Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity.

Introduction: Plasmodium sporozoites (SPZ) inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites. Methods: Considering that IL-6 as a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development. Results and Discussion: Though IL-6 transgenic SPZ developed into exo-erythrocytic forms in hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei SPZ elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious SPZ challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity.

Plasmodium sporozoites require the protein B9 to invade hepatocytes.

Plasmodium sporozoites are transmitted to a mammalian host during blood feeding by an infected mosquito and invade hepatocytes for initial replication of the parasite into thousands of erythrocyte-invasive merozoites. Here we report that the B9 protein, a member of the 6-cysteine domain protein family, is secreted from sporozoite micronemes and is required for productive invasion of hepatocytes. The N-terminus of B9 forms a beta-propeller domain structurally related to CyRPA, a cysteine-rich protein forming an essential invasion complex in Plasmodium falciparum merozoites. The beta-propeller domain of B9 is essential for sporozoite infectivity and interacts with the 6-cysteine proteins P36 and P52 in a heterologous expression system. Our results suggest that, despite using distinct sets of parasite and host entry factors, Plasmodium sporozoites and merozoites may share common structural modules to assemble protein complexes for invasion of host cells.

The AMA1-RON complex drives Plasmodium sporozoite invasion in the mosquito and mammalian hosts.

Plasmodium sporozoites that are transmitted by blood-feeding female Anopheles mosquitoes invade hepatocytes for an initial round of intracellular replication, leading to the release of merozoites that invade and multiply within red blood cells. Sporozoites and merozoites share a number of proteins that are expressed by both stages, including the Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck Proteins (RONs). Although AMA1 and RONs are essential for merozoite invasion of erythrocytes during asexual blood stage replication of the parasite, their function in sporozoites was still unclear. Here we show that AMA1 interacts with RONs in mature sporozoites. By using DiCre-mediated conditional gene deletion in P. berghei, we demonstrate that loss of AMA1, RON2 or RON4 in sporozoites impairs colonization of the mosquito salivary glands and invasion of mammalian hepatocytes, without affecting transcellular parasite migration. Three-dimensional electron microscopy data showed that sporozoites enter salivary gland cells through a ring-like structure and by forming a transient vacuole. The absence of a functional AMA1-RON complex led to an altered morphology of the entry junction, associated with epithelial cell damage. Our data establish that AMA1 and RONs facilitate host cell invasion across Plasmodium invasive stages, and suggest that sporozoites use the AMA1-RON complex to efficiently and safely enter the mosquito salivary glands to ensure successful parasite transmission. These results open up the possibility of targeting the AMA1-RON complex for transmission-blocking antimalarial strategies.

A toolbox for conditional control of gene expression in apicomplexan parasites.

Apicomplexan parasites encompass diverse pathogens for humans and animals, including the causative agents of malaria and toxoplasmosis, Plasmodium spp. and Toxoplasma gondii. Genetic manipulation of these parasites has become central to explore parasite biology, unravel gene function and identify new targets for therapeutic strategies. Tremendous progress has been achieved over the past years with the advent of next generation sequencing and powerful genome editing methods. In particular, various methods for conditional gene expression have been developed in both Plasmodium and Toxoplasma to knockout or knockdown essential genes, or for inducible expression of master developmental regulators or mutant versions of proteins. Conditional gene expression can be achieved at three distinct levels. At the DNA level, inducible site-specific recombinases allow conditional genome editing. At the RNA level, regulation can be achieved during transcription, using stage-specific or regulatable promoters, or post-transcriptionally through alteration of mRNA stability or translation. At the protein level, several systems have been developed for inducible degradation or displacement of a protein of interest. In this review, we provide an overview of current systems for conditional control of gene expression in Plasmodium and Toxoplasma parasites, highlighting the advantages and limitations of each approach.

Conditional Gene Deletion in Mammalian and Mosquito Stages of Plasmodium berghei Using Dimerizable Cre Recombinase.

Genome editing in the malaria parasite Plasmodium relies on homologous recombination and requires parasite transfection in asexual blood stages. Therefore, conditional genetic approaches are needed to delete genes that are essential during blood stage replication. Among these, the dimerizable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene knockout in Plasmodium parasites. In this system, the Cre recombinase is expressed in the form of two separate, enzymatically inactive polypeptides. Rapamycin-induced heterodimerization of the two components restores recombinase activity, leading to site-specific excision of floxed DNA sequences. Here, we describe methods to generate genetically modified DiCre-expressing Plasmodium berghei mutants by introducing Lox sites upstream and downstream of a gene of interest and to induce conditional excision of the floxed gene in different stages of the parasite life cycle. Administration of rapamycin to P. berghei-infected mice allows conditional gene deletion in the asexual erythrocytic stages. Rapamycin-induced gene excision can also be achieved in P. berghei sexual blood stages prior to transmission to mosquitoes, or during sporogony by treating P. berghei-infected mosquitoes, both methods allowing functional studies in P. berghei mosquito stages. Finally, rapamycin can be administered to in vitro cell cultures in order to induce gene excision in P. berghei liver stages. Subsequent phenotyping allows for the analysis of essential gene function across the parasite life cycle stages.

Plasmodium sporozoites on the move: Switching from cell traversal to productive invasion of hepatocytes.

Parasites of the genus Plasmodium, the etiological agent of malaria, are transmitted through the bite of anopheline mosquitoes, which deposit sporozoites into the host skin. Sporozoites migrate through the dermis, enter the bloodstream, and rapidly traffic to the liver. They cross the liver sinusoidal barrier and traverse several hepatocytes before switching to productive invasion of a final one for replication inside a parasitophorous vacuole. Cell traversal and productive invasion are functionally independent processes that require proteins secreted from specialized secretory organelles known as micronemes. In this review, we summarize the current understanding of how sporozoites traverse through cells and productively invade hepatocytes, and discuss the role of environmental sensing in switching from a migratory to an invasive state. We propose that timely controlled secretion of distinct microneme subsets could play a key role in successful migration and infection of hepatocytes. A better understanding of these essential biological features of the Plasmodium sporozoite may contribute to the development of new strategies to fight against the very first and asymptomatic stage of malaria.

The dimerisable Cre recombinase allows conditional genome editing in the mosquito stages of Plasmodium berghei.

Asexual blood stages of the malaria parasite are readily amenable to genetic modification via homologous recombination, allowing functional studies of parasite genes that are not essential in this part of the life cycle. However, conventional reverse genetics cannot be applied for the functional analysis of genes that are essential during asexual blood-stage replication. Various strategies have been developed for conditional mutagenesis of Plasmodium, including recombinase-based gene deletion, regulatable promoters, and mRNA or protein destabilization systems. Among these, the dimerisable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene deletion in P. falciparum. In this system, the bacteriophage Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin-binding protein. Rapamycin-induced heterodimerization of the two components restores recombinase activity. We have implemented the DiCre system in the rodent malaria parasite P. berghei, and show that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle.

A genetically hmgb2 attenuated blood stage P. berghei induces crossed-long live protection.

Due to the lack of efficiency to control malaria elicited by sub-unit vaccine preparations, vaccination with live-attenuated Plasmodium parasite as reported 70 years ago with irradiated sporozoites regained recently a significant interest. The complex life cycle of the parasite and the different stages of development between mammal host and anopheles do not help to propose an easy vaccine strategy. In order to achieve a complete long-lasting protection against Plasmodium infection and disease, we considered a genetically attenuated blood stage parasite in the hmgb2 gene coding for the high-mobility-group-box 2 (HMGB2). This Plasmodium protein belongs to the HMGB family and hold as the mammal proteins, a double life since it acts first as a nuclear factor involved in chromatin remodelling and transcription regulation and second, when secreted as an active pro-inflammatory alarmin protein. Even though the number of reports on whole living attenuated blood stage parasites is limited when compared to attenuated sporozoites, the results reported with Plasmodium KO parasites are very encouraging. In this report, we present a novel strategy based on pre-immunization with Δhmgb2PbNK65 parasitized red blood cells that confer long-lasting protection in a murine experimental cerebral malaria model against two highly pathogenic homologous and heterologous parasites.

Preparing for Transmission: Gene Regulation in Plasmodium Sporozoites.

Plasmodium sporozoites are transmitted to mammals by anopheline mosquitoes and first infect the liver, where they transform into replicative exoerythrocytic forms, which subsequently release thousands of merozoites that invade erythrocytes and initiate the malaria disease. In some species, sporozoites can transform into dormant hypnozoites in the liver, which cause malaria relapses upon reactivation. Transmission from the insect vector to a mammalian host is a critical step of the parasite life cycle, and requires tightly regulated gene expression. Sporozoites are formed inside oocysts in the mosquito midgut and become fully infectious after colonization of the insect salivary glands, where they remain quiescent until transmission. Parasite maturation into infectious sporozoites is associated with reprogramming of the sporozoite transcriptome and proteome, which depends on multiple layers of transcriptional and post-transcriptional regulatory mechanisms. An emerging scheme is that gene expression in Plasmodium sporozoites is controlled by alternating waves of transcription activity and translational repression, which shape the parasite RNA and protein repertoires for successful transition from the mosquito vector to the mammalian host.

Identification of Plasmodium falciparum nuclear proteins by mass spectrometry and proposed protein annotation.

