A comparative study of the localization and membrane topology of members of the RIFIN, STEVOR and PfMC-2TM protein families in Plasmodium falciparum-infected erythrocytes.

BACKGROUND: Variant surface antigens (VSA) exposed on the membrane of Plasmodium falciparum infected erythrocytes mediate immune evasion and are important pathogenicity factors in malaria disease. In addition to the well-studied PfEMP1, the small VSA families RIFIN, STEVOR and PfMC-2TM are assumed to play a role in this process. METHODS: This study presents a detailed comparative characterization of the localization, membrane topology and extraction profile across the life cycle of various members of these protein families employing confocal microscopy, immunoelectron microscopy and immunoblots. RESULTS: The presented data reveal a clear association of variants of the RIFIN, STEVOR and PfMC-2TM proteins with the host cell membrane and topological studies indicate that the semi-conserved N-terminal region of RIFINs and some STEVOR proteins is exposed at the erythrocyte surface. At the Maurer's clefts, the semi-conserved N-terminal region as well as the variable stretch of RIFINs appears to point to the lumen away from the erythrocyte cytoplasm. These results challenge the previously proposed two transmembrane topology model for the RIFIN and STEVOR protein families and suggest that only one hydrophobic region spans the membrane. In contrast, PfMC-2TM proteins indeed seem to be anchored by two hydrophobic stretches in the host cell membrane exposing just a few, variable amino acids at the surface of the host cell. CONCLUSION: Together, the host cell surface exposure and topology of RIFIN and STEVOR proteins suggests members of these protein families may indeed be involved in immune evasion of the infected erythrocyte, whereas members of the PfMC-2TM family seem to bear different functions in parasite biology.

Diverse expression patterns of subgroups of the rif multigene family during Plasmodium falciparum gametocytogenesis.

BACKGROUND: The maturation of Plasmodium falciparum gametocytes in the human host takes several days, during which the parasites need to efficiently evade the host immune system. Like asexual stage parasites, immature gametocytes can sequester at various sites in the human body, and only mature sexual stages are found in the circulation. Although the fundamental mechanisms of gametocyte immune evasion are still largely unknown, candidate molecules that may be involved include variant antigens encoded by multigene families in the P. falciparum genome, such as the PfEMP1, STEVOR and RIFIN proteins. While expression of the former two families in sexual stages has been investigated earlier, we report here RIFIN expression during gametocytogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Variants of two previously characterized RIFIN subfamilies (A- and B-type RIFINs) were found to be synthesized in gametocytes. Immunofluorescence experiments showed A-type RIFINs to be accumulated in a crescent-shaped pattern of discrete punctate structures at the infected erythrocyte membrane, while members of the B-type family were associated with the parasite. Transcription analysis demonstrated the existence of diverse transcriptional regulation patterns during sexual differentiation and indicated variant-specific regulation of B-type RIFINs, in contrast to group-specific regulation for A-type RIFINs. Phylogenetic analysis of 5'-upstream regions showed that the rif-gene family falls into five defined clusters, designated rups (rifupstream) A1, A2, AB, B and C. In trophozoites and early gametocytes, rif variants of the rupsA2-type were preferentially expressed. CONCLUSIONS/SIGNIFICANCE: In this work we demonstrate the expression dynamics of the rif-gene family during sexual differentiation and present indications for subgroup specific regulation patterns. Therefore, our data provide a first foundation and point to new directions for future investigations of the potential role of RIFINs in gametocyte immune evasion.

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion.

BACKGROUND: Plasmodium falciparum STEVOR proteins, encoded by the multicopy stevor gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation. METHODS: Immunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process. RESULTS: Antigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown. CONCLUSION: The localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.

Immune responses after single-dose sulphadoxine-pyrimethamine indicate underestimation of protective efficacy of intermittent preventive treatment in infants.

OBJECTIVE: To assess how intermittent preventive treatment in infants (IPTi) with sulphadoxine-pyrimethamine (SP) affects Immunoglobulin (IgG) immune responses against Plasmodium falciparum in infants from rural Ghana. METHODS: Randomized, placebo-controlled and double-blinded clinical trial with participants randomized in blocks of 10 to receive either 250 mg sulphadoxine/2.5 mg pyrimethamine or placebo at the age of 3 (IPTi-1), 9 (IPTi-2) and 15 (IPTi-3) months and followed-up for 21 months. (i) Anti-P. falciparum IgG levels were measured in 180 children at the age of 9 months. (ii) Longitudinal study of the relationship between IgG levels and P. falciparum infections and/or clinical malaria in 17 naive children until they reached the age of 2 years. RESULTS: IgG antibody levels against crude P. falciparum lysate were dependent on the frequency of preceding infections and significantly lower in children treated with SP. CONCLUSION: Placebo-treated children had an indifferentially higher incidence of P. falciparum infections than clinically observed, which implicates an underestimation of the protective efficacy of IPTi. IgG profiles in 17 children followed up until the age of 2 years provided no evidence for impaired immune responses after a single dose of SP within the framework of IPTi.

Variant proteins of the Plasmodium falciparum RIFIN family show distinct subcellular localization and developmental expression patterns.

In order to avoid immune recognition in favor of a chronic infection, the malaria parasite Plasmodium falciparum has developed means to express clonally variant antigens at the surface of the infected erythrocyte (IE). Proteins of the var and rif multicopy gene families, encoding PfEMP1 and RIFINs, respectively, have been implicated in these processes. Here, we studied members of the latter family and present data revealing different subcellular localization patterns for RIFIN variants belonging to two distinct subgroups, which have been designated A- and B-type RIFINs. While A-type RIFINs were found to be associated with the parasite and transported to the surface of infected erythrocytes via Maurer's clefts, B-type RIFINs appeared to be mostly retained inside the parasite. However, expression of both subtypes does not seem to be mutually exclusive. Moreover, both A- and B-type variants were also expressed in the merozoite, present either in the apical region (A-type) or in the cytosol (B-type). The presence of RIFINs in merozoites suggests that antigenic variation in P. falciparum is not only restricted to parasite-derived proteins at the IE surface, but the phenomenon also prevails in other life cycle stages. Interestingly, some RIFIN variants were detected only in intracellular stages and not in merozoites, pointing to differential developmental expression patterns for distinct members of this large protein family.

The impact of IgG antibodies to recombinant Plasmodium falciparum 732var CIDR-1alpha domain in mothers and their newborn babies.

Different domains of a novel full-length var gene (termed 732var) isolated from a placenta of a malaria-infected woman were expressed in Escherichia coli as recombinant proteins and analysed biochemically and immunologically. Two of these, the Duffy binding-like (DBL)-3gamma domain and the cysteine-rich interdomain region (CIDR)-1alpha were able to bind chondroitin sulfate A and CD36, respectively. The DBL-3gamma domain was investigated in a previous study and confirmed here to exhibit anti-disease characteristics related to pregnancy-associated malaria. Mothers with high anti-DBL-3gamma antibody levels were protected from placental infection. The novel finding in this study is that babies born to mothers carrying anti-CIDR-1alpha antibodies had a delayed time to the first infection.

Receptor-binding studies of the DBLgamma domain of Plasmodium falciparum erythrocyte membrane protein 1 from a placental isolate.

We have previously identified a number of DBLgamma domains in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) transcripts obtained from placental parasite isolates, showing that they bind specifically to chondroitin sulfate A (CSA) (Khattab A, Kun J, Deloron P, Kremsner PG, Klinkert MQ. Variants of Plasmodium falciparum erythrocyte membrane protein 1 expressed by different placental parasites are closely related and adhere to chondroitin sulfate A. J Infect Dis 2001;183:1165-9). Here we give a more detailed physico-chemical and binding characterisation of the soluble, recombinant DBLgamma domain derived from one of these isolates. Results from circular dichroism and limited proteolysis experiments are consistent with the recombinant domain being expressed with the native fold. Specific binding of DBLgamma to placental cryosections was demonstrated by labeling with antibodies raised against the recombinant domain; binding was diminished after treatment of the cryosections with chondroitinase or by blocking with anti-CSA antibody, showing that CSA mediates the interaction. Binding of the DBLgamma domain to purified placental chondroitin sulfate proteoglycan (CSPG) was also studied using surface plasmon resonance techniques, with DBLgamma as analyte and CSPG immobilised on the sensor chip; these quantitative measurements gave an affinity constant in the mu-molar range under the conditions used. The native conformation of the DBLgamma domain is essential for CSPG recognition since binding to the sensor chip is abolished when the protein is irreversibly reduced. As with the placental cryosections, association was significantly reduced after treating the immobilised CSPG with chondroitinase. Together, these results demonstrate specific interaction between the DBLgamma domain and the placental receptor.

Maurer's clefts-restricted localization, orientation and export of a Plasmodium falciparum RIFIN.

