Plasmodium falciparum gametocytes: their longevity and infectivity.

The longevity and infectivity of isolated populations of Plasmodium falciparum gametocytes were studied. Following chloroquine treatment gametocyte numbers fell with a constant rate of loss over a period of 16-24 days; the populations had a half-life of 2-4 days. The sex ratio stayed constant throughout at 4 female: 1 male. The ability of the microgametocytes to exflagellate and the infectivity of the population to mosquitoes persisted for 3 weeks. Antibodies to the gametocytes were detected but not in every patient studied. It was concluded that the gametocytes of P. falciparum are both long-lived and show persistent infectivity to mosquitoes. They can stimulate antibody production but the immune response appears to play no part in their elimination, which probably takes place in the spleen as a part of the normal process of removing old, damaged and malformed red cells.     

The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion

Much of the epidemiology of Plasmodium falciparum in Sub-Saharan Africa focuses on the prevalence patterns of asexual parasites in people of different ages, whereas the gametocytes that propagate the disease are often neglected. One expected benefit of the widespread introduction of artemisinin-based combination therapy for malaria is a reduction in gametocyte carriage. However, the factors that affect the transmission of parasites from humans to mosquitoes show complex dynamics in relation to the intensity and seasonality of malaria transmission, and thus such benefits might not be automatic. Here, we review data on gametocyte carriage in the context of the development of naturally acquired immunity and population infectivity.     

Membrane potential of Plasmodium falciparum gametocytes monitored with rhodamine 123

The membrane potential of Plasmodium falciparum gametocytes was monitored with the cationic permeant fluorescent dye rhodamine 123 (R123) as a probe. Epifluorescence microscopy revealed that R123 at 1 microgram/ml rather selectively partitioned into structure resembling large mitochondria. Treatment of R123-loaded gametocytes with various inhibitors including those of respiration resulted in disappearance of fluorescence from what appeared to be the mitochondria, but not from the cytosol. These results indicate that P. falciparum gametocytes have the mitochondrion maintaining an inside negative membrane potential.     

Organization and function of an actin cytoskeleton in Plasmodium falciparum gametocytes

In preparation for transmission to its mosquito vector, Plasmodium falciparum, the most virulent of the human malaria parasites, adopts an unusual elongated shape. Here we describe a previously unrecognized actin‐based cytoskeleton that is assembled in maturing P. falciparum gametocytes. Differential extraction reveals the presence of a highly stabilized population of F‐actin at all stages of development. Super‐resolution microscopy reveals an F‐actin cytoskeleton that is concentrated at the ends of the elongating gametocyte but extends inward along the microtubule cytoskeleton. Formin‐1 is also concentrated at the gametocyte ends suggesting a role in actin stabilization. Immunoelectron microscopy confirms that the actin cytoskeleton is located under the inner membrane complex rather than in the sub‐alveolar space. In stage V gametocytes, the actin and microtubule cytoskeletons are reorganized in a coordinated fashion. The actin‐depolymerizing agent, cytochalasin D, depletes actin from the end of the gametocytes, whereas the actin‐stabilizing compound, jasplakinolide, induces formation of large bundles and prevents late‐stage disassembly of the actin cytoskeleton. Long‐term treatment with these compounds is associated with disruption of the normal mitochondrial organization and decreased gametocyte viability.     

Modelling the transition of asexual blood stages of Plasmodium falciparum to gametocytes

In this paper, we investigate the transition of asexual blood stages of P. falciparum to gametocytes. The study is based on daily data, collected from 262 individual courses of parasitaemia. We propose several mathematical models that follow biological reasoning. The models are fitted with maximum likelihood and are compared with each other. The models differ in the assumptions made about the mortality of circulating gametocytes and about the transition rate of the asexual parasites. Gametocyte mortality is modelled as being (i) constant over time, (ii) linearly increasing over time, (iii) linearly increasing over gametocyte age, and (iv) exponentially increasing over gametocyte age, respectively. The transition rate is either kept constant per patient or piecewise constant within intervals that correspond to waves of asexual parasitaemia which are assumed to be caused by different Pf(emp1)-variants. According to likelihood ratio tests, the models with age-dependent mortality rate and wave-dependent transition rates are superior to the models with constant transition rate and/or constant or time-dependent mortality rate. The best fits are reached for models with exponentially increasing (Gompertz-type) mortality. Furthermore, an impact of high asexual parasite densities on the survival of gametocytes, interpreted as a cytokine-mediated effect, is evident in some cases.     

Refractoriness of erythrocytes infected with Plasmodium falciparum gametocytes to lysis by sorbitol

Unlike erythrocytes infected with mature asexual parasites of Plasmodium falciparum, those infected with gametoeytes are not lysed by 5% sorbitol solutions. This observation was used to devise a method for producing synchronized cultures of gametocytes, free of asexual stage parasites. The refractoriness to sorbitol suggests that the major anion transport pathway, which appears in the membrane of erythrocytes infected with asexual stage parasites, is not present in cells infected with gametocytes.     

Plasmodium falciparum: mRNA co-expression and protein co-localisation of two gene products upregulated in early gametocytes

Genes encoding Plasmodium falciparum proteins Pfs16 and Pfpeg3/mdv1, specifically appearing in the parasitophorous vacuole of the early gametocytes, are upregulated at the onset of sexual differentiation. Analysis of asexual development in gametocyte producing and non-producing clones of P. falciparum indicated that these genes are also transcribed at a low level in asexual parasites, although their protein products are not detectable in these stages by immunofluorescence. Immunoelectron microscopic analysis of stage II gametocytes indicated that Pfs16 and Pfpeg3/mdv1 proteins co-localise in the parasitophorous vacuole membrane and in all derived membranous structures (such as the multi-laminate membrane whorls of the circular clefts in the infected erythrocyte cytoplasm and the membranes of the gametocyte food vacuoles). In this analysis both proteins were also observed for the first time in the membrane and in the lumen of distinct cleft-like structures in the erythrocyte cytoplasm.     

Fitting hidden Markov models of protein domains to a target species: application to Plasmodium falciparum

Background

The identification of gene sets that are significantly impacted in a given condition based on microarray data is a crucial step in current life science research. Most gene set analysis methods treat genes equally, regardless how specific they are to a given gene set.

Results

In this work we propose a new gene set analysis method that computes a gene set score as the mean of absolute values of weighted moderated gene t-scores. The gene weights are designed to emphasize the genes appearing in few gene sets, versus genes that appear in many gene sets. We demonstrate the usefulness of the method when analyzing gene sets that correspond to the KEGG pathways, and hence we called our method P athway A nalysis with D own-weighting of O verlapping G enes (PADOG). Unlike most gene set analysis methods which are validated through the analysis of 2-3 data sets followed by a human interpretation of the results, the validation employed here uses 24 different data sets and a completely objective assessment scheme that makes minimal assumptions and eliminates the need for possibly biased human assessments of the analysis results.

Conclusions

PADOG significantly improves gene set ranking and boosts sensitivity of analysis using information already available in the gene expression profiles and the collection of gene sets to be analyzed. The advantages of PADOG over other existing approaches are shown to be stable to changes in the database of gene sets to be analyzed. PADOG was implemented as an R package available at: http://bioinformaticsprb.med.wayne.edu/PADOG/or http://www.bioconductor.org.     

Cryptosporidium parvum: the many secrets of a small genome

The coccidium Cryptosporidium parvum is an obligate intracellular parasite of the phylum Apicomplexa. It infects the gastrointestinal tract of humans and livestock, and represents the third major cause of diarrhoeal disease worldwide. Scarcely considered for decades due to its apparently non-pathogenic nature, C. parvum has been studied very actively over the last 15 years, after its medical relevance as a dangerous opportunistic parasite and widespread water contaminant was fully recognised. Despite the lack of an efficient in vitro culture system and appropriate animal models, significant advances have been made in this relatively short period of time towards understanding C. parvum biology, immunology, genetics and epidemiology. Until recently, very little was known about the genome of C. parvum, with even basic issues, such as the number and size of chromosomes, being the object of a certain controversy. With the advent of pulsed field gradient electrophoresis and the introduction of molecular biology techniques, the overall structure and fine organisation of the genome of C. parvum have started to be disclosed. Organised into eight chromosomes distributed in a very narrow range of molecular masses, the genome of C. parvum is one of the smallest so far described among unicellular eukaryotic organisms. Although fewer than 30 C. parvum genes have been cloned so far, information about the overall structure of the parasite genome has increased exponentially over the last 2 years. From the first karyotypic analyses to the recent development of physical maps for individual chromosomes, this review will try to describe the state-of-the-art of our knowledge on the nuclear genome of C. parvum and will discuss the available experimental evidence concerning the presence of extra-chromosomal elements.     

Design of antimalarial transmission blocking agents: Pharmacophore mapping of ligands active against stage-V mature gametocytes of Plasmodium falciparum

Abstract

The discovery of transmission-blocking (T-B) agents is crucial for preventing and complete removal of malaria infection. However, most of the existing antimalarials are only active against the asexual stages of Plasmodium parasite, but ineffective against the sexual stage (gametocytes). In this background, we have developed pharmacophore models against the stage-V mature gametocytes of P. falciparum parasites. The pharmacophore model (Hypo-1) showed five pharmacophoric features namely, one hydrogen bond donor (HBD), one hydrophobic aliphatic (HYAl), one ring aromatic (RA), and two hydrophobic aromatic (HYAr) essential for the anti-gametocytic activity. The amino, methyl, fused phenyl ring of the quinazoline heterocycle, two phenyl rings of biphenyl moiety (HBD, HYAl, HYAr1, HYAr2 and RA) are the crucial features responsible for the non-specific anti-gametocytic activity (PfG). Subsequently, the model (Hypo-2) developed against the stage-V female gametocytes (PffG) showed the contribution of three pharmacophoric features namely, two hydrogen bond acceptor (HYA) and one RA required for the anti-gametocytic activity. The sulfhydryl, imine and pyridyl groups are observed to be essential for anti-gametocytic activity against female gametocytes. Both the models (PfG and PfGG) showed the classification accuracies of 78.26 and 71.64% for training set compounds and 60.80 and 60.18% for the test set compounds, respectively, for classification of compounds into higher and lower active classes. Also, both the models were found to retain the higher active compounds (IC₅₀ <100?nM) in top 1% of total compounds (actives and decoys) as observed after screening the decoy set compounds.

Communicated by Ramaswamy H Sarma     

Early gametocytes of the malaria parasite Plasmodium falciparum specifically remodel the adhesive properties of infected erythrocyte surface

In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains instead obscure how sequestration is established, and how the earliest sexual stages, morphologically similar to asexual trophozoites, modify the infected erythrocytes and their cytoadhesive properties at the onset of gametocytogenesis. Here, purified P. falciparum early gametocytes were used to ultrastructurally and biochemically analyse parasite‐induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do not modify its surface with adhesive ‘knob’ structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface bythese parasites, and the expression of the var gene family, which encodes 50–60 variants of PfEMP1, is dramatically downregulated in the transition from asexual development to gametocytogenesis. Cytoadhesion assays show that such gene expression changes and host cell surface modifications functionally result in the inability of stage I gametocytes to bind the host ligands used by the asexual parasite to bind endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites.