No abstract

No abstract

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.

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.

We previously reported that intraerythrocytic malaria parasites have their development synchronized by melatonin and other products of tryptophan catabolism (i.e. serotonin, N-acetylserotonin and tryptamine). Here, we show that N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), a product of melatonin degradation, synchronizes Plasmodium chabaudi and Plasmodium falciparum. The synchronization is abrogated with a melatonin receptor antagonist, luzindole. We established quantitatively that a differential AFMK production occurred within the intraerythrocytic stages of rodent malaria parasite Plasmodium chabaudi (ring, trophozoite and schizont), when the infected erythrocytes were previously incubated with melatonin. Measurement of AFMK formation in P. chabaudi after incubation with melatonin at a concentration of 500 nmol/L revealed the following values for AFMK production: ring 0.1 +/- 0.1 nmol/L, trophozoite 22.9 +/- 0.5 nmol/L, schizont 29 +/- 5 nmol/L. Confocal and spectrofluorophotometer experiments with isolated parasites and infected-RBC, loaded with calcium indicator Fluo-4 showed that AFMK elicits an increase in the cytosol calcium concentration in these parasites. Our data suggest that AFMK could have an important role in modulating the cell cycle of malaria parasites mainly in the late stages (trophozoite and schizont).

The malaria parasite Plasmodium falciparum is exposed, during its development, to major changes of ionic composition in its surrounding medium. We demonstrate that the P. falciparum serpentine-like receptor PfSR25 is a monovalent cation sensor capable of modulating Ca2+ signaling in the parasites. Changing from high (140 mM) to low (5.4 mM) KCl concentration triggers [Ca2+]cyt increase in isolated parasites and this Ca2+ rise is blocked either by phospholipase C (PLC) inhibition or by depleting the parasite’s internal Ca2+ pools. This response persists even in the absence of free extracellular Ca2+ and cannot be elicited by addition of Na+, Mg2+ or Ca2+. However, when the PfSR25 gene was deleted, no effect on [Ca2+]cyt was observed in response to changing KCl concentration in the knocked out (PfSR25 −) parasite. Finally, we also demonstrate that: i) PfSR25 plays a role in parasite volume regulation, as hyperosmotic stress induces a significant decrease in parasite volume in wild type (wt), but not in PfSR25 − parasites; ii) parasites lacking PfSR25 show decreased parasitemia and metacaspase gene expression on exposure to the nitric oxide donor sodium nitroprusside (SNP) and iii), compared to PfSR25 − parasites, wt parasites showed a better survival in albumax-deprived condition.

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.

Inositol 1,4,5 trisphosphate (IP) signaling plays a crucial role in a wide range of eukaryotic processes. In Plasmodium falciparum, IP elicits Ca release from intracellular Ca stores, even though no IP receptor homolog has been identified to date. The human host hormone melatonin plays a key role in entraining the P. falciparum life cycle in the intraerythrocytic stages, apparently through an IP-dependent Ca signal. The melatonin-induced cytosolic Ca ([Ca]) increase and malaria cell cycle can be blocked by the IP receptor blocker 2-aminoethyl diphenylborinate (2-APB). However, 2-APB also inhibits store-operated Ca entry (SOCE). Therefore, we have used two novel 2-APB derivatives, DPB162-AE and DPB163-AE, which are 100-fold more potent than 2-APB in blocking SOCE in mammalian cells, and appear to act by interfering with clustering of STIM proteins. In the present work we report that DPB162-AE and DPB163-AE block the [Ca] rise in response to melatonin in P. falciparum, but only at high concentrations. These compounds also block SOCE in the parasite at similarly high concentrations suggesting that P. falciparum SOCE is not activated in the same way as in mammalian cells. We further find that DPB162-AE and DPB163-AE affect the development of the intraerythrocytic parasites and invasion of new red blood cells. Our efforts to episomally express proteins that compete with native IP receptor like IP-sponge and an IP sensor such as IRIS proved to be lethal to P. falciparum during intraerythrocytic cycle. The present findings point to an important role of IP-induced Ca release in intraerythrocytic stage of P. falciparum.