Alexandre Budu

PLASMODIUM IN THE POSTGENOMIC ERA : NEW INSIGHTS INTO THE MOLECULAR CELL BIOLOGY OF MALARIA PARASITES

In this review, we bring together some of the approaches toward understanding the cellular and molecular biology of Plasmodium species and their interaction with their host red blood cells. Considerable impetus has come from the development of new methods of molecular genetics and bioinformatics, and it is important to evaluate the wealth of these novel data in the context of basic cell biology. We describe how these approaches are gaining valuable insights into the parasite-host cell interaction, including (1) the multistep process of red blood cell invasion by the merozoite; (2) the mechanisms by which the intracellular parasite feeds on the red blood cell and exports parasite proteins to modify its cytoadherent properties; (3) the modulation of the cell cycle by sensing the environmental tryptophan-related molecules; (4) the mechanism used to survive in a low Ca(2+) concentration inside red blood cells; (5) the activation of signal transduction machinery and the regulation of intracellular calcium; (6) transfection technology; and (7) transcriptional regulation and genome-wide mRNA studies in Plasmodium falciparum.

Genome-Wide Detection of Serpentine Receptor-Like Proteins in Malaria Parasites

Serpentine receptors comprise a large family of membrane receptors distributed over diverse organisms, such as bacteria, fungi, plants and all metazoans. However, the presence of serpentine receptors in protozoan parasites is largely unknown so far. In the present study we performed a genome-wide search for proteins containing seven transmembrane domains (7-TM) in the human malaria parasite Plasmodium falciparum and identified four serpentine receptor-like proteins. These proteins, denoted PfSR1, PfSR10, PfSR12 and PfSR25, show membrane topologies that resemble those exhibited by members belonging to different families of serpentine receptors. Expression of the pfsrs genes was detected by Real Time PCR in P. falciparum intraerythrocytic stages, indicating that they potentially code for functional proteins. We also found corresponding homologues for the PfSRs in five other Plasmodium species, two primate and three rodent parasites. PfSR10 and 25 are the most conserved receptors among the different species, while PfSR1 and 12 are more divergent. Interestingly, we found that PfSR10 and PfSR12 possess similarity to orphan serpentine receptors of other organisms. The identification of potential parasite membrane receptors raises a new perspective for essential aspects of malaria parasite host cell infection.

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

BackgroundPlasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host.MethodsUsing the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated.ResultsThe protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11).ConclusionsThe data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Involvement of TSSA (trypomastigote small surface antigen) in Trypanosoma cruzi invasion of mammalian cells

TSSA (trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca(2+)-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a ~2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T. cruzi stocks.

Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS.

Background: Ca2+ signaling is important for the lytic cycle of T. gondii. Results: Genetically encoded Ca2+ indicators revealed cytosolic Ca2+ changes in real time. Conclusion: New approach highlights important features of the lytic cycle. Significance: Ca2+ influx leads to signaling that results in enhancement of important lytic cycle features. Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca2+ oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca2+ enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca2+ changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca2+ oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca2+ influx. This is the first study showing, in real time, Ca2+ signals preceding egress and their direct link with motility, an essential virulence trait.

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum-infected red blood cells.

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.

Generation of second messengers in Plasmodium.

Signalling in malaria parasites is a field of growing interest as its components may prove to be valuable drug targets, especially when one considers the burden of a disease that is responsible for up to 500 million infections annually. The scope of this review is to discuss external stimuli in the parasite life cycle and the upstream machinery responsible for translating them into intracellular responses, focussing particularly on the calcium signalling pathway.

Encapsulation of metalloporphyrins improves their capacity to block the viability of the human malaria parasite Plasmodium falciparum

UNLABELLED Several synthetic metallated protoporphyrins (M-PPIX) were tested for their ability to block the cell cycle of the lethal human malaria parasite Plasmodium falciparum. After encapsulating the porphyrin derivatives in micro- and nanocapsules of marine atelocollagen, their effects on cultures of red blood cells infected (RBC) with P. falciparum were verified. RBCs infected with synchronized P. falciparum incubated for 48 h showed a toxic effect over a micromolar range. Strikingly, the IC50 of encapsulated metalloporphyrins reached nanomolar concentrations, where Zn-PPIX showed the best antimalarial effect, with an IC50=330 nM. This value is an 80-fold increase in the antimalarial activity compared to the antimalarial effect of non-encapsulated Zn-PPIX. These findings reveal that the incubation of P. falciparum infected-RBCs with 20 μM Zn-PPIX reduced the size of hemozoin crystal by 34%, whereas a 28% reduction was noticed with chloroquine, confirming the importance of heme detoxification pathway in drug therapy. FROM THE CLINICAL EDITOR In this study, synthetic metalloporphyrins were tested as therapeutics that target Plasmodium falciparum. The IC50 of encapsulated metalloporphyrins was found to be in the nanomolar concentration range, with encapsulated Zn-PPIX showing an 80-fold increase in its antimalarial activity compared to the non-encapsulated form.

Melatonin-induced temporal up-regulation of gene expression related to ubiquitin/proteasome system (UPS) in the human malaria parasite Plasmodium falciparum.

There is an increasing understanding that melatonin and the ubiquitin/proteasome system (UPS) interact to regulate multiple cellular functions. Post-translational modifications such as ubiquitination are important modulators of signaling processes, cell cycle and many other cellular functions. Previously, we reported a melatonin-induced upregulation of gene expression related to ubiquitin/proteasome system (UPS) in Plasmodium falciparum, the human malaria parasite, and that P. falciparum protein kinase 7 influences this process. This implies a role of melatonin, an indolamine, in modulating intraerythrocytic development of the parasite. In this report we demonstrate by qPCR analysis, that melatonin induces gene upregulation in nine out of fourteen genes of the UPS, consisting of the same set of genes previously reported, between 4 to 5 h after melatonin treatment. We demonstrate that melatonin causes a temporally controlled gene expression of UPS members.

Strategies for Development of Antimalarials Based on Encapsulated Porphyrin Derivatives

Despite the efforts in controlling the parasite and infection, and the significant progress achieved in recent years in its treatment, malaria is still prevalent in many regions and out of control in others. The repertoire of alternatives to fight malaria is being expanded, not only by designing new drugs but also by developing improved drug delivery systems able to enhance the antimalarial efficiency of conventional and new drugs. Among the new drugs that have been investigated, several publications report the use of porphyrin derivatives as antimalarials but their efficiency is contradictory. The low activity of porphyrins seems to be associated with low dispersibility and bioavailability. In this respect, Nanotechnology can provide efficient solutions to enhance bioavailability and delivery of conventional and new antimalarials, in order to assure high enough efficiency levels to inactivate the parasite. Thus, in this review we highlight the use of drug delivery systems for conventional and new antimalarials and we propose the encapsulation of porphyrins as a promising alternative for development of anti-malarial formulations.