The severity of malarial anaemia in Plasmodium chabaudi infections of BALB/c mice is determined independently of the number of circulating parasites

Background: Severe malarial anaemia is a major complication of malaria infection and is multifactorial resulting from loss of circulating red blood cells (RBCs) from parasite replication, as well as immune-mediated mechanisms. An understanding of the causes of severe malarial anaemia is necessary to develop and implement new therapeutic strategies to tackle this syndrome of malaria infection. Methods: Using analysis of variance, this work investigated whether parasite-destruction of RBCs always accounts for the severity of malarial anaemia during infections of the rodent malaria model Plasmodium chabaudi in mice of a BALB/c background. Differences in anaemia between two different clones of P. chabaudi were also examined. Results: Circulating parasite numbers were not correlated with the severity of anaemia in either BALB/c mice or under more severe conditions of anaemia in BALB/c RAG2 deficient mice (lacking T and B cells). Mice infected with P. chabaudi clone CB suffered more severe anaemia than mice infected with clone AS, but this was not correlated with the number of parasites in the circulation. Instead, the peak percentage of parasitized RBCs was higher in CB-infected animals than in AS-infected animals, and was correlated with the severity of anaemia, suggesting that the availability of uninfected RBCs was impaired in CB-infected animals. Conclusion: This work shows that parasite numbers are a more relevant measure of parasite levels in P. chabaudi infection than % parasitaemia, a measure that does not take anaemia into account. The lack of correlation between parasite numbers and the drop in circulating RBCs in this experimental model of malaria support a role for the host response in the impairment or destruction of uninfected RBC in P. chabaudi infections, and thus development of acute anaemia in this malaria model.

The pathology of Plasmodium chabaudi infection is not ameliorated by the secreted filarial nematode immunomodulatory molecule, ES-62

ES-62 is a phosphorylcholine-containing glycoprotein secreted by filarial nematodes. This molecule has been shown to reduce the severity of inflammation in collagen-induced arthritis (CIA) in mice, a model of rheumatoid arthritis, via down-regulation of anti-collagen type 1 immune responses. Malaria parasites induce a pro-inflammatory host immune response and many of the symptoms of malaria are immune system-mediated. Therefore we have asked whether the immunomodulatory properties of ES-62 can down-regulate the severity of malaria infection in BALB/c mice infected with Plasmodium chabaudi. We have found that ES-62 has no significant effect on the course of P. chabaudi parasitaemia, and does not significantly affect any of the measures of malaria-induced pathology taken throughout infection.

Gradual Decline in Malaria-Specific Memory T Cell Responses Leads to Failure to Maintain Long-Term Protective Immunity to Plasmodium chabaudi AS Despite Persistence of B Cell Memory and Circulating Antibody

The mechanisms responsible for the generation and maintenance of immunological memory to Plasmodium are poorly understood and the reasons why protective immunity in humans is so difficult to achieve and rapidly lost remain a matter for debate. A possible explanation for the difficulty in building up an efficient immune response against this parasite is the massive T cell apoptosis resulting from exposure to high-dose parasite Ag. To determine the immunological mechanisms required for long-term protection against P. chabaudi malaria and the consequences of high and low acute phase parasite loads for acquisition of protective immunity, we performed a detailed analysis of T and B cell compartments over a period of 200 days following untreated and drug-treated infections in female C57BL/6 mice. By comparing several immunological parameters with the capacity to control a secondary parasite challenge, we concluded that loss of full protective immunity is not determined by acute phase parasite load nor by serum levels of specific IgG2a and IgG1. Abs, but appears to be a consequence of the progressive decline in memory T cell response to parasites, which occurs similarly in untreated and drug-treated mice with time after infection. Furthermore, by analyzing adoptive transfer experiments, we confirmed the major role of CD4(+) T cells for guaranteeing long-term full protection against P. chabaudi malaria. The Journal of Immunology, 2008, 181: 8344-8355.

Melatonin triggers PKA activation in the rodent malaria parasite Plasmodium chabaudi

Calcium (Ca2+) is a critical regulator of many aspects of the Plasmodium reproductive cycle. In particular, intra-erythrocyte Plasmodium parasites respond to circulating levels of the melatonin in a process mediated partly by intracellular Ca2+. Melatonin promotes the development and synchronicity of parasites, thereby enhancing their spread and worsening the clinical implications. The signalling mechanisms underlying the effects of melatonin are not fully established, although both Ca2+ and cyclic AMP (cAMP) have been implicated. Furthermore, it is not clear whether different strains of Plasmodium use the same, or divergent, signals to control their development. The aim of this study was to explore the signalling mechanisms engaged by melatonin in P. chabaudi, a virulent rodent parasite. Using parasites at the throphozoite stage acutely isolated from mice erythrocytes, we demonstrate that melatonin triggers cAMP production and protein kinase A (PKA) activation. Interestingly, the stimulation of cAMP/PKA signalling by melatonin was dependent on elevation of Ca2+ within the parasite, because buffering Ca2+ changes using the chelator BAPTA prevented cAMP production in response to melatonin. Incubation with melatonin evoked robust Ca2+ signals within the parasite, as did the application of a membrane-permeant analogue of cAMP. Our data suggest that P. chabaudi engages both Ca2+ and cAMP signalling systems when stimulated by melatonin. Furthermore, there is positive feedback between these messengers, because Ca2+ evokes cAMP elevation and vice versa. Melatonin more than doubled the observed extent of parasitemia, and the increase in cAMP concentration and PKA activation was essential for this effect. These data support the possibility to use melatonin antagonists or derivates in therapeutic approach.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

IFN-γ-induced priming maintains long-term strain-transcending immunity against blood-stage Plasmodium chabaudi malaria

The mechanism by which protective immunity to Plasmodium is lost in the absence of continued exposure to this parasite has yet to be fully elucidated. It has been recently shown that IFN-γ produced during human and murine acute malaria primes the immune response to TLR agonists. In this study, we investigated whether IFN-γ-induced priming is important to maintain long-term protective immunity against Plasmodium chabaudi AS malaria. On day 60 postinfection, C57BL/6 mice still had chronic parasitemia and efficiently controlled homologous and heterologous (AJ strain) challenge. The spleens of chronic mice showed augmented numbers of effector/effector memory (TEM) CD4(+) cells, which is associated with increased levels of IFN-γ-induced priming (i.e., high expression of IFN-inducible genes and TLR hyperresponsiveness). After parasite elimination, IFN-γ-induced priming was no longer detected and protective immunity to heterologous challenge was mostly lost with >70% mortality. Spontaneously cured mice had high serum levels of parasite-specific IgG, but effector T/TEM cell numbers, parasite-driven CD4(+) T cell proliferation, and IFN-γ production were similar to noninfected controls. Remarkably, the priming of cured mice with low doses of IFN-γ rescued TLR hyperresponsiveness and the capacity to control heterologous challenge, increasing the TEM cell population and restoring the CD4(+) T cell responses to parasites. Contribution of TLR signaling to the CD4(+) T cell responses in chronic mice was supported by data obtained in mice lacking the MyD88 adaptor. These results indicate that IFN-γ-induced priming is required to maintain protective immunity against P. chabaudi and aid in establishing the molecular basis of strain-transcending immunity in human malaria.

Trafficking of Plasmodium chabaudi adami-infected erythrocytes within the mouse spleen.

Plasmodium chabaudi adami causes a nonlethal infection in mice. We found that crisis, the time of rapidly dropping parasitemia, was abrogated by splenectomy, indicating the role of spleen in parasite killing. The factors that mediate spleen-dependent immunity are not known. An earlier study in Plasmodium berghei-infected rats showed an association between increased clearance of heat-treated erythrocytes and the onset of crisis [Wyler, D. J., Quinn, T. C. & Chen, L.-T. (1982) J. Clin. Invest. 67, 1400-1404]. To determine the potential effects of different vascular beds in parasite killing, we studied the distribution of parasitized erythrocytes and bacteria in the spleens of P. chabaudi adami-infected mice during precrisis (a period of rising parasitemia) and during crisis. After intravenous injection, bacteria were localized predominantly in the marginal zone. In contrast, parasitized erythrocytes were found in the red pulp. We also found that during precrisis, a time of no immunity, the uptake of radiolabeled infected erythrocytes by the spleen was increased, not decreased. These data imply that no change occurs in the flow of parasitized erythrocytes through the spleen during the transition to an immune state (crisis). Our observations suggest that immune effector mechanisms, not circulatory changes, account for spleen-dependent parasite killing during a P. chabaudi adami infection in mice.

