An Invariant Protein That Colocalizes With VAR2CSA on Plasmodium falciparum-Infected Red Cells Binds to Chondroitin Sulfate A.

BACKGROUND: Plasmodium falciparum-infected red blood cells (iRBCs) bind and sequester in deep vascular beds, causing malaria-related disease and death. In pregnant women, VAR2CSA binds to chondroitin sulfate A (CSA) and mediates placental sequestration, making it the major placental malaria (PM) vaccine target. METHODS: In this study, we characterize an invariant protein associated with PM called P falciparum chondroitin sulfate A ligand (PfCSA-L). RESULTS: Recombinant PfCSA-L binds both placental CSA and VAR2CSA with nanomolar affinity, and it is coexpressed on the iRBC surface with VAR2CSA. Unlike VAR2CSA, which is anchored by a transmembrane domain, PfCSA-L is peripherally associated with the outer surface of knobs through high-affinity protein-protein interactions with VAR2CSA. This suggests that iRBC sequestration involves complexes of invariant and variant surface proteins, allowing parasites to maintain both diversity and function at the iRBC surface. CONCLUSIONS: The PfCSA-L is a promising target for intervention because it is well conserved, exposed on infected cells, and expressed and localized with VAR2CSA.

Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA.

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM.

Beninese children with cerebral malaria do not develop humoral immunity against the IT4-VAR19-DC8 PfEMP1 variant linked to EPCR and brain endothelial binding.

BACKGROUND: Malaria is still one of the most prevalent infectious diseases in the world. Sequestration of infected erythrocytes (IEs) is the prime mediator of disease. Cytoadhesion of IEs is mediated by members of the highly diverse Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). A restricted sub-set of var genes encoding for PfEMP1s possessing the domain cassettes DC8 and DC13 were found to bind to the endothelial protein C receptor (EPCR). These var genes were shown to be highly expressed by parasites from patients with severe malaria clinical outcomes compared to those from patients with uncomplicated symptoms. METHODS: In order to further study the molecular mechanisms underlying DC8/DC13 expressing IEs adhesion to EPCR, a method was developed to produce highly pure recombinant EPCR. The IT4 parasite strain was selected on either anti-IT4-VAR19 purified IgG, EPCR or human brain endothelial cell line and their var gene expression profiles as well as their binding phenotypes were compared. The N-terminal region of IT4-VAR19 comprising a full-length DC8 cassette as well as the single EPCR binding CIDRα1.1 domain were also produced, and their immune recognition (IgG) was assessed using plasma samples from Beninese children presenting acute mild malaria, severe malaria or cerebral malaria at the time of their admission to the clinic, and from convalescent-phase plasma collected 30 days after anti-malarial treatment. RESULTS: The multi-domain VAR19-NTS-DBLγ6 binds to EPCR with a greater affinity than the CIDRα1.1 domain alone and this study also demonstrates that VAR19-NTS-DBLγ6 binding to the EPCR-expressing endothelial cell line (HBEC5i) is more pronounced than that of the CIDRα1.1 domain alone. IT4-VAR19 represents the preferentially expressed-PfEMP1 when FCR3-IEs are selected based on their capability to bind EPCR. Notably, no significant difference in the levels of antibodies towards IT4-VAR19 antigens was observed within all clinical groups between plasma samples collected during the acute malaria phase compared to samples collected 30 days after anti-malaria treatment. CONCLUSIONS: These data indicate that even being the preferentially selected IT4-EPCR-binding variant, the IT4-VAR19-DC8 region does not appear to be associated with the acquisition of antibodies during a single severe paediatric malaria episode in Benin.

Induction of strain-transcending antibodies against Group A PfEMP1 surface antigens from virulent malaria parasites.

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (<10 µg/ml of total IgG) against homologous parasites. Furthermore, the antibodies showed broad cross-reactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four sub-Saharan African countries that showed surface reactivity with either DBLα1.5 antibodies (variant HB3var6) or DBLα1.8 antibodies (variant TM284var1). These data show that parasites with a virulence-associated adhesion phenotype share IE surface epitopes that can be targeted by strain-transcending antibodies to PfEMP1. The existence of shared surface epitopes amongst functionally similar disease-associated P. falciparum parasite isolates suggests that development of therapeutic interventions to prevent severe malaria is a realistic goal.

