The Plasmodium sporozoite journey: a rite of passage

Sporozoites are the most versatile of the invasive stages of the Plasmodium life cycle. During their passage within the mosquito vector and the vertebrate host, sporozoites display diverse behaviors, including gliding locomotion and invasion of, migration through and egress from target cells. At the end of the journey, sporozoites invade hepatocytes and transform into exoerythrocytic stages, marking the transition from the pre-erythrocytic to the erythrocytic part of the life cycle. This article discusses recent work, mostly done with rodent malaria parasites, that has contributed to a better understanding of the sporozoites' complex biology and which has opened up new avenues for future sporozoite research.     

Life Cycles of Leucocytozoon dubreuili Mathis and Leger, 1911 and L. fringillinarum Woodcock, 1910 (Haemosporidia: Leucocytozoidae)*

SYNOPSIS The development of Leucocytozoon dubreuili and L. fringillinarum was studied on successive days in simuliid and avian hosts. Sporogony of both parasites is completed in at least 5 species of sylvatic Simuliidae in a minimum of 4-5 days at 21 C. The pattern of development of the 2 species is similar but the size of the oocysts and the number of sporozoites differ. Sporozoites of L. dubreuili and L. fringillinarum were injected into uninfected robins (Turdus m. migratorius) and grackles (Quiscalus quiscula versicolor), respectively. Hepatic biopsies were performed on some of the injected birds. These and others were killed at intervals following inoculation and their tissues examined to detect stages of schizogony. Blood and macerated tissues from birds injected with sporozoites were transferred to uninfected birds to determine whether asexual stages would develop in the latter as a result of the inoculations. The 1st asexual cycle of L. dubreuili is completed in hepatic parenchymal cells in a minimum of 84 hr. Merozoites produced by the hepatic schizonts apparently follow one of 3 courses: invade hepatic parenchymal cells to initiate another cycle; penetrate blood cells and become gametocytes; penetrate tubular cells of the kidneys and grow into renal schizonts. The minimum prepatent period in infections with L. fringillinarum is 76 hr. The 1st asexual cycle occurs in hepatic parenchymal cells and in tubular cells of the kidney. A schizogonic cycle is completed in a minimum of 72 hr in the former and 96 hr in the kidney. Merozoites from the primary hepatic schizonts apparently give rise to (a) gametocytes; (b) secondary hepatic schizonts; (c) renal schizonts. Thus the schizogonic cycles of L. dubreuili and L. fringillinarum differ from each other and from those of L. simondi in ducks.     

Nomadic or sessile: can Kupffer cells function as portals for malaria sporozoites to the liver?

The initial site of replication for Plasmodium parasites in mammalian hosts are hepatocytes, cells that offer unique advantages for the extensive parasite replication occurring prior to the erythrocytic phase of the life cycle. The liver is the metabolic centre of the body and has an unusual relationship to the immune system. However, to reach hepatocytes, sporozoites must cross the sinusoidal barrier, composed of specialized endothelia and Kupffer cells, the resident macrophages of the liver. Mounting evidence suggests that, instead of taking what would seem a safer route through endothelia, the parasites traverse Kupffer cells yet suffer no harm. Kupffer cells have a broad range of responses towards incoming microorganisms, toxins and antigens which depend on the nature of the intruder, the experimental conditions and the environmental circumstances. Kupffer cells may become activated or remain anergic, produce pro- or anti-inflammatory mediators. Consequently, outcomes are diverse and include development of immunity or tolerance, parenchymal necrosis or regeneration, chronic cirrhotic transformation or acute liver failure. Here we review data concerning the unique structural and functional characteristics of Kupffer cells and their interactions with Plasmodium sporozoites in the context of a model in which these hepatic macrophages function as the sporozoite gate to the liver.     

