Quantitative Bioluminescent Imaging of Pre-Erythrocytic Malaria Parasite Infection Using Luciferase-Expressing Plasmodium yoelii

The liver stages of Plasmodium parasites are important targets for the development of anti-malarial vaccine candidates and chemoprophylaxis approaches that aim to prevent clinical infection. Analyzing the impact of interventions on liver stages in the murine malaria model system Plasmodium yoelii has been cumbersome and requires terminal procedures. In vivo imaging of bioluminescent parasites has previously been shown to be an effective and non-invasive alternative to monitoring liver stage burden in the Plasmodium berghei model. Here we report the generation and characterization of a transgenic P. yoelii parasite expressing the reporter protein luciferase throughout the parasite life cycle. In vivo bioluminescent imaging of these parasites allows for quantitative analysis of P. yoelii liver stage burden and parasite development, which is comparable to quantitative RT-PCR analysis of liver infection. Using this system, we show that both BALB/cJ and C57BL/6 mice show comparable susceptibility to P. yoelii infection with sporozoites and that bioluminescent imaging can be used to monitor protective efficacy of attenuated parasite immunizations. Thus, this rapid, simple and noninvasive method for monitoring P. yoelii infection in the liver provides an efficient system to screen and evaluate the effects of anti-malarial interventions in vivo and in real-time.     

Imaging liver‐stage malaria parasites

Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High‐speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver‐stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver.     

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.     

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.     

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.     

Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden. The liver stage of Plasmodium falciparum infection is a leading target for immunological and pharmacological interventions. Therefore, novel approaches providing specific detection and isolation of live P. falciparum exoerythrocytic forms (EEFs) are warranted. Utilizing a recently generated parasite strain expressing green fluorescent protein (GFP) we established a method which, allows for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. Here, we show that EEFs can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Finally, we validated the practical applications of our approach by re-examining the importance of host ligand CD81 for hepatocyte infection by P. falciparum sporozoites in vitro and assessment of the inhibitory activity of anti-sporozoite antibodies. This methodology provides us with the tools to study both, the basic biology of the P. falciparum liver stage and the effects of host-derived factors on the development of P. falciparum EEFs.     

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 Vivo and In Vitro Characterization of a Plasmodium Liver Stage-Specific Promoter

Little is known about stage-specific gene regulation in Plasmodium parasites, in particular the liver stage of development. We have previously described in the Plasmodium berghei rodent model, a liver stage-specific (lisp2) gene promoter region, in vitro. Using a dual luminescence system, we now confirm the stage specificity of this promoter region also in vivo. Furthermore, by substitution and deletion analyses we have extended our in vitro characterization of important elements within the promoter region. Importantly, the dual luminescence system allows analyzing promoter constructs avoiding mouse-consuming cloning procedures of transgenic parasites. This makes extensive mutation and deletion studies a reasonable approach also in the malaria mouse model. Stage-specific expression constructs and parasite lines are extremely valuable tools for research on Plasmodium liver stage biology. Such reporter lines offer a promising opportunity for assessment of liver stage drugs, characterization of genetically attenuated parasites and liver stage-specific vaccines both in vivo and in vitro, and may be key for the generation of inducible systems.     

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.     

Inhibition of Plasmodium sporozoites infection by targeting the host cell

There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and Plasmodium yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion.     

Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge

The pre-erythrocytic stages of Plasmodium spp. are increasingly recognised as ideal targets for prophylactic vaccines and drug treatments. Intense research efforts in the last decade have been focused on in vitro culture and in vivo detection and quantification of liver stage parasites to assess the effects of candidate vaccines or drugs. Typically, the onset of blood stage parasitaemia is used as a surrogate endpoint to estimate the efficacy of vaccines and drugs targeting pre-erythrocytic parasite stages in animal models. However, this provides no information on the parasite burden in the liver after vaccination or treatment and therefore does not detect partial efficacy of any vaccine or drug candidates. Herein, we describe a quantitative RT-PCR method adapted to detect and quantitate Plasmodium yoelii liver stages in mice with increased sensitivity even after challenge with as few as 50 cryopreserved sporozoites (corresponding to approximately 5-10 freshly isolated sporozoites). We have validated our quantitative RT-PCR assay according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and established high reproducibility and accuracy. Our assay provides a rapid and reproducible assessment of liver stage parasite burden in rodent malaria models, thereby facilitating the evaluation of the efficacy of anti-malarial drugs or prophylactic vaccines with high precision and efficacy.     

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.     

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.     

Going live: A comparative analysis of the suitability of the RFP derivatives RedStar,mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.     

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.     

CSP—A Model for In Vivo Presentation of Plasmodium berghei Sporozoite Antigens by Hepatocytes

One target of protective immunity against the Plasmodium liver stage in BALB/c mice is represented by the circumsporozoite protein (CSP), and mainly involves its recognition by IFN-γ producing specific CD8+T-cells. In a previous in vitro study we showed that primary hepatocytes from BALB/c mice process Plasmodium berghei (Pb) CSP (PbCSP) and present CSP-derived peptides to specific H-2k^d restricted CD8+T-cells with subsequent killing of the presenting cells. We now extend these observations to an in vivo infection model in which infected hepatocytes and antigen specific T-cell clones are transferred into recipient mice inducing protection from sporozoite (SPZ) challenge. In addition, using a similar protocol, we suggest the capacity of hepatocytes in priming of naïve T-cells to provide protection, as further confirmed by induction of protection after depletion of cross-presenting dendritic cells (DCs) by cytochrome c (cyt c) treatment or using traversal deficient parasites. Our results clearly show that hepatocytes present Plasmodium CSP to specific-primed CD8+T-cells, and could also prime naïve T-cells, leading to protection from infection. These results could contribute to a better understanding of liver stage immune response and design of malaria vaccines.     

GFP‐targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites

Background information. The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo‐erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. Results. In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo‐erythrocytic schizogony in vitro, leading to impaired parasite maturation. Conclusions. Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red‐blood‐cell‐infective merozoites.     

Inhibitory effect of TNF-α on malaria pre-erythrocytic stage development: influence of host hepatocyte/parasite combinations

Background

The liver stages of malaria parasites are inhibited by cytokines such as interferon-γ or Interleukin (IL)-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO) and radical oxygen intermediates (ROI), which kill hepatic parasites. However, conflicting results were obtained with TNF-α possibly because of differences in the models used. We have reassessed the role of TNF-α in the different cellular systems used to study the Plasmodium pre-erythrocytic stages.

Methods and Findings

Human or mouse TNF-α were tested against human and rodent malaria parasites grown in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-α treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-α inhibition was mediated by a soluble factor present in the supernatant of TNF-α stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified.

Conclusions

Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection.