Characterization of anti-var2CSA-PfEMP1 cytoadhesion inhibitory mouse monoclonal antibodies

Pregnancy-associated malaria (PAM) is associated with the massive sequestration of erythrocytes infected with CSA-binding parasites in the placenta. Natural protective immunity against PAM is acquired during the course of pregnancies, with the development of anti-PfEMP1 antibodies recognizing placental infected erythrocytes (IEs) from different geographical regions. Mouse monoclonal antibodies (mabs) were raised against Plasmodium falciparum variant surface proteins expressed by CSA-binding parasites. These mabs blocked 0-60% of CSA-binding parasite adhesion and immunoprecipitated a 350 kDa 125I-labeled PfEMP1(CSA). Two var2CSA domains expressed on the surface of CHO cells (DBL5epsilon and DBL6epsilon) were identified as the targets of three of four antibodies inhibiting CSA binding. Two of these antibodies also recognized either DBL2x or DBL3x, suggesting that some epitopes may be common to several var2CSA domains. These mabs also specifically selected CSA-binding IEs and facilitated the purification from IE extracts of the native var2CSA ligand. This purified ligand elicited antibodies in immunized mice inhibiting efficiently IE(CSA) cytoadhesion. Based on our findings, we provide the first demonstration that the parasite var2CSA surface protein can elicit inhibitory antibodies and define here the subunits of the var2CSA ligand suitable for use in vaccine development.     

The solution structure of the adhesion protein Bd37 from Babesia divergens reveals structural homology with eukaryotic proteins involved in membrane trafficking

Babesia divergens is the Apicomplexa agent of the bovine babesiosis in Europe: this infection leads to growth and lactation decrease, so that economical losses due to this parasite are sufficient to require the development of a vaccine. The major surface antigen of B. divergens has been described as a 37 kDa protein glycosyl phosphatidyl inositol (GPI)-anchored at the surface of the merozoite. The immuno-prophylactic potential of Bd37 has been demonstrated, and we present here the high-resolution solution structure of the 27 kDa structured core of Bd37 (Δ-Bd37) using NMR spectroscopy. A model for the whole protein has been obtained using additional small angle X-ray scattering (SAXS) data. The knowledge of the 3D structure of Bd37 allowed the precise epitope mapping of antibodies on its surface. Interestingly, the geometry of Δ-Bd37 reveals an intriguing similarity with the exocyst subunit Exo84p C-terminal region, an eukaryotic protein that has a direct implication in vesicle trafficking. This strongly suggests that Apicomplexa have developed in parallel molecular machines similar in structure and function to the ones used for endo- and exocytosis in eukaryotic cells.     

Structural Comparison of Two CSPG-Binding DBL Domains from the VAR2CSA Protein Important in Malaria during Pregnancy

Severe malaria during pregnancy is associated with accumulation of parasite-infected erythrocytes in the placenta due to interactions between VAR2CSA protein, expressed on the surface of infected-erythrocytes, and placental chondroitin sulfate proteoglycans (CSPG). VAR2CSA contains multiple CSPG-binding domains, including DBL3X and DBL6?. Previous structural studies of DBL3X suggested CSPG to bind to a positively charged patch and sulfate-binding site on the concave surface of the domain. Here we present the structure of the DBL6? domain from VAR2CSA. This domain displays the same overall architecture and secondary structure as that of DBL3X but differs in loop structures, disulfide bond positions and surface charge distribution. In particular, despite binding to CSPG, DBL6? lacks the key features of the CSPG-binding site of DBL3X. Instead DBL6? binds to CSPG through a positively charged surface on the distal side of subdomain 2 that is exposed in intact VAR2CSA on the erythrocyte surface. Finally, unlike intact VAR2CSA, both DBL3X and DBL6? bind to various carbohydrates, with greatest affinity for ligands with high sulfation and negative charge. These studies provide further insight into the structure of DBL domains and suggest a model for the role of individual domains in CSPG binding by VAR2CSA in placental malaria.     

The chemical microenvironment of the symbiotic planktonic foraminifer Orbulina universa

Microsensor measurements of CO₂, O₂, pH and Ca²⁺ in the vicinity of the symbiont-bearing planktonic foraminifer Orbulina universa showed major light-modulated changes in the chemical microenvironment due to symbiont photosynthesis, respiration of the holobiont, and precipitation of the calcite shell. Under saturating light conditions, microprofiles measured towards the shell surface showed an O₂ increase of up to 220% air saturation, a decrease in CO₂ concentration to 4.9 μM, and a pH increase to 8.8 due to symbiont photosynthesis. The Ca²⁺ concentration decreased to ∼9.6 mM in two specimens, while it increased to 10.2-10.8 mM in three other specimens kept in light. In darkness, the respiration of the community decreased the O₂ concentration to 82% of air saturation, CO₂ increased up to 15 μM, the pH decreased to 8.0, and the Ca²⁺ concentration increased up to 10.4 mM. These data, and derived calculations of the distribution of HCO₃^- and CO₃^2- near the shell, showed that the carbonate system in the vicinity of O. universa was significantly different from conditions in the surrounding seawater, both in light and darkness, due to the metabolism of the foraminifer and its associated algae. Experimental light-dark cycles indicated a sufficient CO₂ supply sustaining high carbon fixation rates of the symbiotic algae via conversion of HCO₃^- or via CO₂ release from calcification and host respiration. Our findings on irradiance-dependent CO₂ and pH changes in the vicinity of symbiont-bearing planktonic foraminifera give direct experimental evidence for the predictions of isotope fractionation models used in palaeoclimatology stating that metabolic processes affect the isotopic carbon signal (δ¹³C) in foraminifera.     

低N胁迫下巴西橡胶实生苗叶片SPAD值、N素营养及农艺性状的变化

对3个品种(GT1、RRIM600、PR107)巴西橡胶(Hevea brasiliensis)实生苗进行不同N素(0%N,50%N,100%N)处理,分析比较叶片SPAD值、N素营养和农艺性状的变化及其关系。结果表明:(1)缺N胁迫明显降低了叶片SPAD值、叶片N含量和硝酸还原酶活性,缩小了叶蓬距、叶面积,且影响幅度随胁迫强度的加大而增大;(2)不同品种橡胶树实生苗对缺N胁迫的响应程度不同,总体上GT1较为敏感,PR107次之,RRIM600较不敏感;(3)橡胶树实生苗叶片SPAD值、叶蓬距、叶面积与叶片N含量、硝酸还原酶活性之间均呈显著或极显著正相关。因此,有可能采用测定叶片SPAD值、叶蓬距和叶面积等简易方法判断橡胶树新生叶蓬的N素营养状况。     

Full-Length Recombinant Plasmodium falciparum VAR2CSA Binds Specifically to CSPG and Induces Potent Parasite Adhesion-Blocking Antibodies

Plasmodium falciparum malaria remains one of the world's leading causes of human suffering and poverty. Each year, the disease takes 1-3 million lives, mainly in sub-Saharan Africa. The adhesion of infected erythrocytes (IEs) to vascular endothelium or placenta is the key event in the pathogenesis of severe P. falciparum infection. In pregnant women, the parasites express a single and unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family named VAR2CSA, which is associated with the ability of the IEs to adhere specifically to chondroitin sulphate A (CSA) in the placenta. Several Duffy-binding-like domains from VAR2CSA molecules have been shown in vitro to bind to CSA, but it has also been demonstrated that Duffy-binding-like domains from PfEMP1 proteins other than VAR2CSA can bind CSA. In addition, the specificity of the binding of VAR2CSA domains to glycosaminoglycans does not match that of VAR2CSA-expressing IEs. This has led to speculation that the domains of native VAR2CSA need to come together to form a specific binding site or that VAR2CSA might bind to CSA through a bridging molecule. Here, we describe the expression and purification of the complete extracellular region of VAR2CSA secreted at high yields from insect cells. Using surface plasmon resonance, we demonstrate that VAR2CSA alone binds with nanomolar affinity to human chondroitin sulphate proteoglycan and with significantly weaker affinity to other glycosaminoglycans, showing a specificity similar to that observed for IEs. Antibodies raised against full-length VAR2CSA completely inhibit recombinant VAR2CSA binding, as well as parasite binding to chondroitin sulphate proteoglycan. This is the first study to describe the successful production and functionality of a full-length PfEMP1. The specificity of the binding and anti-adhesion potency of induced IgG, together with high-yield production, encourages the use of full-length PfEMP1 in vaccine development strategies.     

