Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.     

Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis.     

In Vitro Alterations Do Not Reflect a Requirement for Host Cell Cycle Progression during Plasmodium Liver Stage Infection

Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection.     

Host cell death induced by the egress of intracellular <Emphasis Type="Italic">Plasmodium</Emphasis> parasites

Intracellular pathogens are known to inhibit host cell apoptosis efficiently to ensure their own survival. However, following replication within a cell, they typically need to egress in order to infect new cells. For a long time it was assumed that this happens by simply disrupting the host cell and in some cases, such as for Plasmodium-infected erythrocytes, this seems indeed to be true. However, recently it has been shown that in Plasmodium-infected hepatocytes, an ordered form of cell death is initiated. This cell death is parasite-dependent and can clearly be distinguished from apoptosis and necrosis. The key event, and point of no return, appears to be the rupture of the parasitophorous vacuole membrane (PVM). PVM disruption and host cell death depend on the activation of cysteine proteases. Whether these are of parasite or host cell origin seems to rely on the life cycle stage of the Plasmodium parasite and the corresponding host cell.     

Intracellular survival of apicomplexan parasites and host cell modification

The intracellular stages of apicomplexan parasites are known to extensively modify their host cells to ensure their own survival. Recently, considerable progress has been made in understanding the molecular details of these parasite-dependent effects for Plasmodium-, Toxoplasma- and Theileria-infected cells. We have begun to understand how Plasmodium liver stage parasites protect their host hepatocytes from apoptosis during parasite development and how they induce an ordered cell death at the end of the liver stage. Toxoplasma parasites are also known to regulate host cell survival pathways and it has been convincingly demonstrated that they block host cell major histocompatibility complex (MHC)-dependent antigen presentation of parasite epitopes to avoid cell-mediated immune responses. Theileria parasites are the masters of host cell modulation because their presence immortalises the infected cell. It is now accepted that multiple pathways are activated to induce Theileria-dependent host cell transformation. Although it is now known that similar host cell pathways are affected by the different parasites, the outcome for the infected cell varies considerably. Improved imaging techniques and new methods to control expression of parasite and host cell proteins will help us to analyse the molecular details of parasite-dependent host cell modifications.     

LISP1 is important for the egress of Plasmodium berghei parasites from liver cells

Most Apicomplexa are obligatory intracellular parasites that multiply inside a so-called parasitophorous vacuole (PV) formed upon parasite entry into the host cell. Plasmodium , the agent of malaria and the Apicomplexa most deadly to humans, multiplies in both hepatocytes and erythrocytes in the mammalian host. Although much has been learned on how Apicomplexa parasites invade host cells inside a PV, little is known of how they rupture the PV membrane and egress host cells. Here, we characterize a Plasmodium protein, called LISP1 ( li ver- s pecific p rotein 1), which is specifically involved in parasite egress from hepatocytes. LISP1 is expressed late during parasite development inside hepatocytes and locates at the PV membrane. Intracellular parasites deficient in LISP1 develop into hepatic merozoites, which display normal infectivity to erythrocytes. However, LISP1-deficient liver-stage parasites do not rupture the membrane of the PV and remain trapped inside hepatocytes. LISP1 is the first Plasmodium protein shown by gene targeting to be involved in the lysis of the PV membrane.     

Exit from host cells by the pathogenic parasite Toxoplasma gondii does not require motility

The process by which the intracellular parasite Toxoplasma gondii exits its host cell is central to its propagation and pathogenesis. Experimental induction of motility in intracellular parasites results in parasite egress, leading to the hypothesis that egress depends on the parasite's actin-dependent motility. Using a novel assay to monitor egress without experimental induction, we have established that inhibiting parasite motility does not block this process, although treatment with actin-disrupting drugs does delay egress. However, using an irreversible actin inhibitor, we show that this delay is due to the disruption of host cell actin alone, apparently resulting from the consequent loss of membrane tension. Accordingly, by manipulating osmotic pressure, we show that parasite egress is delayed by releasing membrane tension and promoted by increasing it. Therefore, without artificial induction, egress does not depend on parasite motility and can proceed by mechanical rupture of the host membrane.     

