Plasmodium falciparum VAR2CSA binds to chondroitin sulfate A (CSA) on the surface of the syncytiotrophoblast during placental malaria. This interaction facilitates placental sequestration of malaria parasites resulting in severe health outcomes for both the mother and her offspring. Furthermore, CSA is presented by diverse cancer cells and specific targeting of cells by VAR2CSA may become a viable approach for cancer treatment. Here, we determined the Cryo-EM structures of the full-length ectodomain of VAR2CSA from P. falciparum strain NF54 in complex with CSA, and VAR2CSA from a second P. falciparum strain FCR3. The architecture of VAR2CSA is composed of a stable core flanked by a flexible arm. CSA traverses the core domain by binding within two channels and CSA binding does not induce major conformational changes in VAR2CSA. The CSA-binding elements are conserved across VAR2CSA variants and are flanked by polymorphic segments, suggesting immune selection outside the CSA-binding sites. This work provides paths for developing interventions against placental malaria and cancer.

In Gram‐negative bacteria, a wide variety of virulence factors are secreted via the autotransporter (AT) pathway. Intriguingly, there is no significant concentration of ATP in the periplasm, nor a proton gradient across the OM, so the energetic origin of efficient secretion of AT proteins is unknown. More than 97% of AT proteins are predicted to contain right‐handed parallel β‐helical structure, and the three crystal structures available for AT passenger domains each contain a long right‐handed parallel β‐helix. Previous studies have shown that pertactin, an AT from Bordetella pertussis, exhibits three‐state folding and has a C‐terminal stable core structure. Here, we show that Pet, an unrelated AT from Escherichia coli, also exhibits three‐state unfolding and also has a stable core structure. Deletion mutants, mass spectrometry, and N‐terminal sequencing demonstrate that the Pet stable core is also located near the C‐terminus of the passenger domain. Moreover, sequence analysis suggests that three‐state folding and a C‐terminal stable core structure could be important general features of the biogenesis of AT proteins in vivo. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 420–427, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Background. Vaccines that block human-to-mosquito Plasmodium transmission are needed for malaria eradication and clinical trials have targeted zygote antigen Pfs25 for decades. We reported that a Pfs25 protein-protein conjugate vaccine formulated in alum adjuvant induced significant serum functional activity in both US and Malian adults. However, antibody levels declined rapidly, and transmission-reducing activity required four vaccine doses. Functional immunogenicity and durability must be improved before advancing TBV further in clinical development. We hypothesized that the pre-fertilization protein Pfs230 alone or in combination with Pfs25 would improve functional activity. Methods. Transmission-blocking vaccine candidates based on gamete antigen Pfs230 or Pfs25 were conjugated with Exoprotein A, formulated in Alhydrogel ®, and administered to mice, rhesus macaques, and humans. Antibody levels were measured by ELISA and transmission-reducing activity was assess by the Standard Membrane Feeding Assay. Results. Pfs25-EPA/Alhydrogel ® and Pfs230D1-EPA/Alhydrogel ® induced similar serum functional activity in mice, but Pfs230D1-EPA induced significantly greater activity in rhesus monkeys that was enhanced by complement. In U.S. adults, two vaccine doses induced complement-dependent activity in 4 of 5 Pfs230D1-EPA/Alhydrogel® recipients but no significant activity in five Pfs25-EPA recipients, and combination with Pfs25-EPA did not increase activity over Pfs230D1-EPA alone. Conclusion. The complement-dependent functional immunogenicity of Pfs230D1-EPA represents a significant improvement over Pfs25-EPA in this comparative study. The rhesus model is more predictive of the functional human immune response to Pfs230D1 than is the mouse model.

Summary Autotransporter (AT) proteins are the largest class of extracellular virulence proteins secreted from Gram-negative bacteria. The mechanism by which AT proteins cross the bacterial outer membrane (OM), in the absence of ATP or another external energy source, is unknown. Here we demonstrate a linear correlation between localized regions of stability (ΔGfolding) in the mature virulence protein (the AT “passenger”) and OM secretion efficiency. Destabilizing the C-terminal β-helical domain of a passenger reduced secretion efficiency. In contrast, destabilizing the globular N-terminal domain of a passenger produced a linearly correlated increase in secretion efficiency. Thus, C-terminal passenger stability facilitates OM secretion, whereas N-terminal stability hinders it. The contributions of regional passenger stability to OM secretion demonstrate a crucial role for the passenger itself in directing its secretion, suggesting a novel type of ATP-independent, folding-driven transporter.