Nox1 activation by βPix and the role of Ser-340 phosphorylation

Rac is an activating factor for Nox1, an O₂⁻-generating NADPH oxidase, expressed in the colon and other tissues. Rac requires a GDP-GTP exchange factor for activation. Nox1 activation by βPix has been demonstrated in cell lines. We examined the effects of βPix and its phosphomimetic mutant on endogenous Nox1 in Caco-2 cells transfected with Noxo1 and Noxa1. βPix expression enhanced O₂⁻ production in resting cells and cells stimulated with EGF or phorbol ester. βPix(S340E) further enhanced O₂⁻ production, while βPix(S340A) eliminated the βPix effect. βPix(S340E), but not βPix(S340A), had higher affinity and GEF activity for Rac than wild-type βPix. These results suggest that βPix phosphorylation at Ser-340 upregulates Nox1 through Rac activation, confirming Rac as a trigger for acute Nox1-dependent ROS production.     

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton

Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (K_d = 0.60 µM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.     

The requirement for external carbonic anhydrase in diatoms is influenced by the supply and demand for dissolved inorganic carbon

Photosynthesis by marine diatoms contributes significantly to the global carbon cycle. Due to the low concentration of CO₂ in seawater, many diatoms use extracellular carbonic anhydrase (eCA) to enhance the supply of CO₂ to the cell surface. While much research has investigated how the requirement for eCA is influenced by changes in CO₂ availability, little is known about how eCA contributes to CO₂ supply following changes in the demand for carbon. We therefore examined how changes in photosynthetic rate influence the requirement for eCA in three centric diatoms. Modeling of cell surface carbonate chemistry indicated that diffusive CO₂ supply to the cell surface was greatly reduced in large diatoms at higher photosynthetic rates. Laboratory experiments demonstrated a trend of an increasing requirement for eCA with increasing photosynthetic rate that was most pronounced in the larger species, supporting the findings of the cellular modeling. Microelectrode measurements of cell surface pH and O₂ demonstrated that individual cells exhibited an increased contribution of eCA to photosynthesis at higher irradiances. Our data demonstrate that changes in carbon demand strongly influence the requirement for eCA in diatoms. Cell size and photosynthetic rate will therefore be key determinants of the mode of dissolved inorganic carbon uptake.     

Malarial EBA-175 region VI crystallographic structure reveals a KIX-like binding interface

The malaria parasite proliferates in the bloodstream of its vertebrate host by invading and replicating within erythrocytes. To achieve successful invasion, a number of discrete and essential events need to take place at the parasite-host cell interface. Erythrocyte-binding antigen 175 (EBA-175) is a member of a family of Plasmodium falciparum erythrocyte-binding proteins involved in the formation of a tight junction, a necessary step in invasion. Here we present the crystal structure of EBA-175 region VI (rVI), a cysteine-rich domain that is highly conserved within the protein family and is essential for EBA-175 trafficking. The structure was solved by selenomethionine single-wavelength anomalous dispersion at 1.8 Å resolution. It reveals a homodimer, containing in each subunit a compact five-α-helix core that is stabilized by four conserved disulfide bridges. rVI adopts a novel fold that is likely conserved across the protein family, indicating a conserved function. It shows no similarity to the Duffy-binding-like domains of EBA-175 involved in erythrocyte binding, indicating a distinct role. Remarkably, rVI possesses structural features related to the KIX-binding domain of the coactivator CREB-binding protein, supporting the binding and trafficking roles that have been ascribed to it and providing a rational basis for further experimental investigation of its function.