Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites
Introduction
The unifying feature of the zoite stages of the phyllum Apicomplexa consists of a set of secretory organelles, namely the rhoptries, micronemes and dense granules. These organelles are believed to play a major role in the host cell binding and invasion as they disappear during stage conversion from the zoite to the trophozoite. Cryptosporidium parvum is a member of this group of parasites. It has evolved into an important pathogen of humans and mammals which causes severe and prolonged diarrhoea in the immunocompromised host[1]. In immunocompetent individuals the disease is self-limited and symptoms can range from mild to overwhelming enteritis[2]. No reliable antibiotic therapy has been described. Due to the small size and the resistance of the transmission stage, the oocyst, to chemical treatment, C. parvum constitutes a major threat to the water supplies used for human consumption[3]. The infective stages of the parasite, the sporozoites and merozoites, show a narrow host cell specificity, with the epithelial cells of the small intestine being the major cell type for parasite development. In immunocompromised hosts, infections of the epithelia of the bile duct and the lung have been described.
The initial steps in the infection with C. parvum involve the recognition and binding to the intestinal epithelium and the parasite-driven invasion of the host cell. Studies on other members of the Apicomplexa suggest that a fixed sequence of events takes place, with the secretory organelles playing a major role. Secretion of the content of micronemes appears to happen during the gliding activity of the zoites and the host cell binding. During invasion the rhoptries release their content, while components of the dense granules are secreted when the parasite establishes itself within the host cell (reviewed in[4]).
As the molecular basis of the host cell invasion by C. parvum is only poorly understood, we attempted to isolate the apical complex organelles from sporozoites of this parasite after cell disruption and subcellular fractionation by density gradient ultracentrifugation. The data obtained from the analysis of these components should provide the necessary prerequisites for an in-depth study of the host–parasite relationship on a molecular basis.
Section snippets
Parasites
Oocysts of C. parvum passaged in newborn calves (Iowa strain) were purchased from Ms Patricia Mason (Pleasant Hill Farm, Troy, Idaho). According to the supplier, calf faeces were passed through a coarse screen to remove solids and were 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−1 penicillin and 1000 μg ml−1 streptomycin.
Intact
Results
Due to the limited availability of parasite material, structural analysis of the subcellular components of C. parvum sporozoites was performed by TEM of negatively stained specimen. However, for comparison and interpretation of the results from the negatively stained specimen we performed TEM on thin sectioned whole parasites. Fig. 1Fig. 2Fig. 3Fig. 4 show partially excysted oocysts and sporozoites. In Fig. 2. the residual body of the oocyst, containing a lipid body and amylopectin granules of
Discussion
This paper presents the first description of the subcellular fractionation of the components of C. parvum sporozoites. Organelles of zoite stages from other Apicomplexa species have been isolated and characterised. An early work on the zoites of Sarcocystis tenella involving cell disruption by a `French press' and fractionation of the homogenate by ultracentrifugation in sucrose density gradients successfully showed the preparation of highly enriched micronemes and dense granules[5]. In initial
Acknowledgements
We would like to acknowledge the skilled technical assistance provided by Inka Kneib and Elisabeth Sehn. We thank Professor Alain Bonnin (Université de Bourgogne, Dijon) for culture supernatants of hybridoma lines. Electron microscope facilities were made available by Professor Albrecht Fischer, Institute of Zoology, University of Mainz. F.P. is supported by grants from the German Ministry of Research and Technology (BMFT)—Infection Program and the German Research Foundation (DFG).
References (19)
- et al.
Apical organelles and host-cell invasion by Apicomplexa
Int J Parasitol
(1998) - et al.
Characteristic proteins of micronemes and dense granules from Sarcocystis tenella zoites (Protozoa Coccidia)
Mol Biochem Parasitol
(1980) - et al.
Characterization of microneme proteins of Toxoplasma gondii
Mol Biochem Parasitol
(1991) - Griffiths JK. Human cyptosporidiosis: epidemiology, transmission, clinical disease, treatment, and diagnosis. In:...
- et al.
A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply
N Engl J Med
(1994) Cryptosporidiosis: an emerging, highly infectious threat
Emerg Infect Dis
(1997)- Harris JR, Agutter PS. A negative staining study of human erythrocyte ghosts and rat liver nuclear membranes. J...
- Harris JR. Negative staining and cryoelectron microscopy. Oxford: BIOS Scientific Publishers Ltd,...
Cleavage of structural proteins during the assembly of the head of bacteriophage T4
Nature
(1970)
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Parasitology meets cryo-electron tomography – exciting prospects await
2022, Trends in ParasitologyCitation Excerpt :The apical complex houses two of the three types of secretory organelle found in these parasites – small rod-shaped micronemes (50–100 nm wide and 150–300 nm long) and larger club-shaped rhoptries (the third kind of secretory organelle being the dense granules, which are secreted post-invasion to help with establishing infection) [84]. The number of rhoptries varies across different apicomplexan species [85–87]. The rhoptry proteins are subcompartmentalized into the neck and bulb regions; the neck proteins participate in host cell invasion while bulb proteins modulate host cell functions post-invasion.
Statistical comparison of excystation methods in Cryptosporidium parvum oocysts
2016, Veterinary ParasitologyCitation Excerpt :These six methods have not been compared before and therefore this study could be a help for other researchers. From the results of the present study, we can conclude that the most efficient excystation protocols investigated were those described by Rasmussen et al. (1993), Petry and Harris (1999) and Mead et al. (1990). In all these methods, sodium hypochlorite is used as a pre-incubation step in comparison with the remaining, less efficient protocols.
Electron microscopic observation of the early stages of Cryptosporidium parvum asexual multiplication and development in in vitro axenic culture
2016, European Journal of ProtistologyCitation Excerpt :In the present study, the fine features of sporozoites were similar to previous descriptions of the sporozoites of C. parvum and other species (Uni et al., 1987; Lumb et al., 1988; Tetley et al., 1998; Petry and Harris, 1999; O’Hara et al., 2005). Consistent with former TEM studies, the sporozoites had only a single rhoptry (Lumb et al., 1988; Tetley et al., 1998; Petry and Harris, 1999; O’Hara et al., 2005; Valigurová et al., 2007; Fayer, 2008; Melicherová et al., 2014), which is in contrast to the merozoites, which possessed several rhoptries (Current and Reese, 1986; Valigurová et al., 2007; Fayer, 2008; Melicherová et al., 2014). The obvious vesicular nature of the ‘crystalloid body spheres’ (CBs) in sporozoites has been reported, and the size of the vesicular bodies was in agreement with Lumb et al. (1988) (30–37 nm) but was half the size observed by Petry and Harris (1999) (45–60 nm) and twice the size of those described by Keithly et al. (2005) (16–25 nm).
Transport processes in Plasmodium falciparum-infected erythrocytes: Potential as new drug targets
2002, International Journal for Parasitology