RIFINs are clonally variant antigens expressed in Plasmodium falciparum. Transfection and the green fluorescence protein (GFP) tagged either internally or C-terminally to the 3D7 PFI0050c RIFIN gene product were used to investigate protein localization, orientation and trafficking. Green fluorescence pattern emerging from live transfectant parasites expressing each of the RIFIN-GFP chimera was different. The internally GFP-tagged protein was exported to Maurer's clefts (MC) in the erythrocyte cytosol, whereas the C-terminally GFP-tagged full-length RIFIN chimera was not trafficked out of the parasite. Interestingly, when some RIFIN-specific C-terminal amino acid sequences were removed, the resulting truncated molecule reached the MC. Using anti-RIFIN and anti-GFP antibodies to probe both live and fixed transfectants, staining was confined to MC and was not detected on the erythrocyte surface, a location previously suggested for this protein family. From selective permeabilization experiments, the highly variable portion of the RIFIN-GFP-insertion chimera appeared to be exposed to the erythrocyte cytosol, presumably anchored in the MC membrane via the two transmembrane domains. Trafficking of both chimeras in young ring stages was sensitive to Brefeldin A (BFA), although older rings showed differential sensitivity to BFA.

Nonimmune immunoglobulin binding and multiple adhesion characterize Plasmodium falciparum-infected erythrocytes of placental origin.

The harmful effects of pregnancy-associated malaria (PAM) are engendered by the heavy sequestration of Plasmodium falciparum-parasitized RBCs in the placenta. It is well documented that this process is mediated by interactions of parasite-encoded variant surface antigens and placental receptors. A P. falciparum erythrocyte membrane protein 1 variant, VAR2CSA, and the placental receptor chondroitin sulfate A (CSA) are currently the focus of PAM research. A role for immunoglobulins (IgG and IgM) from normal human serum and hyaluronic acid as additional receptors in placental sequestration have also been suggested. We show here (i) that CSA and nonimmune IgG/IgM binding are linked phenotypes of in vitro-adapted parasites, (ii) that a VAR2CSA variant shown to bind CSA also harbors IgG- and IgM-binding domains (DBL2-X, DBL5-epsilon, and DBL6-epsilon), and (iii) that IgG and IgM binding and adhesion to multiple receptors (IgG/IgM/HA/CSA) rather than the exclusive binding to CSA is a characteristic of fresh Ugandan placental isolates. These findings are of importance for the understanding of the pathogenesis of placental malaria and have implications for the ongoing efforts to develop a global PAM vaccine.

Cerebral malaria is associated with IgG2 and IgG4 antibody responses to recombinant Plasmodium falciparum RIFIN antigen.

RIFIN proteins belong to the largest Plasmodium falciparum multicopy family of variant surface antigens (VSA) expressed by infected erythrocytes. VSA antibodies have been shown to be associated with protection against malaria. Here, antibody subclass responses to a recombinant RIFIN protein (RIF-29) in 116 Ghanaian children were determined by ELISA to investigate the relationship between severe malaria and anti-RIF-29 antibodies. The study group was composed of 23 children diagnosed exclusively for cerebral malaria and 35 children who had non-cerebral severe malaria. The remaining 58 individuals were age-, gender- and area-matched asymptomatic controls. The finding that IgG1 and IgG3 responses predominated in severe malaria patients compared to matched controls suggests that these antibodies are not protective, but are most probably induced by a current infection, an observation substantiated by the equally high reactivity to both recombinant RIF-29 protein and to P. falciparum crude lysate proteins. The exclusive detection of IgG2 and IgG4 antibodies to RIF-29 protein only in cerebral malaria children brings to mind the possibility that these antibodies are pathogenic. This is a new finding that may go some way towards explaining why these children are at risk of developing the life-threatening form of cerebral malaria.

Functional and immunological characterization of a duffy binding-like- gamma domain from Plasmodium falciparum erythrocyte membrane protein-1 expressed by a placental isolate.

A recombinant Duffy binding-like (DBL)- gamma domain from a previously identified placental isolate, 732, was expressed by use of the baculovirus/insect cell system and was purified in milligram quantities. The recombinant protein binds specifically to chondroitin sulfate A (CSA) and inhibits CSA binding by placental infected erythrocytes (IEs). Polyclonal antibodies raised against the domain recognized the surfaces of live IEs from CSA-adherent clinical placental isolates. These antibodies also abrogated the in vitro binding of IEs to CSA. The 732 DBL-3 gamma domain was specifically recognized by plasma from pregnant women but not by plasma from control subjects. In addition, the protein was, comparatively, significantly more reactive with plasma from women with infected placentas, strongly suggesting that the 732 DBL-3 gamma domain carries preferentially IE-expressed immunogenic epitopes. High levels of plasma antibodies to the recombinant domain were associated with reduced placental parasite density. This is the first report of a recombinant DBL- gamma domain derived from a placental isolate that shows CSA-binding properties.

Molecular and biochemical characterization of a protein cyclophilin from the nematode Haemonchus contortus( P ).