A Plasmodium α/β‐hydrolase modulates the development of invasive stages

The bud emergence (BEM)46 proteins are evolutionarily conserved members of the α/β‐hydrolase superfamily, which includes enzymes with diverse functions and a wide range of substrates. Here, we identified a Plasmodium BEM46‐like protein (PBLP) and characterized it throughout the life cycle of the rodent malaria parasite Plasmodium yoelii. The Plasmodium BEM46‐like protein is shown to be closely associated with the parasite plasma membrane of asexual erythrocytic stage schizonts and exo‐erythrocytic schizonts; however, PBLP localizes to unique intracellular structures in sporozoites. Generation and analysis of P. yoelii knockout (Δpblp) parasite lines showed that PBLP has an important role in erythrocytic stage merozoite development with Δpblp parasites forming fewer merozoites during schizogony, which results in decreased parasitemia when compared with wild‐type (WT) parasites. Δpblp parasites showed no defects in gametogenesis or transmission to mosquitoes; however, because they formed fewer oocysts there was a reduction in the number of developed sporozoites in infected mosquitoes when compared with WT. Although Δpblp sporozoites showed no apparent defect in mosquito salivary gland infection, they showed decreased infectivity in hepatocytes in vitro. Similarly, mice infected with Δpblp sporozoites exhibited a delay in the onset of blood‐stage patency, which is likely caused by reduced sporozoite infectivity and a discernible delay in exo‐erythrocytic merozoite formation. These data are consistent with the model that PBLP has an important role in parasite invasive‐stage morphogenesis throughout the parasite life cycle.     

Schizogony and Gametogony of Leucocytozoon simondi and Associated Reactions in the Avian Host*

SYNOPSIS. Stages of development of Leucocytozoon simondi in White Pekin ducklings and their reactions to the parasite were studied on successive days after infecting them artificially with sporozoites from Simulium rugglesi. The minimum prepatent period was 5 days. The first asexual cycle occurred exclusively in the parenchymal cells of the liver. Progeny of these hepatic schizonts followed one of 3 courses: (a) invaded parenchymal liver cells to give rise to another hepatic cycle, (b) penetrated blood cells to form round gametocytes, and (c) were phagocytized by macrophages and grew into megaloschizonts thruout the body. The appearance of elongating gametocytes coincided with the period of maturation and release of merozoites from the megaloschizonts. Experimental evidence supports the hypothesis that the round gametocytes arise from the hepatic schizonts and the elongate forms from the megaloschizonts. Mature megaloschizonts released millions of merozoites, but a high 2nd peak in parasitemia did not develop because of retention of developing gametocytes in the deep circulation, particularly the liver and spleen, and a pronounced host reaction.     

An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum erythrocytic schizogony

Aurora kinases compose a family of conserved Ser/Thr protein kinases playing essential roles in eukaryotic cell division. To date, Aurora homologues remain uncharacterized in the protozoan phylum Apicomplexa. In malaria parasites, the characterization of Aurora kinases may help understand the cell cycle control during erythrocytic schizogony where asynchronous nuclear divisions occur. In this study, we revisited the kinome of Plasmodium falciparum and identified three Aurora-related kinases, Pfark-1, -2, -3. Among these, Pfark-1 is highly conserved in malaria parasites and also appears to be conserved across Apicomplexa. By tagging the endogenous Pfark-1 gene with the green fluorescent protein (GFP) in live parasites, we show that the Pfark-1-GFP protein forms paired dots associated with only a subset of nuclei within individual schizonts. Immunofluorescence analysis using an anti-α-tubulin antibody strongly suggests a recruitment of Pfark-1 at duplicated spindle pole bodies at the entry of the M phase of the cell cycle. Unsuccessful attempts at disrupting the Pfark-1 gene with a knockout construct further indicate that Pfark-1 is required for parasite growth in red blood cells. Our study provides new insights into the cell cycle control of malaria parasites and reports the importance of Aurora kinases as potential targets for new antimalarials.     

Stage-dependent inhibition of chloroquine on Plasmodium falciparum in vitro

Morphological observation of the life cycle of the malaria parasite, Plasmodium falciparum, in highly synchronous cultures after an exposure to therapeutic concentrations of chloroquine in ring, trophozoite and schizont stages, respectively, were carried out in order to determine the influence of chloroquine on the growth of the different stages of the malarial parasites. It was found that chloroquine could not affect merozoite invasion of the erythrocytes; the ring stage was more sensitive to chloroquine than the trophozoite and schizont stages; and chloroquine in therapeutic concentrations prevented only the transformation of rings to trophozoites and could not affect the transformations of trophozoites to schizonts and schizonts to new rings. The determination of the IC50 of chloroquine showed that the IC50 of trophozoites was about 6 times as high as that of rings.     

Fine structure of human malaria in vitro.

The erythrocytic cycle of the human malaria parasite, Plasmodium, falciparum, was examined by electron microscopy. Three strains of parasites maintained in continuous culture in human erythrocytes were compared with in vivo infections in Aotus monkeys. The ultrastructure of P. falciparum is not altered by continuous cultivation in vitro. Mitochondria contain DNA-like filaments and some cristae at all stages of the erythrocytic life cycle. The Golgi apparatus is prominent at the schizont stage and may be involved in the formation of rhoptries. In culture, knob-like protrusions first appear on the surface of trophozoite-infected erythrocytes. The time of appearance of knobs on cells in vitro correlates with the life cycle stage of parasites which are sequestered from the peripheral circulation in vivo. Knob material of older parasites coalesces and forms extensions from the erythrocyte surface. Some of this material is sloughed from the host cell surface. The parasitophorous vacuole membrane breaks down in erythrocytes containing mature merozoites both in vitro and in vivo. Merozoite structure is similar to that of P. knowlesi. The immature gametocytes in culture have no knobs.     

The reciprocal influence of the liver and blood stages of the malaria parasite’s life cycle

While the liver and blood stages of the Plasmodium life cycle are commonly regarded as two separate fields of malaria research, several studies have pointed towards the existence of a bidirectional cross-talk, where one stage of mammalian infection may impact the establishment and progression of the other. Despite the constraints in experimentally addressing concurrent liver and blood stage Plasmodium infections, animal models and clinical studies have unveiled a plethora of molecular interactions between the two. Here, we review the current knowledge on the reciprocal influence of hepatic and erythrocytic infection by malaria parasites, and discuss its impacts on immunity, pathology and vaccination against this deadly disease.     

A clash to conquer: the malaria parasite liver infection

All mammalian malaria parasite species have an initial tissue stage in liver cells. The liver stage produces new parasite forms that can enter and live inside red blood cells. Accordingly, the first place of residence provides parasites with a radically different cellular and molecular environment from their subsequent red blood cell home. Liver stages have remained refractory to reveal their secrets, yet the last few years have seen several advances in elucidating their biology. This review looks at the more recent findings concerning the liver stage-host hepatocyte association, some of which may become powerful weapons in the prevention of malaria infection. We also outline areas of liver stage research and technological development that provide promising foci to accelerate a better understanding of this most elusive of the parasites many life cycle stages.     

Falcipain cysteine proteases of malaria parasites: An update

BackgroundThe malaria parasite Plasmodium falciparum expresses four related papain-family cysteine proteases known as falcipains. These proteases play critical roles in the parasite life cycle, and as such are potential targets for new modes of antimalarial chemotherapy, as discussed in this review.Scope of reviewThis review summarizes available knowledge describing falcipain cysteine proteases of malaria parasites.Major conclusionsBased on available data the falcipains can be broken into two sub-families, the falcipain-1 and the falcipain-2/3 sub-families. Falcipain-1 has been difficult to study; it appears to play its most important roles in nonerythrocytic parasites, but not the erythrocytic stage responsible for human disease. Falcipain-2 and falcipain-3 have similar biochemical features, and are expressed sequentially during the erythrocytic cycle. Inhibition of either of these enzymes blocks hemoglobin hydrolysis and completion of the parasite developmental cycle. Knockout of falcipain-2 blocks hemoglobin hydrolysis, but parasites recover, presumably due to subsequent expression of falcipain-3. Knockout of falcipain-3 has not been possible, suggesting that the protease is essential for erythrocytic parasites. Determination of structures of falcipains and extensive chemistry efforts have facilitated identification of numerous small molecule falcipain inhibitors as potential new antimalarial agents. Other malaria parasites express close homologs of falcipain-1 and falcipain-2/3 proteases, suggesting that agents that target the falcipains will also be active against other human malaria parasites.General Significance. Falcipain-2 and falcipain-3 play vital roles during the erythrocytic stage of infection with P. falciparum and thus are promising targets for new agents to treat malaria.     

A malarial parasite of Australian skinks, Plasmodium mackerrasae sp. n.

Plasmodium mackerrasae sp. n. parasitizes the Australian lizards Egernia cunninghami and E. striolata (Sauria: Scincidae). Described from an experimental host, E. whitei, it produces mature schizonts containing 6--12 nuclei arranged peripherally as a rosette, and round to oval gametocytes which are equal to or slightly smaller than host cell nuclei. Both schizonts and gametocytes parasitize all cells in the erythrocyte series. Presence of pigment in both asexual and sexual stages is correlated with maturity of the host cell. Asexual forms contain a single large vacuole, whereas mature gametocytes may show 1--4 vacuoles. Plasmodium mackerrasae resembles most closely P. sasai of Japan and P. tropiduri of tropical America. It differs from P. sasai by lacking fan-shaped schizonts and by having less heavily pigmented gametocytes, and from P. tropiduri by less variability in shape and greater vacuolation of the gametocytes. Host and geographic differences further support its distinction.     

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans‐cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis‐Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages.     

Confocal microscopic findings of cysteine protease calpain in Plasmodium falciparum

Pf-calpain, a cysteine protease of Plasmodium falciparum, is believed to be one of the central mediators for essential parasitic activity. However, the roles of calpain on parasitic activity have not been determined in P. falciparum. In the present study, the localization of Pf-calpain was investigated using polyclonal antibodies (anti-Pf-calpain antibody A and B) against peptides that distinguished it from human calpain-7 and rat calpain-10 protein. Recombinant Pf-calpain (rPf-calpain) was identified as a 46 kDa protein using an anti-Pf-calpain antibody A, which can recognize the Pf-calpain binding site. Confocal microscopy revealed calpain within cytoplasmic localized parasites in the erythrocytic cycle. The findings suggested that the expression of Pf-calpain would be proportional to all different parasites in the erythrocytic cycle. On the other hand, anti-human calpain-7 antibody detected Pf-calpain in schizonts, and the immunofluorescence was stronger than with anti-rat calpain-10 antibody. However, the antibodies reacted with calpains in human red blood cells. These results show that anti-Pf-calpain antibody A and B specifically recognize only Pf-calpain. Taken together, the results suggest that Pf-calpain is expressed in all erythrocytic stages. In particular, the expression of Pf-calpain is increased much more when the late ring matures into the early trophozoite. Moreover, anti-Pf-calpain antibody A and B against synthetic peptides of the catalytic domain of Pf-calpain are useful to specifically detect Pf-calpain in all erythrocytic stages, while human and rat calpain antibody are not useful.     

The species specificity of immunity generated by live whole organism immunisation with erythrocytic and pre-erythrocytic stages of rodent malaria parasites and implications for vaccine development

A promising strategy for the development of a malaria vaccine involves the use of attenuated whole parasites, as these present a greater repertoire of antigens to the immune system than subunit vaccines. The complexity of the malaria parasite's life cycle offers multiple stages on which to base an attenuated whole organism vaccine. An important consideration in the design and employment of such vaccines is the diversity of the parasites that are infective to humans. The most valuable vaccine would be one that was effective against multiple species/strains of malaria parasite. Here we compare the species specificity of pre-erythrocytic and erythrocytic whole organism vaccination using live parasites with anti-malarial drug attenuation. The cross-stage protection afforded by each vaccination strategy, and the possibility that immunity against one stage may be abrogated by exposure to other stages of both homologous and heterologous parasites was also assessed. The rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium vinckei lentum are to address these questions, as they offer the widest possible genetic distance between sub-species of malaria parasites infectious to rodents. It was found that both erythrocytic and pre-erythrocytic stage immunity generated by live, attenuated parasite vaccination have species-specific components, with pre-erythrocytic stage immunity offering a much broader pan-species protection. We show that the protection achieved following sporozoite inoculation with concurrent mefloquine treatment is almost entirely dependent of CD8(+) T-cells. Evidence is presented for cross-stage protection between erythrocytic and pre-erythrocytic stage vaccination. Finally, it is shown that, with these species, an erythrocytic stage infection of either a homologous or heterologous species following immunisation with pre-erythrocytic stages does not abrogate this immunity. This is the first direct comparison of the specificity and efficacy of erythrocytic and pre-erythrocytic stage whole organism vaccination strategies utilising the same parasite species pair.     

A retrospective evaluation of the role of T cells in the development of malaria vaccine

Due to the fact that the life cycle of malaria parasites is complex, undergoing both an extracellular and intracellular phases in its host, the human immune system has to mobilize both the humoral and cellular arms of immune responses to fight against this parasitic infection. Whereas humoral immunity is directed toward the extracellular stages which include sporozoites and merozoites, cell-mediated immunity (CMI), in which T cells play a major role, targets hepatic stages - liver stages - of the parasites. In this review, the role of T cells in protective immunity against liver stages of the malaria infection is being re-evaluated. Furthermore, this review intends to address how to translate the findings regarding the role of T cells obtained in experimental systems to actual development of malaria vaccine for humans.     