Disrupting a Plasmodium berghei putative phospholipase impairs efficient egress of merosomes

Merosome development is a very important part of the Plasmodium life cycle. It constitutes the last step of liver stage development after which merozoites egress to the bloodstream and invade red blood cells. Many parasite proteins have been shown to play key roles in this process. Phospholipases are known to be actively involved in membrane formation and remodeling. At least one phospholipase A2, localized to the parasitophorous vacuolar membrane, is known to be directly important for parasitophorous vacuolar membrane (PVM) rupture and subsequent release of merozoites. Here, we characterize a Plasmodium berghei phosphatidic acid (PA) preferring phospholipase A1 (PbPla1) homolog. C-terminal 3XHA-mCherry tagging revealed that PbPla1 is expressed in all life cycle stages except sporozoites and is present in the parasite’s cytoplasm. Targeted disruption of PbPla1 revealed its non-essentiality for the blood and mosquito stages. Pla1^? sporozoites were found to be late in their ability to establish successful blood stage infection in mice. While exoerythrocytic form development was found to be normal in vitro, a decrease in the number of merosomes was observed. PVM rupture in late exo-erythrocytic forms (EEFs) was quantified by counting the number of parasites with intact PVMs, and was found to be significantly higher in Pla1^?. Our findings indicate the role of PbPla1 in merosome release.     

A crucial role for the C‐terminal domain of exported protein 1 during the mosquito and hepatic stages of the Plasmodium berghei life cycle

Intracellular Plasmodium parasites develop inside a parasitophorous vacuole (PV), a specialised compartment enclosed by a membrane (PVM) that contains proteins of both host and parasite origin. Although exported protein 1 (EXP1) is one of the earliest described parasitic PVM proteins, its function throughout the Plasmodium life cycle remains insufficiently understood. Here, we show that whereas the N‐terminus of Plasmodium berghei EXP1 (PbEXP1) is essential for parasite survival in the blood, parasites lacking PbEXP1's entire C‐terminal (CT) domain replicate normally in the blood but cause less severe pathology than their wild‐type counterparts. Moreover, truncation of PbEXP1's CT domain not only impairs parasite development in the mosquito but also abrogates PbEXP1 localization to the PVM of intrahepatic parasites, severely limiting their replication and preventing their egress into the blood. Our findings highlight the importance of EXP1 during the Plasmodium life cycle and identify this protein as a promising target for antiplasmodial intervention.     

Plasmodium berghei sporozoites in nonreplicative vacuole are eliminated by a PI3P‐mediated autophagy‐independent pathway

The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium‐associated autophagy‐related (PAAR) response. This is characterised by a long‐lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3‐phosphate (PI3P)‐labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P‐labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3‐labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.     

Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito

Malaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.     

Toxoplasma gondii: induction of egress by the potassium ionophore nigericin

The obligate intracellular parasite Toxoplasma gondii is an important pathogen of humans and animals. Some of the devastating consequences of toxoplasmosis are in part due to the lysis of the host cell during parasite egress. The process of egress is poorly understood and since it is asynchronous in tissue culture its study has been limited to those conditions that induce it, such as artificial permeabilisation of the host cell and induction of calcium fluxes with ionophores. Given that permeabilisation leads to egress by the activation of motility upon a drop in host cell potassium concentration, we investigated whether the ionophore nigericin, which selectively causes efflux of potassium from the cell without the need for permeabilisation, would cause egress. Nigericin effectively causes intracellular parasites to exit their host cell within 30 min of treatment with the drug. Our results show that nigericin-induced egress depends on an efflux of potassium from the cell and requires phospholipase C function and parasite motility. This novel method of inducing and synchronising egress mimics the effect of artificial permeabilisation in all respects. Nevertheless, since the membrane remains intact during the treatment, in our nigericin-induced egress we are able to detect parasite-dependent permeabilisation of the host cell, a known step in induced egress. In addition, consistent with the model that loss of host cell potassium leads to egress through the activation of intraparasitic calcium fluxes, a previously isolated Toxoplasma mutant lacking a sodium hydrogen exchanger and defective in responding to calcium fluxes does not undergo nigericin-induced egress. Thus, the discovery that nigericin induces egress presents a novel assay that allows for the genetic and biochemical analysis of the signalling mechanisms that lead to the induction of motility and egress.     