The nuclear proteome of Plasmodium falciparum results from the continual shuttle of proteins between the cell cytoplasm-nucleus and vice versa. Using shotgun proteomics tools, we explored the nuclear proteins of mixed populations of Plasmodium falciparum extracted from infected erythrocytes. We combined GeLC-MS/MS and 2D-LC-MS/MS with a peptide ion exclusion procedure in order to increase the detection of low abundant proteins such as those involved in gene expression. We have identified 446 nuclear proteins covering all expected nuclear protein families involved in gene regulation. All structural ribosomal (40S and 60S) proteins were identified which is consistent with the nuclear localization of ribosomal biogenesis. Proteins involved in the translation machinery were also found suggesting that translational events might occur in the nucleus in P. falciparum as previously hypothesized in eukaryotes. These data were compared to the protein list established by PlasmoDB and submitted to Plasmobase a recently reported Plasmodium annotation website to propose new functional putative annotation of several unknown proteins found in the nuclear extracts.

Plasmodium P36 determines host cell receptor usage during sporozoite invasion.

Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, first infect the liver for an initial round of replication before the emergence of pathogenic blood stages. Sporozoites represent attractive targets for antimalarial preventive strategies, yet the mechanisms of parasite entry into hepatocytes remain poorly understood. Here we show that the two main species causing malaria in humans, Plasmodium falciparum and Plasmodium vivax, rely on two distinct host cell surface proteins, CD81 and the Scavenger Receptor BI (SR-BI), respectively, to infect hepatocytes. By contrast, CD81 and SR-BI fulfil redundant functions during infection by the rodent parasite P. berghei. Genetic analysis of sporozoite factors reveals the 6-cysteine domain protein P36 as a major parasite determinant of host cell receptor usage. Our data provide molecular insights into the invasion pathways used by different malaria parasites to infect hepatocytes, and establish a functional link between a sporozoite putative ligand and host cell receptors.

Malaria Sporozoites Traverse Host Cells within Transient Vacuoles.

Plasmodium sporozoites are deposited in the host skin by Anopheles mosquitoes. The parasites migrate from the dermis to the liver, where they invade hepatocytes through a moving junction (MJ) to form a replicative parasitophorous vacuole (PV). Malaria sporozoites need to traverse cells during progression through host tissues, a process requiring parasite perforin-like protein 1 (PLP1). We find that sporozoites traverse cells inside transient vacuoles that precede PV formation. Sporozoites initially invade cells inside transient vacuoles by an active MJ-independent process that does not require vacuole membrane remodeling or release of parasite secretory organelles typically involved in invasion. Sporozoites use pH sensing and PLP1 to exit these vacuoles and avoid degradation by host lysosomes. Next, parasites enter the MJ-dependent PV, which has a different membrane composition, precluding lysosome fusion. The malaria parasite has thus evolved different strategies to evade host cell defense and establish an intracellular niche for replication.

Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity.

Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (Δhmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in Δhmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4(+) and CD8(+) T cells, including CD8(+) granzyme B(+) and CD8(+) IFN-γ(+) cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria.

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.

A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites.

Experimental genetics have been widely used to explore the biology of the malaria parasites. The rodent parasites Plasmodium berghei and less frequently P. yoelii are commonly utilised, as their complete life cycle can be reproduced in the laboratory and because they are genetically tractable via homologous recombination. However, due to the limited number of drug-selectable markers, multiple modifications of the parasite genome are difficult to achieve and require large numbers of mice. Here we describe a novel strategy that combines positive-negative drug selection and flow cytometry-assisted sorting of fluorescent parasites for the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutant parasites expressing a GFP or a GFP-luciferase cassette, using minimal numbers of mice. We further illustrate how this new strategy facilitates phenotypic analysis of genetically modified parasites by fluorescence and bioluminescence imaging of P. berghei mutants arrested during liver stage development.

Post-transcriptional silencing of UIS4 in Plasmodium berghei sporozoites is important for host switch.

Plasmodium sporozoites are transmitted by mosquitoes and first infect hepatocytes of their mammalian host, wherein they develop as liver stages, surrounded by the parasitophorous vacuole membrane (PVM). The parasite must rapidly adapt to its changing environment after switching host. Shortly after invasion, the PVM is remodelled by insertion of essential parasite proteins of the early transcribed membrane protein family such as UIS4. Here, using the rodent malaria model Plasmodium berghei, we show that transcripts encoding UIS4 are stored in a translationally repressed state in sporozoites, allowing UIS4 protein synthesis only after host cell invasion. Using a series of reporter transgenic parasite lines we could demonstrate that the open reading frame of UIS4 mRNA is critical for gene silencing, whereas the 5' and 3' untranslated regions are dispensable. Our data further indicate that the UIS4 translational repression machinery is present only in mature sporozoites in the mosquito salivary glands, and that premature expression of UIS4 protein results in a loss of parasite infectivity. Our findings reveal the importance of specific post-transcriptional control in sporozoites, and establish that host switch requires high levels of translationally silent UIS4 RNA, which permits stage conversion, yet avoids premature expression of this liver stage-specific protein.