We have cloned, sequenced and expressed a gene of Haemonchus contortus that encodes a protein (termed HcCYP) consisting of a cyclophilin domain and an RNA recognition motif (RRM). An antiserum raised against the recombinant protein showed that HcCYP was present in the insoluble fraction (mostly nuclear) of the parasite homogenate. The recombinant protein possessed the typical cis-trans peptidyl-prolyl isomerase activity of cyclophilins and this activity was inhibited by the immunosuppressant cyclosporin A. The N-terminal portion of the molecule, carrying the RRM, was able to bind to nucleic acids, whereas the C-terminal portion did not have any binding activity. The possible function of HcCYP in the parasite is discussed on the basis of information available on similar proteins in other organisms.

Cytological and biochemical evidence for a gonad-preferential interplay of SmFKBP12 and SmTbetaR-I in Schistosoma mansoni.

In eukaryotes, FK506-binding proteins with a molecular weight of 12 kDa (FKBP12s) influence a variety of signal transduction pathways that regulate cell division, differentiation, and ion homeostasis. Amongst these, TGFbeta signaling and calcineurin (CN) phosphatase activity is modulated by FKBP12 via binding to TGFbeta-family type I receptors (TbetaR-Is) or to the CN subunit A, respectively. In this work, we demonstrate the tissue-specific expression of the Schistosoma mansoni FKBP12 homologue (SmFKBP12) in the gonads of female parasites as well as in the tegument of both genders. Components of the TGFbeta pathway have been characterized in schistosomes and their roles in mediating host-parasite or male-female interactions proposed. We show that a schistosome TGFbeta-family type I receptor (SmTbetaR-I, SmRK-1) is expressed in the female gonads, suggesting that SmFKBP12 may regulate its activity in this tissue. This hypothesis is supported by yeast two-hybrid analyses showing a direct binding of SmFKBP12 and SmTbetaR-I, which was specifically inhibited by the drug FK506. Our data provide the first evidence for the activity of a transmembrane receptor in the vitellarium of schistosome females and indicate that FKBP12-meditated regulation of the TGFbeta pathway is evolutionarily conserved in a primitive metazoan such as Schistosoma. Furthermore, we show that the schistosome CN (SmCN) is not expressed in the female gonads, but co-localizes with SmFKBP12 only in the tegument. From these data we conclude an SmFKBP12/SmTbetaR-I, but not an SmCN/SmFKBP12 interplay in the female gonads.

Schistosoma mansoni fatty acid binding protein: specificity and functional control as revealed by crystallographic structure.

Schistosoma mansoni fatty acid binding protein (Sm14) was crystallized with bound oleic acid (OLA) and arachidonic acid (ACD), and their structures were solved at 1.85 and 2.4 A resolution, respectively. Sm14 is a vaccine target for schistosomiasis, the second most prevalent parasitic disease in humans. The parasite is unable to synthesize fatty acids depending on the host for these nutrients. Moreover, arachidonic acid (ACD) is required to synthesize prostaglandins employed by schistosomes to evade the host's immune defenses. In the complex, the hydrocarbon tail of bound OLA assumes two conformations, whereas ACD adopts a unique hairpin-looped structure. ACD establishes more specific interactions with the protein, among which the most important is a pi-cation bond between Arg78 and the double bond at C8. Comparison with homologous fatty acid binding proteins suggests that the binding site of Sm14 is optimized to fit ACD. To test the functional implications of our structural data, the affinity of Sm14 for 1,8-anilinonaphthalenesulfonic acid (ANS) has been measured; moreover the binding constants of six different fatty acids were determined from their ability to displace ANS. OLA and ACD exhibited the highest affinities. To determine the rates of fatty acid binding and dissociation we carried out stopped flow kinetic experiments monitoring displacement by (and of) ANS. The binding rate constant of ligands is controlled by a slow pH dependent conformational change, which we propose to have physiological relevance.

Submicroscopic Plasmodium falciparum infections and multiplicity of infection in matched peripheral, placental and umbilical cord blood samples from Gabonese women.

Summary In malaria-endemic regions, pregnant women are more susceptible to malarial infections than non-pregnant women. The main objective of this study, which was conducted in the malaria hyperendemic town of Lambaréné (Gabon, Central Africa), was to characterize Plasmodium falciparum infections in peripheral, placental and cord blood from women of different gravidities with submicroscopic infections. Using the P. falciparum merozoite surface protein 2 (MSP 2)* gene as a polymorphic marker in polymerase chain reactions, we analysed genetic diversity and multiplicity of infection in isolates from all three kinds of samples of 184 pregnant women at delivery. We detected infection in 44% of the women who were originally negative by microscopy. Equally important was the finding that the placenta had the highest prevalence of infection (P < 0.001). There was no correlation with gravidity status or age of the patients. The multiplicities of infection in the peripheral and placental blood samples did not differ and single infection was observed in cord blood, independently of the gravidity. The FC27/MSP 2 was the predominant allelic family. The major FC27 alleles detected in the peripheral, placental and cord blood were sequenced and found to be closely related to the published K1 form sequence. Below microscopy level, the placenta remains the most infected organ and this submicroscopic carriage of parasites may contribute to the development and maintenance of immunity to malaria during pregnancy.

Analysis of IgG with specificity for variant surface antigens expressed by placental Plasmodium falciparum isolates.

BACKGROUND: Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes that can sequester in placental intervillous space by expressing particular variant surface antigens (VSA) that can mediate adhesion to chondroitin sulfate A (CSA) in vitro. IgG antibodies with specificity for the VSA expressed by these parasites (VSAPAM) are associated with protection from maternal anaemia, prematurity and low birth weight, which is the greatest risk factor for death in the first month of life. METHODS: In this study, the development of anti-VSAPAM antibodies in a group of 151 women who presented to the maternity ward of Albert Schweitzer Hospital in Lambaréné, Gabon for delivery was analysed using flow cytometry assays. Plasma samples from placenta infected primiparous women were also investigated for their capacity to inhibit parasite binding to CSA in vitro. RESULTS: In the study cohort, primiparous as well as secundiparous women had the greatest risk of infection at delivery as well as during pregnancy. Primiparous women with infected placentas at delivery showed higher levels of VSAPAM-specific IgG compared to women who had no malaria infections at delivery. Placental isolates of Gabonese and Senegalese origin tested on plasma samples from Gabon showed parity dependency and gender specificity patterns. There was a significant correlation of plasma reactivity as measured by flow cytometry between different placental isolates. In the plasma of infected primiparous women, VSAPAM-specific IgG measured by flow cytometry could be correlated with anti-adhesion antibodies measured by the inhibition of CSA binding. CONCLUSION: Recognition of placental parasites shows a parity- and sex- dependent pattern, like that previously observed in laboratory strains selected to bind to CSA. Placental infections at delivery in primiparous women appear to be sufficient to induce functional antibodies which can both recognize the surface of the infected erythrocytes as well as block their binding to CSA. The correlation between serum reactivities of placental field isolates from different geographic locations and collected at different times is indicative of the conserved nature of the antigen(s) mediating PAM.

Antibodies to Plasmodium falciparum rifin proteins are associated with rapid parasite clearance and asymptomatic infections.

Plasmodium falciparum rifin proteins, belonging to the largest known family of variable infected-erythrocyte surface-expressed proteins encoded by rif genes, were recently shown to be capable of inducing a strong immune response in P. falciparum-infected adults living in an area in Gabon where malaria is endemic. In the present study, the levels of antirifin antibodies were analyzed in serum obtained from 60 children from the same area who were admitted to hospital and diagnosed with severe malaria. High antirifin antibody concentrations in these individuals correlated significantly with their capacity to rapidly clear their parasites from the circulation after the start of chemotherapy. A doubling of antirifin antibody concentrations reduced the clearance time by 5 h (95% confidence interval, 4.1 to 6.9 h). In the same group of children, who were followed up for 2 years, antirifin antibody levels did not correlate with a reduced rate of reinfection or with a delay in the time to the first reinfection. However, the initial antirifin antibody levels were sustained over the study period. The likelihood that these antibodies could confer a certain degree of protection against malaria is supported by our findings of statistically higher levels of antirifin antibodies to all four rifin proteins in a group of 42 asymptomatic parasitemic children.

Commonly recognised Plasmodium falciparum parasites cause cerebral malaria.

Cerebral malaria (CM) is a devastating form of Plasmodium falciparum malaria, in which adherence and sequestration of infected red blood cells in cerebral blood vessels play a major role. In order to determine whether a distinct parasite phenotype favours the development of this severe complication, P. falciparum isolates from Gabonese children suffering from CM or uncomplicated malaria (UM) were analysed for their binding phenotypes and their recognition in flow cytometry. CM isolates exhibited the ability to form rosettes and to bind ICAM-1, in line with previous studies correlating these phenotypes with CM disease pathology. CM isolates were more reactive with plasma from our cohort than UM parasites. This observation, together with the finding that some CM isolates were highly correlated with each other in their immunoreactivities, confirms that common parasites bearing conserved epitopes, which are capable of inducing cross-reactive antibodies, can cause CM in children.