Arthur M. Talman

Gametocytogenesis : the puberty of Plasmodium falciparum

The protozoan Plasmodium falciparum has a complex life cycle in which asexual multiplication in the vertebrate host alternates with an obligate sexual reproduction in the anopheline mosquito. Apart from the apparent recombination advantages conferred by sex, P. falciparum has evolved a remarkable biology and adaptive phenotypes to insure its transmission despite the dangers of sex. This review mainly focuses on the current knowledge on commitment to sexual development, gametocytogenesis and the evolutionary significance of various aspects of gametocyte biology. It goes further than pure biology to look at the strategies used to improve successful transmission. Although gametocytes are inevitable stages for transmission and provide a potential target to fight malaria, they have received less attention than the pathogenic asexual stages. There is a need for research on gametocytes, which are a fascinating stage, responsible to a large extent for the success of P. falciparum.

Evaluation of the intra- and inter-specific genetic variability of Plasmodium lactate dehydrogenase.

BackgroundMalaria diagnosis is vital to efficient control programmes and the recent advent of malaria rapid diagnostic tests (RDTs) provides a reliable and simple diagnostic method. However a characterization of the efficiency of these tests and the proteins they detect is needed to maximize RDT sensitivity.MethodsPlasmodial lactate dehydrogenase (pLDH) gene of wild isolates of the four human species of Plasmodium from a variety of malaria endemic settings were sequenced and analysed.ResultsNo variation in nucleotide was found within Plasmodium falciparum, synonymous mutations were found for Plasmodium malariae and Plasmodium. vivax; and three different types of amino acid sequence were found for Plasmodium ovale. Conserved and variable regions were identified within each species.ConclusionThe results indicate that antigen variability is unlikely to explain variability in performance of RDTs detecting pLDH from cases of P. falciparum, P. vivax or P. malariae malaria, but may contribute to poor detection of P. ovale.

PbGEST mediates malaria transmission to both mosquito and vertebrate host.

The malaria life cycle relies on the successful transfer of the parasite between its human and mosquito hosts. We identified a Plasmodium berghei secreted protein (PBANKA_131270) that plays distinct roles in both the mammal‐to‐mosquito and the mosquito‐to‐mammal transitions. This protein, here named gamete egress and sporozoite traversal (GEST), plays an important role in the egress of male and female gametes from the vertebrate red blood cell. Interestingly, GEST is also required following the bite of the infected mosquito, for sporozoite progression through the skin. We found PbGEST to be secreted shortly after activation of the intraerythrocytic gametocyte, and during sporozoite migration. These findings indicate that a single malaria protein may have pleiotropic roles in different parasites stages mediating transmission between its insect and mammalian hosts.

A Plasmodium falciparum Strain Expressing GFP throughout the Parasite's Life-Cycle

The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito.

Use of a Selective Inhibitor To Define the Chemotherapeutic Potential of the Plasmodial Hexose Transporter in Different Stages of the Parasite's Life Cycle

ABSTRACT During blood infection, malarial parasites use d-glucose as their main energy source. The Plasmodium falciparum hexose transporter (PfHT), which mediates the uptake of d-glucose into parasites, is essential for survival of asexual blood-stage parasites. Recently, genetic studies in the rodent malaria model, Plasmodium berghei, found that the orthologous hexose transporter (PbHT) is expressed throughout the parasites development within the mosquito vector, in addition to being essential during intraerythrocytic development. Here, using a d-glucose-derived specific inhibitor of plasmodial hexose transporters, compound 3361, we have investigated the importance of d-glucose uptake during liver and transmission stages of P. berghei. Initially, we confirmed the expression of PbHT during liver stage development, using a green fluorescent protein (GFP) tagging strategy. Compound 3361 inhibited liver-stage parasite development, with a 50% inhibitory concentration (IC50) of 11 μM. This process was insensitive to the external d-glucose concentration. In addition, compound 3361 inhibited ookinete development and microgametogenesis, with IC50s in the region of 250 μM (the latter in a d-glucose-sensitive manner). Consistent with our findings for the effect of compound 3361 on vector parasite stages, 1 mM compound 3361 demonstrated transmission blocking activity. These data indicate that novel chemotherapeutic interventions that target PfHT may be active against liver and, to a lesser extent, transmission stages, in addition to blood stages.

Heritability of the Human Infectious Reservoir of Malaria Parasites

Background Studies on human genetic factors associated with malaria have hitherto concentrated on their role in susceptibility to and protection from disease. In contrast, virtually no attention has been paid to the role of human genetics in eliciting the production of parasite transmission stages, the gametocytes, and thus enhancing the spread of disease. Methods and Findings We analysed four longitudinal family-based cohort studies from Senegal and Thailand followed for 2–8 years and evaluated the relative impact of the human genetic and non-genetic factors on gametocyte production in infections of Plasmodium falciparum or P. vivax. Prevalence and density of gametocyte carriage were evaluated in asymptomatic and symptomatic infections by examination of Giemsa-stained blood smears and/or RT-PCR (for falciparum in one site). A significant human genetic contribution was found to be associated with gametocyte prevalence in asymptomatic P. falciparum infections. By contrast, there was no heritability associated with the production of gametocytes for P. falciparum or P. vivax symptomatic infections. Sickle cell mutation, HbS, was associated with increased gametocyte prevalence but its contribution was small. Conclusions The existence of a significant human genetic contribution to gametocyte prevalence in asymptomatic infections suggests that candidate gene and genome wide association approaches may be usefully applied to explore the underlying human genetics. Prospective epidemiological studies will provide an opportunity to generate novel and perhaps more epidemiologically pertinent gametocyte data with which similar analyses can be performed and the role of human genetics in parasite transmission ascertained.

Proteomic analysis of Plasmodium in the mosquito: progress and pitfalls

SUMMARY Here we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studies.

The Armadillo repeat protein PF16 is essential for flagellar structure and function in Plasmodium male gametes.

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the worlds population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasites flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite.

Bacterial spread from cell to cell: beyond actin-based motility.

Several intracellular pathogens display the ability to propagate within host tissues by displaying actin-based motility in the cytosol of infected cells. As motile bacteria reach cell-cell contacts they form plasma membrane protrusions that project into adjacent cells and resolve into vacuoles from which the pathogen escapes, thereby achieving spread from cell to cell. Seminal studies have defined the bacterial and cellular factors that support actin-based motility. By contrast, the mechanisms supporting the formation of protrusions and their resolution into vacuoles have remained elusive. Here, we review recent advances in the field showing that Listeria monocytogenes and Shigella flexneri have evolved pathogen-specific mechanisms of bacterial spread from cell to cell.

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility

BackgroundGametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown.MethodsPlasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163.Results615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway.ConclusionsThis study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.