High-mobility-group box nuclear factors of Plasmodium falciparum.

In eukaryotes, the high-mobility-group (HMG) nuclear factors are highly conserved throughout evolution and are divided into three families, including HGMB, characterized by an HMG box domain. Some HMGB factors are DNA structure specific and preferentially interact with distorted DNA sequences, trigger DNA bending, and hence facilitate the binding of nucleoprotein complexes that in turn activate or repress transcription. In Plasmodium falciparum, two HMGB factors were predicted: PfHMGB1 and PfHMGB2. They are small proteins, under 100 amino acids long, encompassing a characteristic HMG box domain closely related to box B of metazoan factors, which comprises two HMG box domains, A and B, in tandem. Computational analyses supported the conclusion that the Plasmodium proteins were genuine architectural HMGB factors, and in vitro analyses performed with both recombinant proteins established that they were able to interact with distorted DNA structures and bend linear DNA with different affinities. These proteins were detected in both asexual- and gametocyte-stage cells in Western blotting experiments and mainly in the parasite nuclei. PfHMGB1 is preferentially expressed in asexual erythrocytic stages and PfHMGB2 in gametocytes, in good correlation with transcript levels of expression. Finally, immunofluorescence studies revealed differential subcellular localizations: both factors were observed in the nucleus of asexual- and sexual-stage cells, and PfHMGB2 was also detected in the cytoplasm of gametocytes. In conclusion, in light of differences in their levels of expression, subcellular localizations, and capacities for binding and bending DNA, these factors are likely to play nonredundant roles in transcriptional regulation of Plasmodium development in erythrocytes.

PfMyb1, a Plasmodium falciparum transcription factor, is required for intra-erythrocytic growth and controls key genes for cell cycle regulation.

During the complex life cycle of Plasmodium falciparum, divided between mosquito and human hosts, the regulation of morphologic changes implies a fine control of transcriptional regulation. Transcriptional control, however, and in particular its molecular actors, transcription factors and regulatory motifs, are as yet poorly described in Plasmodium. In order to decipher the molecular mechanisms implicated in transcriptional regulation, a transcription factor belonging to the tryptophan cluster family was studied. In a previous work, the PfMyb1 protein, contained in nuclear extracts, was shown to have DNA binding activity and to interact specifically with myb regulatory elements. We used long pfmyb1 double-stranded RNA (dsRNA) to interfere with the cognate messenger expression. Parasite cultures treated with pfmyb1 dsRNA exhibited a 40% growth inhibition when compared with either untreated cultures or cultures treated with unrelated dsRNA, and parasite mortality occurred during trophozoite to schizont transition. In addition, the pfmyb1 transcript and protein decreased by as much as 80% in treated trophozoite cultures at the time of their maximum expression. The global effect of this partial loss of transcript and protein was investigated using a thematic DNA microarray encompassing genes involved in signal transduction, cell cycle and transcriptional regulation. SAM software enabled us to identify several genes that were differentially expressed and probably directly or indirectly under the control of PfMyb1. Using chromatin immuno-precipitation, we demonstrated that PfMyb1 binds, within the parasite nuclei, to several promoters and therefore participates directly in the transcriptional regulation of the corresponding genes. This study provides the first evidence of a regulation network involving a Plasmodium transcription factor.

Transcriptome of 3D7 and its gametocyte-less derivative F12 Plasmodium falciparum clones during erythrocytic development using a gene-specific microarray assigned to gene regulation, cell cycle and transcription factors.

During the complex life cycle of Plasmodium falciparum, through mosquito and human, the erythrocytic cycle is responsible for malarial disease and transmission. The regulation of events that occur during parasite development, such as proliferation and differentiation, implies a fine control of transcriptional activities that in turn governs the expression profiles of sets of genes. Pathways that underline gametocyte commitment are yet poorly understood even though kinases and transcription factors have been assumed to play a crucial role in this event. In order to understand the molecular mechanisms controlling the variation of gene expression profiles that might participate in early gametocytogenesis, the transcriptome of two clones, 3D7 and its gametocyte-less derivative F12, was compared at five time points of the erythrocytic asexual development. We have used a thematic DNA microarray containing 150 PCR fragments, representative of P. falciparum genes involved in signal transduction, cell cycle and transcriptional regulation. We identified several genes eliciting different expression profiles among which some implicated in gene regulation or encoding putative transcription factors. The differential expression of transcription factor and kinase transcripts observed in the two clones may enlighten genes that might have a role in impairment of the early gametocytogenesis of the F12 clone.