Aotus nancymaae model predicts human immune response to the placental malaria vaccine candidate VAR2CSA.

Placental malaria vaccines (PMVs) are being developed to prevent severe sequelae of placental malaria (PM) in pregnant women and their offspring. The leading candidate vaccine antigen VAR2CSA mediates parasite binding to placental receptor chondroitin sulfate A (CSA). Despite promising results in small animal studies, recent human trials of the first two PMV candidates (PAMVAC and PRIMVAC) generated limited cross-reactivity and cross-inhibitory activity to heterologous parasites. Here we immunized Aotus nancymaae monkeys with three PMV candidates (PAMVAC, PRIMVAC and ID1-ID2a_M1010) adjuvanted with Alhydrogel, and exploited the model to investigate boosting of functional vaccine responses during PM episodes as well as with nanoparticle antigens. PMV candidates induced high levels of antigen-specific IgG with significant cross-reactivity across PMV antigens by enzyme-linked immunosorbent assay. Conversely, PMV antibodies recognized native VAR2CSA and blocked CSA adhesion of only homologous parasites and not of heterologous parasites. PM episodes did not significantly boost VAR2CSA antibody levels or serum functional activity; nanoparticle and monomer antigens alike boosted serum reactivity but not functional activities. Overall, PMV candidates induced functional antibodies with limited heterologous activity in Aotus monkeys, similar to responses reported in humans. The Aotus model appears suitable for preclinical downselection of PMV candidates and assessment of antibody boosting by PM episodes.

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes.

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.

Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles.

Plasmodium falciparum, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs). ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space. Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases. RBCs were cultured with P. falciparum for 24-48 h prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs. Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP. Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria. The comparison of eATP homeostasis in infected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of P. falciparum infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.

BMFPs, a versatile therapeutic tool for redirecting a preexisting Epstein-Barr virus antibody response toward defined target cells.

Industrial production of therapeutic monoclonal antibodies is mostly performed in eukaryotic-based systems, allowing posttranslational modifications mandatory for their functional activity. The resulting elevated product cost limits therapy access to some patients. To address this limitation, we conceptualized a novel immunotherapeutic approach to redirect a preexisting polyclonal antibody response against Epstein-Barr virus (EBV) toward defined target cells. We engineered and expressed in bacteria bimodular fusion proteins (BMFPs) comprising an Fc-deficient binding moiety targeting an antigen expressed at the surface of a target cell, fused to the EBV-P18 antigen, which recruits circulating endogenous anti-P18 IgG in EBV+ individuals. Opsonization of BMFP-coated targets efficiently triggered antibody-mediated clearing effector mechanisms. When assessed in a P18-primed mouse tumor model, therapy performed with an anti-huCD20 BMFP significantly led to increased survival and total cancer remission in some animals. These results indicate that BMFPs could represent potent and useful therapeutic molecules to treat a number of diseases.

Malaria: targeting parasite and host cell kinomes.

Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases.

Plasmodium falciparum NIMA-related kinase Pfnek-1: sex specificity and assessment of essentiality for the erythrocytic asexual cycle.

The Plasmodium falciparum kinome includes a family of four protein kinases (Pfnek-1 to -4) related to the NIMA (never-in-mitosis) family, members of which play important roles in mitosis and meiosis in eukaryotic cells. Only one of these, Pfnek-1, which we previously characterized at the biochemical level, is expressed in asexual parasites. The other three (Pfnek-2, -3 and -4) are expressed predominantly in gametocytes, and a role for nek-2 and nek-4 in meiosis has been documented. Here we show by reverse genetics that Pfnek-1 is required for completion of the asexual cycle in red blood cells and that its expression in gametocytes in detectable by immunofluorescence in male (but not in female) gametocytes, in contrast with Pfnek-2 and Pfnek-4. This indicates that the function of Pfnek-1 is non-redundant with those of the other members of the Pfnek family and identifies Pfnek-1 as a potential target for antimalarial chemotherapy. A medium-throughput screen of a small-molecule library provides proof of concept that recombinant Pfnek-1 can be used as a target in drug discovery.

Identification of residues in the Cmu4 domain of polymeric IgM essential for interaction with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).

The binding of nonspecific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Herein we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4beta domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H chain and requires the IgM Cmu4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4beta domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cmu4 domain are equivalent to those regions of IgG and IgA recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.

VAR2CSA-Mediated Host Defense Evasion of Plasmodium falciparum Infected Erythrocytes in Placental Malaria.

Over 30 million women living in P. falciparum endemic areas are at risk of developing malaria during pregnancy every year. Placental malaria is characterized by massive accumulation of infected erythrocytes in the intervillous space of the placenta, accompanied by infiltration of immune cells, particularly monocytes. The consequent local inflammation and the obstruction of the maternofetal exchanges can lead to severe clinical outcomes for both mother and child. Even if protection against the disease can gradually be acquired following successive pregnancies, the malaria parasite has developed a large panel of evasion mechanisms to escape from host defense mechanisms and manipulate the immune system to its advantage. Infected erythrocytes isolated from placentas of women suffering from placental malaria present a unique phenotype and express the pregnancy-specific variant VAR2CSA of the Plasmodium falciparum Erythrocyte Membrane Protein (PfEMP1) family at their surface. The polymorphic VAR2CSA protein is able to mediate the interaction of infected erythrocytes with a variety of host cells including placental syncytiotrophoblasts and leukocytes but also with components of the immune system such as non-specific IgM. This review summarizes the described VAR2CSA-mediated host defense evasion mechanisms employed by the parasite during placental malaria to ensure its survival and persistence.

Analysis of erythrocyte signalling pathways during Plasmodium falciparum infection identifies targets for host-directed antimalarial intervention.

Intracellular pathogens mobilize host signaling pathways of their host cell to promote their own survival. Evidence is emerging that signal transduction elements are activated in a-nucleated erythrocytes in response to infection with malaria parasites, but the extent of this phenomenon remains unknown. Here, we fill this knowledge gap through a comprehensive and dynamic assessment of host erythrocyte signaling during infection with Plasmodium falciparum. We used arrays of 878 antibodies directed against human signaling proteins to interrogate the activation status of host erythrocyte phospho-signaling pathways at three blood stages of parasite asexual development. This analysis reveals a dynamic modulation of many host signalling proteins across parasite development. Here we focus on the hepatocyte growth factor receptor (c-MET) and the MAP kinase pathway component B-Raf, providing a proof of concept that human signaling kinases identified as activated by malaria infection represent attractive targets for antimalarial intervention.

An exported kinase family mediates species-specific erythrocyte remodelling and virulence in human malaria.

The most severe form of human malaria is caused by Plasmodium falciparum. Its virulence is closely linked to the increase in rigidity of infected erythrocytes and their adhesion to endothelial receptors, obstructing blood flow to vital organs. Unlike other human-infecting Plasmodium species, P. falciparum exports a family of 18 FIKK serine/threonine kinases into the host cell, suggesting that phosphorylation may modulate erythrocyte modifications. We reveal substantial species-specific phosphorylation of erythrocyte proteins by P. falciparum but not by Plasmodium knowlesi, which does not export FIKK kinases. By conditionally deleting all FIKK kinases combined with large-scale quantitative phosphoproteomics we identified unique phosphorylation fingerprints for each kinase, including phosphosites on parasite virulence factors and host erythrocyte proteins. Despite their non-overlapping target sites, a network analysis revealed that some FIKKs may act in the same pathways. Only the deletion of the non-exported kinase FIKK8 resulted in reduced parasite growth, suggesting the exported FIKKs may instead support functions important for survival in the host. We show that one kinase, FIKK4.1, mediates both rigidification of the erythrocyte cytoskeleton and trafficking of the adhesin and key virulence factor PfEMP1 to the host cell surface. This establishes the FIKK family as important drivers of parasite evolution and malaria pathology.

PRIMVAC vaccine adjuvanted with Alhydrogel or GLA-SE to prevent placental malaria: a first-in-human, randomised, double-blind, placebo-controlled study.

BACKGROUND: PRIMVAC is a VAR2CSA-derived placental malaria vaccine candidate aiming to prevent serious clinical outcomes of Plasmodium falciparum infection during pregnancy. We assessed the safety and immunogenicity of PRIMVAC adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) in French and Burkinabe women who were not pregnant. METHODS: This first-in-human, randomised, double-blind, placebo-controlled, dose escalation trial was done in two staggered phases, a phase 1A trial in 18-35-year-old women who were malaria naive in a hospital in France and a subsequent phase 1B trial in women who were naturally exposed to P falciparum and nulligravid in the clinical site of a research centre in Burkina Faso. Volunteers were recruited into four sequential cohorts receiving PRIMVAC intramuscularly at day 0, 28, and 56: two cohorts in France receiving 20 µg or 50 µg of PRIMVAC and then two in Burkina Faso receiving 50 µg or 100 µg of PRIMVAC. Volunteers were randomly assigned (1:1) to two groups (PRIMVAC adjuvanted with either Alhydrogel or GLA-SE) in France and randomly assigned (2:2:1) to three groups (PRIMVAC adjuvanted with either Alhydrogel, GLA-SE, or placebo) in Burkina Faso. Randomisation was centralised, using stratification by cohort and blocks of variable size, and syringes were masked by opaque labels. The primary endpoint was the proportion of participants with any grade 3 or higher adverse reaction to vaccination up until day 35. Safety at later time points as well as humoral and cellular immunogenicity were assessed in secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02658253. FINDINGS: Between April 19, 2016, and July 13, 2017, 68 women (18 in France, 50 in Burkina Faso) of 101 assessed for eligibility were included. No serious adverse event related to the vaccine occurred. PRIMVAC antibody titres increased with each dose and seroconversion was observed in all women vaccinated with PRIMVAC (n=57). PRIMVAC antibody titres reached a peak (geometric mean 11 843·0, optical density [OD] 1·0, 95% CI 7559·8-18 552·9 with 100 µg dose and GLA-SE) 1 week after the third vaccination (day 63). Compared with Alhydrogel, GLA-SE tended to improve the PRIMVAC antibody response (geometric mean 2163·5, OD 1·0, 95% CI 1315·7-3557·7 with 100 µg dose and Alhydrogel at day 63). 1 year after the last vaccination, 20 (71%) of 28 women who were vaccinated with PRIMVAC/Alhydrogel and 26 (93%) of 28 women who were vaccinated with PRIMVAC/GLA-SE still had anti-PRIMVAC antibodies, although antibody magnitude was markedly lower (452·4, OD 1·0, 95% CI 321·8-636·1 with 100 µg dose and GLA-SE). These antibodies reacted with native homologous VAR2CSA expressed by NF54-CSA infected erythrocytes (fold change from baseline at day 63 with 100 µg dose and GLA-SE: 10·74, 95% CI 8·36-13·79). Limited cross-recognition, restricted to sera collected from women that received the 100 µg PRIMVAC dose, was observed against heterologous VAR2CSA variants expressed by FCR3-CSA (fold change from baseline at day 63: 1·49, 95% CI 1·19-1·88) and 7G8-CSA infected erythrocytes (1·2, 1·08-1·34). INTERPRETATION: PRIMVAC adjuvanted with Alhydrogel or GLA-SE had an acceptable safety profile, was immunogenic, and induced functional antibodies reacting with the homologous VAR2CSA variant expressed by NF54-CSA infected erythrocytes. Cross-reactivity against heterologous VAR2CSA variants was limited and only observed in the higher dose group. An alternate schedule of immunisation, antigen dose, and combinations with other VAR2CSA-based vaccines are envisaged to improve the cross-reactivity against heterologous VAR2CSA variants. FUNDING: Bundesministerium für Bildung und Forschung, through Kreditanstalt für Wiederaufbau, Germany; Inserm, and Institut National de Transfusion Sanguine, France; Irish Aid, Department of Foreign Affairs and Trade, Ireland.

Modular architecture and evolution of the map-1 gene family in the root-knot nematode Meloidogyne incognita.

In eukaryotes, repeat proteins (i.e. proteins that contain a tandem arrangement of repeated structural elements) are often considered as an extra source of variability, and gains and losses of repeats may be an important force driving the evolution and diversification of such proteins, that could allow fast adaptation to new environments. Here, we report genomic sequences of the MAP-1 protein family from of the asexual, plant-parasitic nematode Meloidogyne incognita. The encoded proteins exhibited highly conserved repeats of 13 and 58 aa, and variation in the number and arrangement of these repeats in the MAP-1 proteins was correlated with nematode (a)virulence, suggesting a possible role in the specificity of the plant-nematode interaction. Search in the complete genome sequence of M. incognita confirmed that a small gene family encoding proteins harboring conserved 58 and 13 aa-repeats is present in this nematode, and that the repetitive region of these proteins is modular. Both gene duplication and intragenic gain and loss of repeats have contributed to the complex evolutionary history of the map-1 gene family, and active selection pressure of the plant host probably induced recent additional gene loss, finally resulting in the present-day gene and repeat diversity observed among nematode lines. The genomic differences characterized here between avirulent and virulent individuals are assumed to reflect, at the DNA level, the adaptive capacity of these asexual root-knot nematodes.

An enhancer-like region regulates hrp3 promoter stage-specific gene expression in the human malaria parasite Plasmodium falciparum.

The asexual blood stage of Plasmodium falciparum is comprised of morphologically distinct ring, trophozoite and schizont stages. Each of these developmental stages possesses a distinct pattern of gene expression. Regulation of P. falciparum gene expression is thought to occur, at least in part, at the promoter level. Previously, we have found that although the hrp3 mRNA is only seen in ring-stage parasites, deletion of a specific sequence in the 5' end of the promoter region decreased ring-stage expression of hrp3 and enabled detection of its transcripts in trophozoite-stage parasites. In order to investigate this stage specific regulation of gene expression, we employed a series of nested deletions of the 1.7-kb hrp3 promoter. Firefly luciferase gene was used as a reporter to evaluate the role of promoter sequences in gene regulation. Using this approach, we identified a ring-stage specific regulatory region on the hrp3 promoter located between -1.7 kb and -1.1 kb from the ATG initiation codon. Small 100-150 bp truncations on this enhancer-like region failed to uncover discrete regulatory sequences, suggesting the multipartite nature of this element. The data presented in this study demonstrate that stage specific promoter activity of the hrp3 gene in P. falciparum blood stage parasites is supported, at least in-part, by a small promoter region that can function in the absence of a larger chromosomal context.

Subverting Host Cell P21-Activated Kinase: A Case of Convergent Evolution across Pathogens.

Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases.

Identification of Plasmodium falciparum var1CSA and var2CSA domains that bind IgM natural antibodies.

Malaria in pregnancy is responsible for maternal anaemia, low-birth-weight babies and infant deaths. Plasmodium falciparum infected erythrocytes are thought to cause placental pathology by adhering to host receptors such as chondroitin sulphate A (CSA). CSA binding infected erythrocytes also bind IgM natural antibodies from normal human serum, a process that may facilitate placental adhesion or promote immune evasion. The parasite ligands that mediate placental adhesion are thought to be members of the variant erythrocyte surface antigen family P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var genes. Two var gene sub-families, var1CSA and var2CSA, have been identified as parasite CSA binding ligands and are leading candidates for a vaccine to prevent pregnancy-associated malaria. We investigated whether these two var gene subfamilies implicated in CSA binding are also the molecules responsible for IgM natural antibody binding. By heterologous expression of domains in COS-7 cells, we found that both var1CSA and var2CSA PfEMP1 variants bound IgM, and in both cases the binding region was a DBL epsilon domain occurring proximal to the membrane. None of the domains from a control non-IgM-binding parasite (R29) bound IgM when expressed in COS-7 cells. These results show that PfEMP1 is a parasite ligand for non-immune IgM and are the first demonstration of a specific adhesive function for PfEMP1 epsilon type domains.