The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of `naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments.
We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria.Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.
A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal.
BackgroundThe availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to α-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative α-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB.Methodology/Principal Findings14 α-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 α-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative α-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for α-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved α-helical coiled coil motifs.Conclusion/SignificanceWe conclude that the selection of α-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation.
In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1). In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.