro. Extracted macrogametocytes were stained using cytochemical and immune-labelling methods, and morphological changes of the developing zygote characterised by bright-field, scanning electron and 3D confocal microscopy. Additionally, the WFBs of macrogametocytes were enriched by subcellular fractionation and fractions containing these organelles were analysed by microscopy, western blot and label-free quantitative shotgun proteomics. Data from these studies has shown that gametocytes and early stage oocysts contain surface pores and are capable of actively taking up and internalizing nano beads via endocytosis. In addition, microscopic analyses shows that E. maxima is selective in compartmentalizing neutral lipids to the type 1, and glycoproteins to the type 2 wall forming bodies during gametocytogenesis. Furthermore, it became possible to visualise both neutral lipids and glycoproteins during outer and inner oocyst wall formation. Thus, a model of outer oocyst wall formation was proposed and suggests that neutral lipids found in the WFB1s are translocated to the parasite’s surface where they deposit their cargo via exocytosis. The released molecules fuse with the parasite’s limiting membrane for incorporation into the neutral lipid rich outer oocyst wall. Finally, biochemical and proteomic methods were employed to identify and analyse vesicular trafficking proteins and other putative regulators of endocytosis and transport. The results reported here reveal valuable insights into the mechanisms by which the parasite is able to acquire nutrients essential for development, transport organelles and at the same time synthesise the impervious oocyst wall.
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