The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii

The ability of intracellular parasites to monitor the viability of their host cells is essential for their survival. The protozoan parasite Toxoplasma gondii actively invades nucleated animal cells and replicates in their cytoplasm. Two to 3 days after infection, the parasite-filled host cell breaks down and the parasites leave to initiate infection of a new cell. Parasite egress from the host cell is triggered by rupture of the host plasma membrane and the ensuing reduction in the concentration of cytoplasmic potassium. The many other changes in host cell composition do not appear be used as triggers. The reduction in the host cell [K(+)] appears to activate a phospholipase C activity in Toxoplasma that, in turn, causes an increase in cytoplasmic [Ca(2+)] in the parasite. The latter appears to be necessary and sufficient for inducing egress, as buffering of cytoplasmic Ca(2+) blocks egress and calcium ionophores circumvent the need for a reduction of host cell [K(+)] and parasite phospholipase C activation. The increase in [Ca(2+)](C) brings about egress by the activation of at least two signaling pathways: the protein kinase TgCDPK1 and the calmodulin-dependent protein phosphatase calcineurin.     

Shared elements of host‐targeting pathways among apicomplexan parasites of differing lifestyles

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal‐mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host‐targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal‐mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal‐mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.     

The Plasmodium rhoptry associated protein complex is important for parasitophorous vacuole membrane structure and intraerythrocytic parasite growth

Plasmodium parasites must invade erythrocytes in order to cause the disease malaria. The invasion process involves the coordinated secretion of parasite proteins from apical organelles that include the rhoptries. The rhoptry is comprised of two compartments: the neck and the bulb. Rhoptry neck proteins are involved in host cell adhesion and formation of the tight junction that forms between the invading parasite and erythrocyte, whereas the role of rhoptry bulb proteins remains ill‐defined due to the lack of functional studies. In this study, we show that the rhoptry‐associated protein (RAP) complex is not required for rhoptry morphology or erythrocyte invasion. Instead, post‐invasion when the parasite is bounded by a parasitophorous vacuolar membrane (PVM), the RAP complex facilitates the survival of the parasite in its new intracellular environment. Consequently, conditional knockdown of members of the RAP complex leads to altered PVM structure, delayed intra‐erythrocytic growth, and reduced parasitaemias in infected mice. This study provides evidence that rhoptry bulb proteins localising to the parasite–host cell interface are not simply by‐products of the invasion process but contribute to the growth of Plasmodium in vivo.     

A perforin‐like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes

Successful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin‐like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore‐forming toxin, we conclude that rupture of the erythrocyte membraneis blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforin‐like protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.     

Ca2+‐mediated exocytosis of subtilisin‐like protease 1: a key step in egress of Plasmodium falciparum merozoites

Egress of Plasmodium falciparum merozoites from host erythrocytes is a critical step in multiplication of blood‐stage parasites. A cascade of proteolytic events plays a major role in degradation of membranes leading to egress of merozoites. However, the signals that regulate the temporal activation and/or secretion of proteases upon maturation of merozoites in intra‐erythrocytic schizonts remain unclear. Here, we have tested the role of intracellular Ca²⁺ in regulation of egress of P. falciparum merozoites from schizonts. A sharp rise in intracellular Ca²⁺ just before egress, observed by time‐lapse video microscopy, suggested a role for intracellular Ca²⁺ in this process. Chelation of intracellular Ca²⁺ with chelators such as BAPTA‐AM or inhibition of Ca²⁺ release from intracellular stores with a phospholipase C (PLC) inhibitor blocks merozoite egress. Interestingly, chelation of intracellular Ca²⁺ in schizonts was also found to block the discharge of a key protease PfSUB1 (subtilisin‐like protease 1) from exonemes of P. falciparum merozoites to parasitophorous vacuole (PV). This leads to inhibition of processing of PfSERA5 (serine repeat antigen 5) and a block in parasitophorous vacuolar membrane (PVM) rupture and merozoite egress. A complete understanding of the steps regulating egress of P. falciparum merozoites may provide novel targets for development of drugs that block egress and limit parasite growth.     

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

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.