Application of the serial sectioning and tridimensional reconstruction techniques to the morphological study of the Plasmodium falciparum mitochondrion

The use of serial sectioning followed by tridimensional reconstruction is a convenient way to study the spatial morphology of any structure (cell or organelle). This method was applied to the study of organelles of Plasmodium falciparum (FCR3) and enabled clarification of morphological features of the mitochondrion. The mitochondrion is polymorphic; in single sections it may be rounded, elongated or branched in shape. Its matrix may be dense or transparent, and it may or may not possess cristae. The 3-D reconstruction indicated that the mitochondrion is single in P. falciparum. Its form varies according to the age of the trophozoite. It becomes branched, and each lobe of the mitochondrion follows a daughter nucleus during the formation of merozoites.     

The Plasmodium falciparum Maurer's clefts in 3D

In 1902, the German physician Georg Maurer discovered a dotted staining pattern within the cytoplasm of Plasmodium falciparum infected erythrocytes that, according to the tradition at the time, was named in his honour. The significance of Georg Maurer's discovery remained unrecognized for almost a century. Only recently are Maurer's clefts appreciated as a novel type of secretory organelle. Established by the malaria parasite within its host cell, Maurer's clefts play an essential role in directing proteins from the parasite to the erythrocyte surface. In this issue of Molecular Microbiology, Hanssen et al. report on the three dimensional structure of Maurer's clefts, as determined by electron tomography. The data presented suggest that Maurer's clefts are connected to both the parasitophorous vacuolar and the erythrocyte plasma membrane, however, no continuum exists that would allow lipids or proteins to freely flow between these three compartments. This seminal work, which stands in the tradition of Georg Maurer's original discovery, represents a milestone in our understanding of the structure and function of this fascinating organelle.     

Plasmodium falciparum: analysis of karyotype polymorphism using chromosome-specific probes

Chromosome size variation in Plasmodium falciparum has been examined using a double heterogenous pulse field gradient electrophoresis apparatus and a series of chromosome-specific probes. In the 11 different isolates analyzed the chromosomal markers always hybridized to the corresponding chromosome, indicating that translocations do not significantly contribute to chromosome size variations. Furthermore, despite probes specific for chromosomes 5 and 6 no evidence was obtained to support the hypothesis of a chromosome duplication involving these chromosomes. The double heterogenous electric field combined with longer pulse times allowed the genome to be resolved into a larger number of chromosomal bands and as a result permitted the more precise mapping of cloned genes.     

Three-dimensional reconstruction of a pathogenic yeast Exophiala dermatitidis cell by freeze-substitution and serial sectioning electron microscopy

The structure of a budding cell of the pathogenic yeast Exophiala dermatitidis was observed in three dimensions after freeze-substitution, serial ultrathin sectioning and computer reconstruction. The nucleus occupied about 10% of the cell volume. The spindle pole body was composed of two disk elements connected by an intervening midpiece, and occupied about 0.01% of the cell volume. The cell wall consisted of an inner transparent layer, a middle electron-opaque layer, and an outer fibrous layer. The mitochondria occupied about 10% of the cell volume. There were numerous mitochondria in the mother cell and the bud, but no 'giant mitochondrion' was seen. The ratio of mitochondrial volume within the bud to the mitochondrial volume of the cell was close to the ratio of bud:cell cytoplasmic volume. The results emphasize the importance of good cryofixation for 'perfect' preservation of yeast cell structure.     

The plastid in Plasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis

The plastid in Plasmodium falciparum asexual stages is a tubular structure measuring about 0.5 micron x 0.15 micron in the merozoite, and 1.6 x 0.35 microns in trophozoites. Each parasite contains a single plastid until this organelle replicates in late schizonts. The plastid always adheres to the (single) mitochondrion, along its whole length in merozoites and early rings, but only at one end in later stages. Regions of the plastid are also closely related to the pigment vacuole, nuclear membrane and endoplasmic reticulum. In merozoites the plastid is anchored to a band of 2-3 subpellicular microtubules. Reconstructions show the plastid wall is characteristically three membranes thick, with regions of additional, complex membranes. These include inner and outer membrane complexes. The inner complex in the interior lumen is probably a rolled invagination of the plastid's inner membrane. The outer complex lies between the outer and middle wall membranes. The interior matrix contains ribosome-like granules and a network of fine branched filaments. Merozoites of P. berghei and P. knowlesi possess plastids similar in structure to those of P. falciparum. A model is proposed for the transfer of membrane lipid from the plastid to other organelles in the parasite.     

Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue

Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.     

The production of antigens by Plasmodium falciparum in vitro

La, Lb, R¹ and S-antigens in extracts of human and Aotus monkey blood infected with Plasmodium falciparum were inactivated by several proteolytic enzymes but were not affected by nucleases and selected carbohydrases. S-antigens also survived oxidation by periodate under conditions which inactivated a known carbohydrate-associated antigen. It was concluded that the L, R and S-antigens are proteins.Autoradiographic studies of extracts of parasitized red cells which had been maintained in vitro in the presence of radioactive amino acids, showed that various La, Lb and R-antigens were labelled but S-antigens were not. The incorporation of labelled amino acids into antigens derived from parasitized mature mammalian red cells was taken as evidence that these antigens were of parasite origin whcreas the unlabelled S-antigens might be of host origin.     

Accuracy of computer-aided geometric 3D reconstruction based on histological serial microgrinding preparation

Purpose: For our research on computer-optimised and automated cochlear implant surgery, we pursue a model-based approach to overcome the limitations of currently available clinical imaging modalities. A serial cross section preparation procedure has been developed and evaluated concerning accuracy to serve for modelling of a digital anatomic atlas to make delicate soft tissue structures available for pre-operative planning.

Methods: A special grinding tool was developed allowing the setting of a specific amount of abrasion as equidistant slice thickness was considered a crucial step. Additionally, each actual abrasion was accurately measured and used during three-dimensional reconstruction of the serial cross-sectional images obtained via digital photo documentation after each microgrinding step. A well-known reference object was prepared using this procedure and evaluated in terms of accuracy.

Results: Reconstruction of the whole sample was achieved with an error less than 0.4%, and the edge lengths in the direction of abrasion could be reconstructed with an average error of 0.6 ± 0.3 mm; both prove the realisation of equidistant abrasion. Using artificial registration fiducials and a custom-made algorithm for image alignment, parallelism and rectangularity could be preserved with average errors less than 0.4° ± 0.3°.

Conclusion: We present a systematic, practicable and reliable method for the geometrically accurate reconstruction of anatomical structures, which is especially suitable for the middle and inner ear anatomy including soft tissue structures. For the first time, the quality of such a reconstruction process has been quantified and successfully proven for its usability.     

The karyotype and ultrastructural characteristics of spontaneous preimplantation mouse parthenotes

Karyotypic and light and electron microscopical analyses were made of spontaneous preimplantation mouse parthenotes from the LT/Sv inbred strain. It was found that the activated oocyte and developing embryos were diploid. We believe that diploidization is achieved by the oogonium undergoing a premeiotic mitosis without cytokinesis followed by two meiotic divisions, thus producing diploid parthenotes. The developmental events with respect to membrane specialization, such as junctional complexes, were similar to those observed in fertilized embryos. A unique feature of the developing parthenote was the failure of the mitochondria to change during the morula stage. The mitochondria retained a few irregularly oriented cristae rather than many transversely oriented ones observed in morulae developing from fertilized eggs. The significance of this observation is discussed.     

The mitochondrion of Plasmodium falciparum visualized by rhodamine 123 fluorescence

Rhodamine 123 (Rh123) has been used to probe the functional status of the mitochondrion present within the asexual, intraerythrocytic stages of the malarial parasite Plasmodium falciparum. This cationic fluorescent dye accumulates specifically in negatively charged cellular compartments, such as mitochondria. Using epifluorescence microscopy the development of what appears to be a single mitochondrion has been followed through the intraerythrocytic cycle. Mitochondrial development progresses from a fine thread-like organelle that becomes longer and eventually branched. Each daughter merozoite receives a branch or piece of the parent organelle. Cytoplasmic Rh123 accumulation was also observed, indicating that there exists a transmembrane potential across the outer plasma and parasitophorous vacuolar membranes of the parasite. The effects of uncouplers (protonophores), ionophores, and inhibitors were examined by monitoring Rh123 accumulation and retention. Our results demonstrate that the mitochondrion of P. falciparum actively maintains a high transmembrane potential, the function of which is as yet undefined.     

The putative mitochondrial genome of Plasmodium falciparum

Intraerythrocytic stages of mammalian malarial parasites employ glycolysis for energy production but some aspects of mitochondrial function appear crucial to their survival since inhibitors of mitochondrial protein synthesis and electron transport have antimalarial effects. Investigations of the putative mitochondrial genome of Plasmodium falciparum have detected organellar rRNAs and tRNAs encoded by a 35 kb circular DNA. Some features of the organization and sequence of the rRNA genes are reminiscent of chloroplast DNAs. The 35 kb DNA also encodes open reading frames for proteins normally found in chloroplast but not mitochondrial genomes. An apparently unrelated 6 kb tandemly repeated element which encodes two mitochondrial protein coding genes and fragments of rRNA genes is also found in malarial parasites. The malarial mitochondrial genome thus appears quite unusual. Further investigations are expected to provide insights into the possible functional relationships between these molecules and perhaps their evolutionary history.     

The Dynamics of Natural Plasmodium falciparum Infections

Background

Natural immunity to Plasmodium falciparum has been widely studied, but its effects on parasite dynamics are poorly understood. Acquisition and clearance rates of untreated infections are key elements of the dynamics of malaria, but estimating these parameters is challenging because of frequent super-infection and imperfect detectability of parasites. Consequently, information on effects of host immune status or age on infection dynamics is fragmentary.

Methods

An age-stratified cohort of 347 individuals from Northern Ghana was sampled six times at 2 month intervals. High-throughput capillary electrophoresis was used to genotype the msp-2 locus of all P. falciparum infections detected by PCR. Force of infection (FOI) and duration were estimated for each age group using an immigration-death model that allows for imperfect detection of circulating parasites.

Results

Allowing for imperfect detection substantially increased estimates of FOI and duration. Effects of naturally acquired immunity on the FOI and duration would be reflected in age dependence in these indices, but in our cohort data FOI tended to increase with age in children. Persistence of individual parasite clones was characteristic of all age-groups. Duration peaked in 5–9 year old children (average duration 319 days, 95% confidence interval 318;320).

Conclusions

The main age-dependence is on parasite densities, with only small age-variations in the FOI and persistence of infections. This supports the hypothesis that acquired immunity controls transmission mainly by limiting blood-stage parasite densities rather than changing rates of acquisition or clearance of infections.     

The Putative Mitochondrial Genome of Plasmodium falciparum

Intraerythrocytic stages of mammalian malarial parasites employ glycolysis for energy production but some aspects of mitochondrial function appear crucial to their survival since inhibitors of mitochondrial protein synthesis and electron transport have antimalarial effects. Investigations of the putative mitochondrial genome of Plasmodium falciparum have detected organellar rRNAs and tRNAs encoded by a 35 kb circular DNA. Some features of the organization and sequence of the rRNA genes are reminiscent of chloroplast DNAs. The 35 kb DNA also encodes open reading frames for proteins normally found in chloroplast but not mitochondrial genomes. An apparently unrelated 6 kb tandemly repeated element which encodes two mitochondrial protein coding genes and fragments of rRNA genes is also found in malarial parasites. The malarial mitochondrial genome thus appears quite unusual. Further investigations are expected to provide insights into the possible functional relationships between these molecules and perhaps their evolutionary history.     

The paradoxical population genetics of Plasmodium falciparum

Among the leading causes of death in African children is cerebral malaria caused by the parasitic protozoan Plasmodium falciparum. Endemic forms of this disease are thought to have originated in central Africa 5000-10000 years ago, coincident with the innovation of slash-and-burn agriculture and the diversification of the Anopheles gambiae complex of mosquito vectors. Population genetic studies of P. falciparum have yielded conflicting results. Some evidence suggests that today's population includes multiple ancient lineages pre-dating human speciation. Other evidence suggests that today's population derives from only one, or a small number, of these ancient lineages. Resolution of this issue is important for the evaluation of the long-term efficacy of drug and immunological control strategies.