Structure of sinuses in the human lymph node

Summary A casting technique has been employed to display in three dimensions, the lymphatic microcirculation within the human lymph node. The casting compound filled the marginal sinus, and diffusely permeated the cortical lymphoid parenchyma. However, deep within the lymph node in the medullary region, the medium remained within the limits of the sinus walls. The casts showed well-defined channels appearing similar to vessels. These converged into larger vessels, which drained into efferent lymphatics leaving the node at the hilus.Electron microscopic examination showed that the outer wall of the marginal sinus and the trabecular side of trabecular sinuses had an intact, continuous endothelium with a basement membrane. However, gaps were present in the inner wall of the marginal sinus, as well as in the parenchymal wall of the trabecular sinus. In the medulla, the sinuses were lined by endothelial cells which appeared similar to macrophages. The sinus lining was incomplete and possessed numerous perforations. These observations indicated that sinus walls adjacent to connective tissue served as a barrier to cell movement, but those adjacent to a large lymphoid cell population had gaps, with cells in apparent transit between sinus lumen and parenchyma.     

Expression profiles of peroxiredoxin proteins of the rodent malaria parasite Plasmodium yoelii

Patterns of expression of the 2-Cys and 1-Cys peroxiredoxin (Prx) proteins of the rodent malaria parasite Plasmodium yoelii during its life cycle were observed by immunofluorescent antibody staining and confocal laser scanning microscopy. 2-Cys Prx was expressed in the parasite cytoplasm throughout the life cycle, and the thioredoxin (Trx)-peroxidase activity of 2-Cys Prx revealed with the recombinant protein suggested that the Prx is constitutively expressed and, thus, likely plays a housekeeping role in the parasite's intracellular redox control. In contrast, 1-Cys Prx showed stage-specific expression in blood-stage parasites. The limited expression of 1-Cys Prx in the trophozoite cytoplasm suggests that 1-Cys Prx may be involved in haemoglobin metabolism by the parasite, which generates a prooxidative haem iron and increases intracellular oxidative stress. The antioxidant activity of 1-Cys Prx was tested for its ability to protect yeast enolase against inactivation of the mixed-function oxidation system. Differential expression of the two Prx proteins during the erythrocytic and insect stages suggests the importance of these proteins in protecting parasites against oxidative stress, which is generated by the parasite's metabolism and also from the environment.     

Intralymphocytic schizonts associated with an initial infection of Saurocytozoon tupinambi in Tupinambis teguixin

An initial natural infection of Saurocytozoon tupinambi in a juvenile Tupinambis teguixin from Venezuela was studied for 131 days following capture of the host. Intralymphocytic parasites appeared in this sequence: small uninucleate and binucleate stages (days 1–31 and again on day 41); schizonts with 3–102 nuclei (days 8–14 and 29–35); immature gametocytes (days 29–35) and apparently mature gametocytes of Saurocytozoon tupinambi from day 41. Maximum parasitemia of trophozoites and binucleate schizonts occurred on day 4 when 11% of lymphocytes were infected. Maximum parasitemia by larger schizonts occurred on day 8 at 0.13% of lymphocytes, while maximum gametocytemia was found on day 49 with 16.4% of lymphocytes parasitized. Two types of schizonts were observed: intralymphocytic and the same type free of host cells, and fragments of varying size which may have been torn from capillary endothelium.Due to presence of concurrent infection by a small Plasmodium species, identity of intralymphocytic asexual stages with S. tupinambi cannot be established. Presence of asexual and sexual stages in the same type of host cells (lymphocytes and close derivatives), sequential appearance of trophozoites, schizonts and gametocytes over a period of 40 days, and correlated fluctuations in lymphocyte density suggest they are conspecific, and that Saurocytozoon, which has a plasmodiid type of sporogony may prove to further differ from leucocytozoids by presence of an asexual cycle in circulating blood cells.     

The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny,duplication rate and parasite virulence

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora‐related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub‐cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock‐down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3‐depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target.