Studies on the protective immune response to Plasmodium chabaudi in mice

Inbred strains of C5731 and NIH nice infected with the A/S strain of Plasmodium chaubaudi usually developed high parasitaemias but infections were rarely fatal in immunocompetent mice and in most mice the parasites could be eradicated within 53 days or less. The immune response of C57B1 and NTH mice to infection with the A/S strain of P. chabaudi was studied. The principle method used in this study for investigating the immune response of the mice was to examine the immunity conferred on syngeneic mice, either X-irradiated or non-irradiated, by transferring to them lymphoid cells or serum from immune or semi-immune donors. The lymphoid cell populations examined were unfractionated spleen cells, nylon wool column enriched subpopulations of thymus-derived lymphocytes (T cells) and the so-called bursa-derived lymphocytes (B cells), bone marrow cells and phagocytic cells. In the course of these experiments observations were made on the effect of X-irradiation on the subsequent growth and multiplication of the parasite. In addition, an in vitro assay for antibody-dependent cell mediated cytotoxicity was used to investigate the activity of splenic K cells during malaria infection. K cells are lymphoid cells which may include lymphocytes of an undefined category, but possess receptors for the Fc portion of antibody on their surface and have the ability to non-specifically lyse target cells coated in antibodies. a) The adoptive transfer of immunity to P.chabaudi with immune spleen cells. Spleen cells from mice which had previously been infected with P.chabaudi were able to confer some immunity on syngeneic mice which had been irradiated with 600 or 800 rads. The protection was detected as a shortened patent parasitaemia in immune cell recipients compared to controls. The early experiments indicated the value of using irradiated recipients rather than non-irradiated recipients. In irradiated mice, a) smaller numbers of immune cells were required to promote detectable immunity than in non-irradiated mice, b) there was an amplification of the difference in the duration of primary parasitaemias in recipients of immune cells and normal cells compared to non-irradiated mice and c) as the irradiated host is immunodepressed, the protective effect of donor cells can be examined with a reduced contribution by the hosts own immune system. An initial non-specific resistance to P.chabaudi infection was observed in irradiated mice, although the infection in most of these mice was subsequently more severe than in non-irradiated mice. The non-specific resistance could be reduced or abolished by injecting lymphoid cells into mice shortly after irradiation or by infecting irradiated mice more than 15 days after irradiation. Other workers suggest that following irradiation, the reticulo-endothelial system is stimulated at the time that the non-specific resistance to P.chabaudi was observed. b) the adoptive transfer of immunity in syngeneic mice with enriched subpopulations of splenic immune T cells, B. cells, bone marrow cells and phagocytes. Immunity to P.chabaudi could be adoptively transferred with enriched spleen subpopulations of immune T cells or immune B cells in mice which had been irradiated 600 or 300 rads. The protective effects of unfractionated immune cells was, however, usually better than that of either immune T or F cell subpopulations. In most experiments enriched immune T cell recipients were more likely to suffer relapsing patent parasitaemias than either enriched immune B cell recipients or unfractionated immune cell recipients. In one experiment a comparison was made of the course of P.chabaudi infection in mice which had been irradiated with either 600 rads or 300 rads and which received injections of different immune cells. A dose of 600 rads permits the immune system of mice to recover from the effects of irradiation, but a dose of 800 rads is lethal to mice unless lymphoid cells are injected after irradiation. It was found that in recipients of enriched immune T or B cells, which had been irradiated with 600 rads, the parasitaemia became subpatent before their equivalents irradiated with 800 rads, but that there was little difference in parasitaemias between recipients of unfractionated immune cells given 600 or 800 rads. Experiments in which enriched immune T cells and B cells were recombined and injected into syngeneic mice gave inconclusive results as to whether the immune subpopulations acted synergistically. Similar experiments in which immune subpopulations of lymphoid cells were recombined with normal subpopulations of lymphoid cells demonstrated that the latter cells did not enhance the protective effect of the former cells. Bone marrow cells from immune mice were able to confer some protection on syngeneic recipients, but were not as protective as enriched immune T cells or B cells. The results obtained in adoptive transfer experiments using phagocytic cells from the spleen of immune mice depended on the length of time spleen cells were incubated in petri-dishes at 37° C before harvesting the phagocytes. Using C57B1 mice, phagocytes harvested after 15 hours incubation were as protective as unfractionated immune cells in a cell transfer experiment, but phagocytes harvested after 16 hours incubation were not protective. Examination of NIH phagocytic cells after 2.5 hours incubation at 37°C, which were as protective as unfractionated immune spleen cells in a cell transfer experiment, demonstrated that the petri-dish adherent cells may have contained B lymphocytes. c) The passive transfer of immunity with serum from P.chabaudi infected mice. The passive transfer of serum from C57B1 mice which had been previously infected with P.chabaudi to normal or irradiated syngeneic mice demonstrated that the serum recipients were initially protected from infection. Irradiated mice, however, were delayed longer in the onset of parasitaemia compared to non-irradiated mice. Using NIH mice, sera were collected from unfractionated immune spleen cell recipients, enriched immune T cell recipients and normal spleen recipients on the 11th day of a P.chabaudi infection, just after peak parasitaemia, and also on the 14th day of infection. On day 14, all immune cells recipients and most of the enriched immune T cell recipients had become subpatent but all normal cell recipients still had patent infections. Sera collected from the different spleen cell recipients on the 11th day of infection and passively transferred to irradiated mice demonstrated little protection. Sera collected on the 14th day of infect ion, however, reflected the immune status of the donors in their protective properties in mice infected with P.chabaudi. The serum from unfractionated immune cell recipients was the most protective of the 3 sera when compared to normal NIH serum and the serum from enriched immune T cell recipients was slightly protective, but the serum from normal cell recipients produced an enhanced infection in mice infected with P.chabaudi. d) Antibody-dependent cell-mediated cytotoxicity of spleen cells in P.chabaudi infected mice. In a preliminary investigation of K cell activity in the spleens of P.chabaudi infected mice, it was found that there was an increased activity of K cells collected at around peak parasitaemia compared to the activity of K cells in non-infected mice, and that this increased activity could also be found in mice which had recently become subpatent. As the target cell for antibody-dependent cell-mediated cytotoxicity employed was the thick red blood cell, it is not known whether the K cell is involved in the killing of P.chabaudi parasites. These results suggest that both T cells and B cells and antibody may be important in the immune response to P.chabaudi in mice. Primed T cells may act as helper cells in the production of malarial antibodies, but, as enriched primed T cells could confer protection on immunodepressed mice, it is possible that a cell-mediated mechanism of immunity may also exist.

