Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites

doi:10.1016/S0020-7519(99)00080-6

International Journal for Parasitology

Volume 29, Issue 8, August 1999, Pages 1249-1260

https://doi.org/10.1016/S0020-7519(99)00080-6 Get rights and content

Abstract

Sporozoites of the apicomplexan parasite Cryptosporidium parvum were subjected to cell disruption and subcellular fractionation using a sucrose density step gradient. With this procedure, highly enriched preparations of the parasite membrane, the micronemes, dense granules and amylopectin granules were produced. No separate fraction containing rhoptries was obtained, however this organelle was found in defined fractions of the gradient, still associated with the apical tip of the sporozoites. Using negative staining, the internal structure of the micronemes was revealed by transmission electron microscopy. Micronemes and dense granules showed characteristic protein compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The micronemes contained three major proteins of approximately 30, 120 and 200 kDa and the dense granules contain five major proteins in the 120–180 kDa range.

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).

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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.
Cryo-electron tomography (cryo-ET) is a cryo-electron microscopy (EM) approach that allows 3D imaging of cellular structures in near-native, frozen-hydrated conditions with molecular resolution. Continued development of technologies, including direct electron detectors, phase plates, and energy filters, has improved the information yield from cellular samples, which is further extended by newly developed workflows for data collection and analyses. Moreover, advanced sample-thinning techniques, such as cryogenic focused ion-beam (cryo-FIB) milling, provide access to parasitic events and structures that were previously inaccessible for cryo-ET. Cryo-ET has therefore become more versatile and capable of transforming our understanding of parasite biology, particularly that of apicomplexans. This review discusses cryo-ET’s implementation, its recent contributions, and how it can reveal pathogenesis mechanisms in the near future using apicomplexans as a case study.
Statistical comparison of excystation methods in Cryptosporidium parvum oocysts
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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.
Excystation of sporozoites of Cryptosporidium parvum from oocysts is essential for successful in vitro assays. It has also been traditionally used as a measure for oocyst viability and infectivity. Laboratories use various excystation protocols so there is a need to clarify which method is the best. In this study, six different protocols for in vitro excystation of C. parvum oocysts were compared to find the most efficient excystation method (expressed as percentage excystation). Tested protocols differed in chemical pre-incubation steps, excystation media or time of incubation.
There were significant differences in percentage of excysted oocysts among groups excysted by different methods. There were also significant differences in percentage of excysted oocysts between methods using pre-incubation with sodium hypochlorite and those without. The other variables examined; the presence of trypsin, kind of excystation medium and the incubation time, did not show statistical differences in percentage excystation among groups.
Pre-incubation steps which included sodium hypochlorite, enhancing the permeability of the oocysts were found to increase the excystation ratio and methods using this step were the most effective.
Electron microscopic observation of the early stages of Cryptosporidium parvum asexual multiplication and development in in vitro axenic culture
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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).
The stages of Cryptosporidium parvum asexual exogenous development were investigated at high ultra-structural resolution in cell-free culture using transmission electron microscopy (TEM). Early C. parvum trophozoites were ovoid in shape, 1.07 × 1.47 μm² in size, and contained a large nucleus and adjacent Golgi complex. Dividing and mature meronts containing four to eight developing merozoites, 2.34 × 2.7 μm² in size, were observed within the first 24 h of cultivation. An obvious peculiarity was found within the merozoite pellicle, as it was composed of the outer plasma membrane with underlying middle and inner membrane complexes. Further novel findings were vacuolization of the meront's residuum and extension of its outer pellicle, as parasitophorous vacuole-like membranes were also evident. The asexual reproduction of C. parvum was consistent with the developmental pattern of both eimerian coccidia and Arthrogregarinida (formerly Neogregarinida). The unique cell-free development of C. parvum described here, along with the establishment of meronts and merozoite formation, is the first such evidence obtained from in vitro cell-free culture at the ultrastructural level.
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Cryptosporidium parvum is a protozoan parasite (Apicomplexa) that causes gastrointestinal disease in animals and humans. Whereas immunocompetent hosts can limit the infection within 1 or 2 weeks, immunocompromised individuals develop a chronic, life-threatening disease. The importance of the adaptive cellular immune response, with CD4+ T-lymphocytes being the major players, has been clearly demonstrated. Several non-adaptive immune mechanisms have been suggested to contribute to the host defence, such as interferon-γ (IFN-γ) from NK cells, certain chemokines, β-defensins and pro-inflammatory cytokines, but the influence of the complement systems has been less well studied. We analysed the in vitro binding and activation of the human and mouse complement systems and tested the susceptibility to infection in complement-deficient mouse strains. We found that C. parvum can activate both the classical and lectin pathways, leading to the deposition of C3b on the parasite. Using real-time PCR, parasite development could be demonstrated in adult mice lacking mannan-binding lectin (MBL-A/C^−/−) but not in mice lacking complement factor C1q (C1qA^−/−) or in wild type C57BL/6 mice. The contribution of the complement system and the lectin pathway in particular to the host defence against cryptosporidiosis may become apparent in situations of immunodeficiency such as HIV infections or in early childhood.
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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.
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Plasmodium falciparum infection induces alterations in the transport properties of infected erythrocytes that have recently been defined using electrophysiological techniques. Mechanisms responsible for transport of substrates into intraerythrocytic parasites have also been clarified by studies of three substrate-specific (hexose, nucleoside and aquaglyceroporin) parasite plasma membrane transporters. These have been characterised functionally using the Xenopus laevis oocyte heterologous expression system. The same expression system is currently being used to define the function of parasite ‘P’ type ATPases responsible for intraparasitic [Ca²⁺] homeostasis. We review studies on these transport processes and examine their potential as novel drug targets.

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International Journal for Parasitology

Abstract

Introduction

Section snippets

Parasites

Results

Discussion

Acknowledgements

References (19)

Apical organelles and host-cell invasion by Apicomplexa

Int J Parasitol

Characteristic proteins of micronemes and dense granules from Sarcocystis tenella zoites (Protozoa Coccidia)

Mol Biochem Parasitol

Characterization of microneme proteins of Toxoplasma gondii

Mol Biochem Parasitol

A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply

N Engl J Med

Cryptosporidiosis: an emerging, highly infectious threat

Emerg Infect Dis

Cleavage of structural proteins during the assembly of the head of bacteriophage T4

Nature

Cited by (36)

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Statistical comparison of excystation methods in Cryptosporidium parvum oocysts

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Binding and activation of human and mouse complement by Cryptosporidium parvum (Apicomplexa) and susceptibility of C1q- and MBL-deficient mice to infection

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

Transport processes in Plasmodium falciparum-infected erythrocytes: Potential as new drug targets