Elsevier

Micron

Volume 34, Issue 2, February 2003, Pages 65-78
Micron

Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile apicomplexan organelle

https://doi.org/10.1016/S0968-4328(03)00020-9Get rights and content

Abstract

From an EM study of thin sections, the rod-like microneme organelles within conventionally glutaraldehyde fixed Cryptosporidium parvum sporozoites have been shown to undergo a shape change to a more spherical structure when the sporozoites age in vitro for a period of ∼12 to 24 h. This correlates with the shape change of intact sporozoites, from motile hence viable thin banana-shaped cells to swollen pear-shaped cells, shown by differential interference contrast light microscopy of unstained unfixed and glutaraldehyde-fixed samples, as well as by thin section EM of fixed sporozoites. From negatively stained EM specimens of unfixed and fixed sporozoites the cellular shape change has been confirmed as has the rod to sphere micronemal shape change. Intact micronemes released directly from sporozoites exclude negative stain and appear as smooth-surfaced electron transparent particles. Biochemically purified rod-shaped C. parvum micronemes are shown to be fragile organelles that inevitably undergo variable damage during isolation, storage and subsequent specimen preparation for EM study. In the absence of glutaraldehyde fixation, damaged micronemes allow the negative stain to enter and loose their contents and during storage undergo a rod-to-sphere shape transformation. Glutaraldehyde-fixed micronemes maintain the rod shape; intact fixed micronemes still exclude negative stain but damaged micronemes reveal a complex quasi-helical arrangement of internal protein within the rod-like micronemes. Loss of this internal organized structure appears to be responsible for the micronemal shape change. This interpretation has been advanced from mutually supportive data obtained from cryoelectron microscopy of unstained vitrified samples, conventional air-dry negative staining and cryo-negative staining. Attempts to biochemically solubilize the micronemal content by lysis and ultrasonication, and separate it from the micronemal membranes, have so far met with limited success as the internal material tends to remain as a disorganized cluster of particles upon release.

Introduction

Much is now known about the organelles of the apicomplexan parasites (reviewed by Blackman and Bannister, 2001). More specifically, considerable attention has been devoted to the micronemes, small membrane-bounded organelles, often located immediately beneath the cell membrane close to the anterior end of the zoite stages of the parasite (Mehlhorn et al., 1988, Fayer et al., 1997, Petry and Harris, 1999). The secretory organelles, micronemes, rhoptries and dense granules, and structural elements such as the conoid and the polar rings form the apical complex, a characteristic feature of the whole phylum, hence its name Apicomplexa. The microneme proteins from the Apicomplexa are thought to be involved in the cellular invasion mechanism of the sporozoites, specifically in relation to sporozoite motility, secretion, cell attachment and penetration (Dubremetz et al., 1998, Klein et al., 1998, Langer and Riggs, 1999, Rabenau et al., 2001, Sonda et al., 2000, Watanabe et al., 2001). Most thin section TEM studies on sporozoites show the micronemes to be slightly elongated membrane-bounded structures, ∼60–80 nm in diameter (Bonnin et al., 1991, Bonnin et al., 2001, Heise et al., 1999, Lumb et al., 1988, McDonald et al., 1995, Scholtysek and Mehlhorn, 1970, Tzipori, 1988). Nevertheless, Tetley et al. (1998) using cryofixation rather than glutaraldehyde fixation, claimed that Cryptosporidium parvum micronemes are spherical rather than rod-like structures. Whilst chemical fixation of cells does not generally induce structural alterations to cellular organelles, such changes cannot be ruled out. Glutaraldehyde fixation of intact oocysts presents considerable problems, because of restricted penetration of the fixative through the relatively rigid impervious cyst wall (Harris and Petry, 1999), but it is generally agreed that in C. parvum oocysts four sporozoites cluster and bend around a central residual body, rich in lipid and carbohydrate. Upon excystation, the sporozoites are elongated banana-shaped cells, with a clearly defined apical end and polar rings, with a nucleus and crystalloid body located towards the opposite end.

Knowledge of micronemes from apicomplexan parasites has expanded by the growing volume of data from protein characterization, genome sequencing within both molecular biological and immunological projects. Although the available literature on micronemes dwells heavily upon the Eimeria, Plasmodium and Toxoplasma species, much medical, veterinary and biological interest also centres around C. parvum. C. parvum is a parasite of cattle and sheep, but it also infects man via contaminated drinking water and food. Whereas C. parvum infection leads to self-limiting diarrhoea in the immuno-competent host, patients with an impaired immune defence system are prone to develop a chronic disease. Thus, cryptosporidiosis is one of the associated opportunistic infections of HIV/AIDS and can contribute to the mortality of patients.

By negative staining of spontaneously disrupting unfixed C. parvum sporozoites, micronemes appear to be rod-like (Petry and Harris, 1999). Following controlled sporozoite disruption in a French press, we have also isolated intact C. parvum micronemes by sucrose gradient centrifugation and shown by SDS-PAGE that they contain a diverse yet characteristic population of proteins (Petry and Harris, 1999). The purity and integrity of the microneme fraction was assessed by transmission electron microscopy. C. parvum micronemes were found to be metabolically and osmotically labile organelles, often undergoing spontaneous lysis/disruption during conventional negative staining on carbon support films, unless pre-fixation with glutaraldehyde was included.

A more detailed ultrastructural study by conventional thin sectioning of C. parvum micronemes in situ has now been undertaken. Also, following microneme isolation an improved negative staining procedure on holey carbon support films has been used (Harris and Scheffler, 2002), as well as cryoelectron microscopy of unstained specimens (Adrian et al., 1984) and cryonegative staining (Adrian et al., 1998).

Section snippets

C. parvum oocysts and sporozoites

Oocysts of C. parvum (Iowa strain) passaged in new born calves were obtained from Patricia Mason (Pleasent Hill Farm, Troy, Idaho, USA). According to the supplier, calf faeces were passed through a coarse screen to remove solids and extracted twice with ethyl ether to remove lipids. Oocysts were concentrated by sucrose density centrifugation, washed and resuspended in PBS. The parasite suspension was stored at 4 °C in the presence of 1000 U/ml penicillin and 1000 μg/ml streptomycin. Intact

The total Cryptosporidium parvum excystate

Within intact C. parvum oocysts, the tightly packed sporozoites possess an elongated curved morphology (Fig. 1a), which is maintained following excystation as the characteristic banana-shaped sporozoite cell (Fig. 1b). However, whilst retaining this overall shape, viable sporozoites are very flexible when moving, as can be observed by light microscopy. Structural definition of the micronemes within the freshly excysted intact sporozoite shows that they have a tendency to stack as rows of short

Discussion

In order to obtain reproducible results the storage time of the parasite batches that were used for organelle preparation was kept to a minimum. As infected calves shed parasites over a period of approximately 7 days the starting material contains oocysts of variable ages. In some experiments batches from different animal passages had to be used, due to low yields, leading to even higher variation within oocyst preparation and subsequently of the microneme preparations. The prime parameter used

Acknowledgements

Part of this work has been supported by DFG grant SFB 490 C5 (FP). We acknowledge the skilled technical assistance of Inka Kneib and Elisabeth Sehn. Electron microscope facilities were made available by Professor Albrecht Fischer, Institute of Zoology, University of Mainz.

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