No impact of malaria infection or immunisation on the expression of the immune GTPase GIMAP1 at the protein or mRNA levels (Article no. 53)

GIMAP (GTPase of the immunity-associated protein family) proteins are a family of putative GTPases believed to be regulators of cell death in lymphomyeloid cells. GIMAP1 was the first reported member of this gene family, identified as a gene up-regulated at the RNA level in the spleens of mice infected with the malarial parasite, Plasmodium chabaudi. Methods A monoclonal antibody against mouse GIMAP1 was developed and was used to analyse the expression of the endogenous protein in tissues of normal mice and in defined sub-populations of cells prepared from lymphoid tissues using flow cytometry. It was also used to assess the expression of GIMAP1 protein after infection and/or immunization of mice with P. chabaudi. Real-time PCR analysis was employed to measure the expression of GIMAP1 for comparison with the protein level analysis. Results GIMAP1 protein expression was detected in all lineages of lymphocytes (T, B, NK), in F4/80+ splenic macrophages and in some lymphoid cell lines. Additional evidence is presented suggesting that the strong expression by mature B cells of GIMAP1 and other GIMAP genes and proteins seen in mice may be a species-dependent characteristic. Unexpectedly, no increase was found in the expression of GIMAP1 in P. chabaudi infected mice at either the mRNA or protein level, and this remained so despite applying a number of variations to the protocol. Conclusion The model of up-regulation of GIMAP1 in response to infection/immunization with P. chabaudi is not a robustly reproducible experimental system. The GIMAP1 protein is widely expressed in lymphoid cells, with an interesting increase in expression in the later stages of B cell development. Alternative approaches will be required to define the functional role of this GTPase in immune cells.

FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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. (C) 2012 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Anti-IL-2 Treatment Impairs the Expansion of T-reg Cell Population during Acute Malaria and Enhances the Th1 Cell Response at the Chronic Disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T-reg) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T-reg cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4+ T cell activation and IFN-gamma production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-alpha and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T-reg cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.

Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.

Potencies of human immunodeficiency virus protease inhibitors in vitro against Plasmodium falciparum and in vivo against murine malaria

Parasite resistance to antimalarial drugs is a serious threat to human health, and novel agents that act on enzymes essential for parasite metabolism, such as proteases, are attractive targets for drug development. Recent studies have shown that clinically utilized human immunodeficiency virus (HIV) protease inhibitors can inhibit the in vitro growth of Plasmodium falciparum at or below concentrations found in human plasma after oral drug administration. The most potent in vitro antimalarial effects have been obtained for parasites treated with saquinavir, ritonavir, or lopinavir, findings confirmed in this study for a genetically distinct P. falciparum line (3D7). To investigate the potential in vivo activity of antiretroviral protease inhibitors (ARPIs) against malaria, we examined the effect of ARPI combinations in a murine model of malaria. In mice infected with Plasmodium chabaudi AS and treated orally with ritonavir-saquinavir or ritonavir-lopinavir, a delay in patency and a significant attenuation of parasitemia were observed. Using modeling and ligand docking studies we examined putative ligand binding sites of ARPIs in aspartyl proteases of P. falciparum (plasmepsins II and IV) and P. chabaudi (plasmepsin) and found that these in silico analyses support the antimalarial activity hypothesized to be mediated through inhibition of these enzymes. In addition, in vitro enzyme assays demonstrated that P. falciparum plasmepsins II and IV are both inhibited by the ARPIs saquinavir, ritonavir, and lopinavir. The combined results suggest that ARPIs have useful antimalarial activity that may be especially relevant in geographical regions where HIV and P. falciparum infections are both endemic.

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 by P. 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.