Structural insights into chondroitin sulphate A binding Duffy-binding-like domains from Plasmodium falciparum: implications for intervention strategies against placental malaria

<p>Abstract</p> <p>Background</p> <p>Placental malaria is typified by selective clustering of <it>Plasmodium falciparum </it>in the intervillous blood spaces of the placenta. Sequestration of malaria parasite in the human placenta is mediated by interactions between chondroitin sulphate A (CSA) on the syncytiotrophoblasts and proteins expressed on the surface of infected human erythrocytes. <it>Plasmodium falciparum </it>Erythrocyte Membrane Protein 1 (PfEMP1) encoded by the <it>var2CSA </it>gene is believed to be the main parasite ligand for CSA-mediated placental binding.</p> <p>Methods</p> <p>Extensive sequence and structure comparisons of the various CSA-binding and non-binding DBL domains from the <it>var2CSA </it>gene from A4 and 3D7 strains of <it>P. falciparum </it>were performed. Three-dimensional structural models of various DBL domains were built and analysed with a view to assessing conservation of CSA interaction sites across various DBL domains.</p> <p>Results</p> <p>Each of the six DBL domains from <it>var2CSA </it>are likely to retain the disulfide linkages evident from previously published DBL domain crystal structures. The number of disulfide linkages between the various DBL domains analysed varies from three to seven, of which two are conserved across all DBL domains. The conserved disulfide linkages are distributed within the respective three sub-domains and only one linkage is shared by sub-domains I and II. Major differences between CSA-binding DBL domains are in the loop regions, which tie the alpha helices together, and in variable length terminal extensions. Intriguingly, a crucial loop from A4 DBL 3X which provides the important Gly and Lys residues that chelate the bound sulphate is missing or significantly altered in all other DBL domains that interact with CSA. Further analysis of the proposed sulphate and predicted CSA-binding site indicates either none or very low level of conservation among the critical interacting residues.</p> <p>Conclusion</p> <p>Structural comparisons of the three-dimensional structures of CSA-binding DBL domains indicates that the proposed CSA interaction site on A4 DBL 3X is unlikely to be conserved across the other CSA-binding DBL domains from <it>var2CSA</it>. Therefore, the 4 CSA-binding DBL domains encoded by <it>var2CSA </it>are unlikely to have common architectures to their CSA recognition sites. These structural insights have clear implications in using CSA-binding DBL domains for vaccines against placental malaria as it is proposed that the various CSA-binding DBL domains on <it>var2CSA </it>will recognize their CSA ligands differently.</p

Plasmodium falciparum uses gC1qR/HABP1/p32 as a receptor to bind to vascular endothelium and for platelet-mediated clumping

The ability of Plasmodium falciparum-infected red blood cells (IRBCs) to bind to vascular endothelium, thus enabling sequestration in vital host organs, is an important pathogenic mechanism in malaria. Adhesion of P. falciparum IRBCs to platelets, which results in the formation of IRBC clumps, is another cytoadherence phenomenon that is associated with severe disease. Here, we have used in vitro cytoadherence assays to demonstrate, to our knowledge for the first time, that P. falciparum IRBCs use the 32-&lt;Da human protein gC1qR/HABP1/p32 as a receptor to bind to human brain microvascular endothelial cells. In addition, we show that P. falciparum IRBCs can also bind to gC1qR/HABP1/p32 on platelets to form clumps. Our study has thus identified a novel host receptor that is used for both adhesion to vascular endothelium and platelet-mediated clumping. Given the association of adhesion to vascular endothelium and platelet-mediated clumping with severe disease, adhesion to gC1qR/HABP1/p32 by P. falciparum IRBCs may play an important role in malaria pathogenesis

Receptor-binding residues lie in central regions of duffy-binding-like domains involved in red cell invasion and cytoadherence by malaria parasites

Erythrocyte invasion by malaria parasites and cytoadherence of Plasmodium falciparum-infected erythrocytes to host capillaries are 2 key pathogenic mechanisms in malaria. The receptor-binding domains of erythrocyte-binding proteins (EBPs) such as Plasmodium falciparum EBA-175, which mediate invasion, and P falciparum erythrocyte membrane protein 1 (PfEMP-1) family members, which are encoded by var genes and mediate cytoadherence, have been mapped to conserved cysteine-rich domains referred to as Duffy-binding-like (DBL) domains. Here, we have mapped regions within DBL domains from EBPs and PfEMP-1 that contain receptor-binding residues. Using biochemical and molecular methods we demonstrate that the receptor-binding residues of parasite ligands that bind sialic acid on glycophorin A for invasion as well as complement receptor-1 and chondroitin sulfate A for cytoadherence map to central regions of DBL domains. In contrast, binding to intercellular adhesion molecule 1 (ICAM-1) requires both the central and terminal regions of DBLβC2 domains. Determination of functional regions within DBL domains is the first step toward understanding the structure-function bases for their interaction with diverse host receptors

Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vaccine candidates of Plasmodium vivax

In previous immuno-epidemiological studies of the naturally acquired antibody responses to merozoite surface protein-1 (MSP-1) of Plasmodium vivax, we had evidence that the responses to distinct erythrocytic stage antigens could be differentially regulated. The present study was designed to compare the antibody response to three asexual erythrocytic stage antigens vaccine candidates of P. vivax. Recombinant proteins representing the 19 kDa C-terminal region of MSP-1(PvMSP19), apical membrane antigen n-1 ectodomain (PvAMA-1), and the region II of duffy binding protein (PvDBP-RII) were compared in their ability to bind to IgG antibodies of serum samples collected from 220 individuals from the state of Pará, in the North of Brazil. During patent infection with P. vivax, the frequency of individuals with IgG antibodies to PvMSP1(19), PvAMA-1, and PvDBP-RII were 95, 72.7, and 44.5% respectively. Although the frequency of responders to PvDBP-RII was lower, this frequency increased in individuals following multiple malarial infections. Individually, the specific antibody levels did not decline significantly nine months after treatment, except to PvMSP1(19). Our results further confirm a complex regulation of the immune response to distinct blood stage antigens. The reason for that is presently unknown but it may contribute to the high risk of re-infection in individuals living in the endemic areas.Universidade de São Paulo Faculdade de Ciências Farmacêuticas Departamento de Análises Clínicas e ToxicológicasUniversidade Federal do Pará Centro de Ciências Biológicas Departamento de PatologiaInternational Centre for Genetic Engineering and Biotechnology Centro de Ciências Biológicas Malaria Research GroupUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de Microbiologia, Imunologia e ParasitologiaUNIFESP, EPM, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

cAMP-Dependent Signaling Pathways as Potential Targets for Inhibition of Plasmodium falciparum Blood Stages

We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites

Host age and expression of genes involved in red blood cell invasion in Plasmodium falciparum field isolates

Plasmodium falciparum proteins involved in erythrocyte invasion are main targets of acquired immunity and important vaccine candidates. We hypothesized that anti-parasite immunity acquired upon exposure would limit invasion-related gene (IRG) expression and affect the clinical impact of the infection. 11 IRG transcript levels were measured in P. falciparum isolates by RT-PCR, and IgG/IgM against invasion ligands by Luminex(R), in 50 Mozambican adults, 25 children with severe malaria (SM) and 25 with uncomplicated malaria (UM). IRG expression differences among groups and associations between IRG expression and clinical/immunologic parameters were assessed. IRG expression diversity was higher in parasites infecting children than adults (p = 0.022). eba140 and ptramp expression decreased with age (p = 0.003 and 0.007, respectively) whereas p41 expression increased (p = 0.022). pfrh5 reduction in expression was abrupt early in life. Parasite density decreased with increasing pfrh5 expression (p < 0.001) and, only in children, parasite density increased with p41 expression (p = 0.007), and decreased with eba175 (p = 0.013). Antibody responses and IRG expression were not associated. In conclusion, IRG expression is associated with age and parasite density, but not with specific antibody responses in the acute phase of infection. Our results confirm the importance of multi-antigen vaccines development to avoid parasite immune escape when tested in malaria-exposed individuals

Analysis of factors affecting the variability of a quantitative suspension bead array assay measuring IgG to multiple Plasmodium antigens

Reducing variability of quantitative suspension array assays is key for multi-center and large sero-epidemiological studies. To maximize precision and robustness of an in-house IgG multiplex assay, we analyzed the effect of several conditions on variability to find the best combination. The following assay conditions were studied through a fractional factorial design: antigen-bead coupling (stock vs. several), sample predilution (stock vs. daily), temperature of incubation of sample with antigen-bead (22°C vs. 37°C), plate washing (manual vs. automatic) and operator expertise (expert vs. apprentice). IgG levels against seven P. falciparum antigens with heterogeneous immunogenicities were measured in test samples, in a positive control and in blanks. We assessed the variability and MFI quantification range associated to each combination of conditions, and their interactions, and evaluated the minimum number of samples and blank replicates to achieve good replicability. Results showed that antigen immunogenicity and sample seroreactivity defined the optimal dilution to assess the effect of assay conditions on variability. We found that a unique antigen-bead coupling, samples prediluted daily, incubation at 22°C, and automatic washing, had lower variability. However, variability increased when performing several couplings and incubating at 22°C vs. 37°C. In addition, no effect of temperature was seen with a unique coupling. The expertise of the operator had no effect on assay variability but reduced the MFI quantification range. Finally, differences between sample replicates were minimal, and two blanks were sufficient to capture assay variability, as suggested by the constant Intraclass Correlation Coefficient of three and two blanks. To conclude, a single coupling was the variable that most consistently reduced assay variability, being clearly advisable. In addition, we suggest having more sample dilutions instead of replicates to increase the likelihood of sample MFIs falling in the linear part of the antigen-specific curve, thus increasing precision