Quantitative Proteomic Analysis of Simian Primary Hepatocytes Reveals Candidate Molecular Markers for Permissiveness to Relapsing Malaria Plasmodium cynomolgi

A major obstacle impeding malaria research is the lack of an in vitro system capable of supporting infection through the entire liver stage cycle of the parasite, including that of the dormant forms known as hypnozoites. Primary hepatocytes lose their liver specific functions in long‐term in vitro culture. The malaria parasite Plasmodium initiates infection in hepatocyte. This corresponds to the first step of clinically silent infection and development of malaria parasite Plasmodium in the liver. Thus, the liver stage is an ideal target for development of novel antimalarial interventions and vaccines. However, drug discovery against Plasmodium liver stage is severely hampered by the poor understanding of host–parasite interactions during the liver stage infection and development. In this study, tandem mass tag labeling based quantitative proteomic analysis is performed in simian primary hepatocytes cultured in three different systems of susceptibility to Plasmodium infection. The results display potential candidate molecular markers, including asialoglycoprotein receptor, apolipoproteins, squalene synthase, and scavenger receptor B1 (SR‐BI) that facilitate productive infection and full development in relapsing Plasmodium species. The identification of these candidate proteins required for constructive infection and development of hepatic malaria liver stages paves the way to explore them as therapeutic targets.     

Secreted protein with altered thrombospondin repeat (SPATR) is essential for asexual blood stages but not required for hepatocyte invasion by the malaria parasite Plasmodium berghei

Upon entering its mammalian host, the malaria parasite productively invades two distinct cell types, that is, hepatocytes and erythrocytes during which several adhesins/invasins are thought to be involved. Many surface-located proteins containing thrombospondin Type I repeat (TSR) which help establish host–parasite molecular crosstalk have been shown to be essential for mammalian infection. Previous reports indicated that antibodies produced against Plasmodium falciparum secreted protein with altered thrombospondin repeat (SPATR) block hepatocyte invasion by sporozoites but no genetic evidence of its contribution to invasion has been reported. After failing to generate Spatr knockout in Plasmodium berghei blood stages, a conditional mutagenesis system was employed. Here, we show that SPATR plays an essential role during parasite's blood stages. Mutant salivary gland sporozoites exhibit normal motility, hepatocyte invasion, liver stage development and rupture of the parasitophorous vacuole membrane resulting in merosome formation. But these mutant hepatic merozoites failed to establish a blood stage infection in vivo. We provide direct evidence that SPATR is not required for hepatocyte invasion but plays an essential role during the blood stages of P. berghei.     

AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver

The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA‐like protein, merozoite apical erythrocyte‐binding ligand (MAEBL). MAEBL‐deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages.     

Plasmodium yoelii inhibitor of cysteine proteases is exported to exomembrane structures and interacts with yoelipain‐2 during asexual blood‐stage development

Plasmodium falciparum (Pf) blood stages express falstatin, an inhibitor of cysteine proteases (ICP), which is implicated in regulating proteolysis during red blood cell infection. Recent data using the Plasmodium berghei rodent malaria model suggested an additional role for ICP in the infection of hepatocytes by sporozoites and during liver‐stage development. Here we further characterize the role of ICP in vivo during infection with Plasmodium yoelii (Py) and Pf. We found that Py‐ICP was refractory to targeted gene deletion indicating an essential function during asexual blood‐stage replication, but significant downregulation of ICP using a regulated system did not impact blood‐stage growth. Py‐ICP localized to vesicles within the asexual blood‐stage parasite cytoplasm, as well as the parasitophorous vacuole, and was exported to dynamic exomembrane structures in the infected RBC. In sporozoites, expression was observed in rhoptries, in addition to intracellular vesicles distinct from TRAP containing micronemes. During liver‐stage development, Py‐ICP was confined to the parasite compartment until the final phase of liver‐stage development when, after parasitophorous vacuolemembrane breakdown, it was released into the infected hepatocyte. Finally, we identified the cysteine protease yoelipain‐2 as a binding partner of Py‐ICP during blood‐stage infection. These data show that ICP may be important in regulating proteolytic processes during blood‐stage development, and is likely playing a role in liver stage‐hepatocyte interactions at the time of exoerythrocytic merozoite release.     

Phosphothioate oligodeoxynucleotides inhibit Plasmodium sporozoite gliding motility

Plasmodium sporozoites, transmitted to the mammalian host through a mosquito bite, travel to the liver, where they invade hepatocytes, and develop into a form that is then able to infect red blood cells. In spite of the importance of innate immunity in controlling microbial infections, almost nothing is known about its role during the liver stage of a malaria infection. Here, we tested whether synthetic CpG phosphothioate (PS) oligodeoxynucleotides (ODNs), which bind to Toll‐like receptor 9 (Tlr9), could have a protective effect on Plasmodium berghei infection in hepatocytes. Surprisingly, CpG PS‐ODNs potently impair P. berghei infection in hepatoma cell lines independently of Tlr9 activation. Indeed, not only CpG but also non‐CpG PS‐ODNs, which do not activate Tlr9, decreased parasite infection. Moreover, the ability of PS‐ODNs to impair infection was not due to an effect on the host but rather on the parasite itself. In fact, CpG PS‐ODNs, as well as non‐CpG PS‐ODNs, impair parasite gliding motility. Furthermore, our analysis reveals that PS‐ODNs inhibit parasite migration and invasion due to their negative charge, whereas development inside hepatocytes is undisturbed. Altogether, PS‐ODNs might represent a new class of prophylactic anti‐malaria agents, which hamper hepatocyte entry by Plasmodium sporozoites.     

L-FABP is a critical host factor for successful malaria liver stage development

The malaria parasite liver stage produces tens of thousands of red cell-infectious forms within its host hepatocyte. It is thought that the vacuole-enclosed parasite completely depends on the host cell for successful development but the molecular parasite-host cell interactions underlying this remarkable growth have remained elusive. Using a yeast two-hybrid screen and a yeast overexpression system we show that UIS3, a parasite protein essential for liver stage development, interacts directly with liver-fatty acid binding protein, L-FABP. Down-regulation of L-FABP expression in hepatocytes severely impairs parasite growth and overexpression of L-FABP promotes growth. This is the first identified direct liver stage-host cell protein interaction, providing a possible explanation for the importance of UIS3 in liver infection.     

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.     

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.     

A Plasmodium Phospholipase Is Involved in Disruption of the Liver Stage Parasitophorous Vacuole Membrane

The coordinated exit of intracellular pathogens from host cells is a process critical to the success and spread of an infection. While phospholipases have been shown to play important roles in bacteria host cell egress and virulence, their role in the release of intracellular eukaryotic parasites is largely unknown. We examined a malaria parasite protein with phospholipase activity and found it to be involved in hepatocyte egress. In hepatocytes, Plasmodium parasites are surrounded by a parasitophorous vacuole membrane (PVM), which must be disrupted before parasites are released into the blood. However, on a molecular basis, little is known about how the PVM is ruptured. We show that Plasmodium berghei phospholipase, PbPL, localizes to the PVM in infected hepatocytes. We provide evidence that parasites lacking PbPL undergo completely normal liver stage development until merozoites are produced but have a defect in egress from host hepatocytes. To investigate this further, we established a live-cell imaging-based assay, which enabled us to study the temporal dynamics of PVM rupture on a quantitative basis. Using this assay we could show that PbPL-deficient parasites exhibit impaired PVM rupture, resulting in delayed parasite egress. A wild-type phenotype could be re-established by gene complementation, demonstrating the specificity of the PbPL deletion phenotype. In conclusion, we have identified for the first time a Plasmodium phospholipase that is important for PVM rupture and in turn for parasite exit from the infected hepatocyte and therefore established a key role of a parasite phospholipase in egress.     

In vivo imaging of malaria parasites in the murine liver

The form of the malaria parasite inoculated by the mosquito, called the sporozoite, transforms inside the host liver into thousands of a new form of the parasite, called the merozoite, which infects erythrocytes. We present here a protocol to visualize in vivo the behavior of Plasmodium berghei parasites in the hepatic tissue of the murine host. The use of GFP-expressing parasites and a high-speed spinning disk confocal microscope allows for the acquisition of four-dimensional images, which provide a time lapse view of parasite displacement and development in tissue volumes. These data can be analyzed to give information on the early events of sporozoite penetration of the hepatic tissue, that is, sporozoite gliding in the liver sinusoids, crossing the sinusoidal barrier, gliding in the parenchyma and traversal of hepatocytes, and invasion of a final hepatocyte, as well as the terminal events of merosome and merozoite release from infected hepatocytes. Combined with the use of mice expressing fluorescent cell types or cell markers, the system will provide useful information not only on the primary infection process, but also on parasite interactions with the host immune cells in the liver.     

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite''s life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc_con, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.     

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.     

Feeling at home from arrival to departure: protein export and host cell remodelling during Plasmodium liver stage and gametocyte maturation

Obligate intracellular pathogens actively remodel their host cells to boost propagation, survival, and persistence. Plasmodium falciparum, the causative agent of the most severe form of malaria, assembles a complex secretory system in erythrocytes. Export of parasite factors to the erythrocyte membrane is essential for parasite sequestration from the blood circulation and a major factor for clinical complications in falciparum malaria. Historic and recent molecular reports show that host cell remodelling is not exclusive to P. falciparum and that parasite‐induced intra‐erythrocytic membrane structures and protein export occur in several Plasmodia. Comparative analyses of P. falciparum asexual and sexual blood stages and imaging of liver stages from transgenic murine Plasmodium species show that protein export occurs in all intracellular phases from liver infection to sexual differentiation, indicating that mammalian Plasmodium species evolved efficient strategies to renovate erythrocytes and hepatocytes according to the specific needs of each life cycle phase. While the repertoireof identified exported proteins is remarkably expanded in asexual P. falciparum blood stages, the putative export machinery and known targeting signatures are shared across life cycle stages. A better understanding of the molecular mechanisms underlying Plasmodium protein export could assist in designing novel strategies to interrupt transmission between Anopheles mosquitoes and humans.     

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.     

Calcium signal regulates temperature-dependent transformation of sporozoites in malaria parasite development

The infection by the malaria parasite of its mammalian host is initiated by the asexual reproduction of the parasite within the host hepatocyte. Before the reproduction, the elongated sporozoites undergo a depolarizing morphogenesis to the spherical exo-erythrocytic form (EEF). This change can be induced in vitro by shifting the environmental conditions, in the absence of host hepatocytes. Using rodent malaria parasites expressing a FRET-based calcium sensor, YC3.60, we observed that the intracellular calcium increased at the center of the bulbous structure during sporozoite transformation. Modulators of intracellular calcium signaling (A23187 and W-7) accelerated the sporozoite-rounding process. These data suggest that calcium signaling regulates the morphological development of the malaria parasite sporozoite to the EEF, and support a fundamental role for calcium as a universal transducer of external stimuli in the parasitic life cycle.     

Malaria parasite pre-erythrocytic stage infection: gliding and hiding

In malaria, the red blood cell-infectious form of the Plasmodium parasite causes illness and the possible death of infected hosts. The initial infection in the liver caused by the mosquito-borne sporozoite parasite stage, however, causes little pathology and no symptoms. Nevertheless, pre-erythrocytic parasite stages are attracting passionate research efforts not least because they are the most promising targets for malaria vaccine development. Here, we review how the infectious sporozoite makes its way to the liver and subsequently develops within hepatocytes. We discuss the factors, both parasite and host, involved in the interactions that occur during this "silent" phase of infection.     

Importance of infection of haemosporidia blood parasites during different life history stages for long‐term reproductive fitness of collared flycatchers

The interaction between birds and haemosporidia blood parasites is a well‐used system in the study of parasite biology. However, where, when and how parasites are transmitted is often unclear and defining parasite transmission dynamics is essential because of how they influence parasite‐mediated costs to the host. In this study, we used cross‐sectional and longitudinal data taken from a collared flycatcher Ficedula albicollis population to investigate the temporal dynamics of haemosporidia parasite infection and parasite‐mediated costs to host fitness. We investigated host–parasite interactions starting at the nestling stage of the bird's life‐cycle and then followed their progress over three breeding attempts to quantify their fitness – measured as the number of offspring they produced that recruited back into the breeding population. We found that the majority of haemosporidia blood parasite infections occurred within the first year of life and that the most common parasite lineages that infected the breeding population also infected juvenile birds in the natal environment. Moreover, our findings suggest that collared flycatcher nestlings in poorer condition could be at a higher risk of haemosporidia blood parasite infection. In this study, only female and not male bird fitness was adversely affected by parasite infection and the cost of infection on female fitness depended on the timing of transmission. In conclusion, our study indicates that in collared flycatchers, early‐life is potentially important for many of the interactions with haemosporidia parasite lineages, and evidence of parasite‐mediated costs to fitness suggest that these parasites may have influenced the host population dynamics.