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.     

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.     

Plasmodium 6-cysteine proteins determine the commitment of sporozoites to liver-infection

Plasmodium sporozoites travel a long way from the site where they are released by a mosquito bite to the liver, where they infect hepatocytes and develop into erythrocyte-invasive forms. The success of this infection depends on the ability of the sporozoites to correctly recognize the hepatocyte as a target and change their behavior from migration to infection. However, how this change is accomplished remains incompletely understood. In this paper, we report that 6-cysteine protein family members expressed in sporozoites including B9 are responsible for this ability. Experiments on parasites using double knockouts of B9 and SPECT2, which is essential for sporozoite to migrate through the hepatocyte, showed that the parasites lacked the capacity to stop migration. This finding suggests that interactions between these parasite proteins and hepatocyte-specific cell surface ligands mediate correct recognition of hepatocytes by sporozoites, which is an essential step in malaria transmission to humans.     

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 Cysteine Protease Inhibitor of Plasmodium berghei Is Essential for Exo-erythrocytic Development

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.     

Development of an In Vitro Assay and Demonstration of Plasmodium berghei Liver-Stage Inhibition by TRAP-Specific CD8+ T Cells

The development of an efficacious vaccine against the Plasmodium parasite remains a top priority. Previous research has demonstrated the ability of a prime-boost virally vectored sub-unit vaccination regimen, delivering the liver-stage expressed malaria antigen TRAP, to produce high levels of antigen-specific T cells. The liver-stage of malaria is the main target of T cell-mediated immunity, yet a major challenge in assessing new T cell inducing vaccines has been the lack of a suitable pre-clinical assay. We have developed a flow-cytometry based in vitro T cell killing assay using a mouse hepatoma cell line, Hepa1-6, and Plasmodium berghei GFP expressing sporozoites. Using this assay, P. berghei TRAP-specific CD8⁺ T cell enriched splenocytes were shown to inhibit liver-stage parasites in an effector-to-target ratio dependent manner. Further development of this assay using human hepatocytes and P. falciparum would provide a new method to pre-clinically screen vaccine candidates and to elucidate mechanisms of protection in vitro.     

CD81 is required for rhoptry discharge during host cell invasion by Plasmodium yoelii sporozoites

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and first infect the liver of their mammalian host, where they develop as liver stages before the onset of erythrocytic infection and malaria symptoms. Sporozoite entry into hepatocytes is an attractive target for anti‐malarial prophylactic strategies but remains poorly understood at the molecular level. Apicomplexan parasites invade host cells by forming a parasitophorous vacuole that is essential for parasite development, a process that involves secretion of apical organelles called rhoptries. We previously reported that the host membrane protein CD81 is required for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 acts at an early stage of infection, possibly at the entry step, but the mechanisms involved are still unknown. To investigate the role of CD81 during sporozoite entry, we generated transgenic P. yoelii parasites expressing fluorescent versions of three known rhoptry proteins, RON2, RON4 and RAP2/3. We observed that RON2 and RON4 are lost following rhoptry discharge during merozoite and sporozoite entry. In contrast, our data indicate that RAP2/3 is secreted into the parasitophorous vacuole during infection. We further show that sporozoite rhoptry discharge occurs only in the presence of CD81, providing the first direct evidence for a role of CD81 during sporozoite productive invasion.     

Role of Plasmodium berghei cGMP-dependent Protein Kinase in Late Liver Stage Development

The liver is the first organ infected by Plasmodium sporozoites during malaria infection. In the infected hepatocytes, sporozoites undergo a complex developmental program to eventually generate hepatic merozoites that are released into the bloodstream in membrane-bound vesicles termed merosomes. Parasites blocked at an early developmental stage inside hepatocytes elicit a protective host immune response, making them attractive targets in the effort to develop a pre-erythrocytic stage vaccine. Here, we generated parasites blocked at a late developmental stage inside hepatocytes by conditionally disrupting the Plasmodium berghei cGMP-dependent protein kinase in sporozoites. Mutant sporozoites are able to invade hepatocytes and undergo intracellular development. However, they remain blocked as late liver stages that do not release merosomes into the medium. These late arrested liver stages induce protection in immunized animals. This suggests that, similar to the well studied early liver stages, late stage liver stages too can confer protection from sporozoite challenge.     

Multidrug ATP‐binding cassette transporters are essential for hepatic development of Plasmodium sporozoites

Multidrug resistance‐associated proteins (MRPs) belong to the C‐family of ATP‐binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug‐sensitivity profiles as wild type parasites. We show that MRP1‐deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2‐deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development.     

Research Article: Characterization of the ATG8-conjugation system in 2 Plasmodium species with special focus on the liver stage: Possible linkage between the apicoplastic and autophagic systems?

Plasmodium parasites successfully colonize different habitats within mammals and mosquitoes, and adaptation to various environments is accompanied by changes in their organelle composition and size. Previously, we observed that during hepatocyte infection, Plasmodium discards organelles involved in invasion and expands those implicated in biosynthetic pathways. We hypothesized that this process is regulated by autophagy. Plasmodium spp. possess a rudimentary set of known autophagy-related proteins that includes the ortholog of yeast Atg8. In this study, we analyzed the activity of the ATG8-conjugation pathway over the course of the lifecycle of Plasmodium falciparum and during the liver stage of Plasmodium berghei. We engineered a transgenic P. falciparum strain expressing mCherry-PfATG8. These transgenic parasites expressed mCherry-PfATG8 in human hepatocytes and erythrocytes, and in the midgut and salivary glands of Anopheles mosquitoes. In all observed stages, mCherry-PfATG8 was localized to tubular structures. Our EM and colocalization studies done in P. berghei showed the association of PbATG8 on the limiting membranes of the endosymbiont-derived plastid-like organelle known as the apicoplast. Interestingly, during parasite replication in hepatocytes, the association of PbATG8 with the apicoplast increases as this organelle expands in size. PbATG3, PbATG7 and PbATG8 are cotranscribed in all parasitic stages. Molecular analysis of PbATG8 and PbATG3 revealed a novel mechanism of interaction compared with that observed for other orthologs. This is further supported by the inability of Plasmodium ATG8 to functionally complement atg8Δ yeast or localize to autophagosomes in starved mammalian cells. Altogether, these data suggests a unique role for the ATG8-conjugation system in Plasmodium parasites.     

Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection

Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases. PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACalpha and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.     

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.     

HIV Treatments Reduce Malaria Liver Stage Burden in a Non-Human Primate Model of Malaria Infection at Clinically Relevant Concentrations In Vivo

We have previously shown that the HIV protease inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim sulfamethoxazole (TMP-SMX) inhibit Plasmodium liver stages in rodent malarias and in vitro in P. falciparum. Since clinically relevant levels are better achieved in the non-human-primate model, and since Plasmodium knowlesi is an accepted animal model for the study of liver stages of malaria as a surrogate for P. falciparum infection, we investigated the antimalarial activity of these drugs on Plasmodium knowlesi liver stages in rhesus macaques. We demonstrate that TMP-SMX and TMP-SMX+LPV-RTV (in combination), but not LPV-RTV alone, inhibit liver stage parasite development. Because drugs that inhibit the clinically silent liver stages target parasites when they are present in lower numbers, these results may have implications for eradication efforts.     

d-Glucose concentration is the key factor facilitating liver stage maturation of Plasmodium

The course of malaria infection in mammals begins with transmission of Plasmodium sporozoites into the skin by Anopheles mosquitoes, followed by migration of the sporozoites to the liver. As no symptoms present until hepatic merozoites are released and until they infect erythrocytes in the blood vessels, sporozoites and liver-stage (LS) parasites are promising targets for anti-malaria drugs aiming to prevent mosquito-to-mammal transmission. In vitro LS parasite development system is useful in the screening of candidate drugs on LS parasite development and the elucidation of its underlying molecular mechanisms, which remain unclear. Using rodent malaria parasites (Plasmodium berghei) as a model, this study aimed to develop an optimal in vitro LS culture system for the full maturation of the LS parasite into the hepatic merozoite, the next infective stage in parasite development. As the development of this system required measurement of maturation, a novel quantitative index of LS parasite maturation based on the expression pattern of liver-specific protein 2 (LISP2) was first developed. The use of this index for comparing the effect of incubation in different culture media on LS maturation revealed that the d-glucose concentration of the culture medium is the key factor promoting parasite development in hepatocytes and that a d-glucose concentration of 2000 mg/L/day is the threshold concentration at which the maturation of P. berghei into infective hepatic merozoites is achieved. These findings can be utilized to optimize a human malaria LS culture system for drug discovery.     

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.     

A mating-induced reproductive gene promotes Anopheles tolerance to Plasmodium falciparum infection

Anopheles mosquitoes have transmitted Plasmodium parasites for millions of years, yet it remains unclear whether they suffer fitness costs to infection. Here we report that the fecundity of virgin and mated females of two important vectors—Anopheles gambiae and Anopheles stephensi—is not affected by infection with Plasmodium falciparum, demonstrating that these human malaria parasites do not inflict this reproductive cost on their natural mosquito hosts. Additionally, parasite development is not impacted by mating status. However, in field studies using different P. falciparum isolates in Anopheles coluzzii, we find that Mating-Induced Stimulator of Oogenesis (MISO), a female reproductive gene strongly induced after mating by the sexual transfer of the steroid hormone 20-hydroxyecdysone (20E), protects females from incurring fecundity costs to infection. MISO-silenced females produce fewer eggs as they become increasingly infected with P. falciparum, while parasite development is not impacted by this gene silencing. Interestingly, previous work had shown that sexual transfer of 20E has specifically evolved in Cellia species of the Anopheles genus, driving the co-adaptation of MISO. Our data therefore suggest that evolution of male-female sexual interactions may have promoted Anopheles tolerance to P. falciparum infection in the Cellia subgenus, which comprises the most important malaria vectors.     

Direct Tests of Enzymatic Heme Degradation by the Malaria Parasite Plasmodium falciparum

Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO''s lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection.