Vesicle Targeting In Plasmodium Falciparum: The Identification and Molecular Characterization of Plasmodium Falciparum Family of of Snare Proteins

Proteins of the SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) super-family have been characterized as playing an essential role in vesicle targeting and fusion in all eukaryotes. The intracellular malaria parasite Plasmodium falciparum exhibits an unusual endomembrane system that is characterized by an unstacked Golgi apparatus, a developmentally induced apical complex, and various organellar structures of parasite origin in the infected host cells. How malaria parasites target nuclear-encoded proteins to these novel compartments is a central question in Plasmodium cell biology. Ultrastructural studies elsewhere have implicated the participation of specialized vesicular elements in transport of virulence proteins, including various cytoadherance and host cell remodeling factors, into the infected erythrocyte cytoplasm. However, little is known about the machineries that define the directionality of vesicle trafficking in malaria parasites. We hypothesized that the P. falciparum SNARE proteins would exhibit novel features required for vesicle targeting to the parasite-specific compartments. We then identified for the first time and confirmed the expression of eighteen SNARE genes in P. falciparum. Members of the PfSNAREs exhibit atypical structural features (Ayong et al., 2007, Molecular & Biochemical Parasitology, 152(2), 113-122). Among the atypical PfSNAREs, PfSec22 contains an unusual insertion of the Plasmodium export element (PEXEL) within its profilin-like longin domain, preceded by an N-terminal hydrophobic segment. Localization analyses suggest that PfSec22 is predominantly a vesicle-associated SNARE of the ER/Golgi interface, but which associates partially with mobile extraparasitic vesicles in P. falciparum-infected erythrocytes at trophozoite stages. We showed that PfSec22 export into host cells occurs via a two-step model that involves extraparasitic vesicle budding from the parasite plasma membrane and fusion with the parasitophorous vacuolar membrane. Export of PfSec22 was independent of its membrane-insertion suggesting that this protein might cross the vacuolar space as a single-pass type IV membrane protein. We demonstrated that the atypical longin domain dictates the steady-state localization of PfSec22, regulating its ER/Golgi trafficking and export into host cells. Our study provides the first experimental evidence for SNARE protein export in P. falciparum, and suggests a role of PfSec22 in vesicle trafficking within the infected host cell (Ayong et al, Eukaryotic Cell, Epub Jul 17, 2009) Next, to define the physiological function of the PfSec22 protein in Plasmodium parasites, we investigated its cognate partners. Using purified recombinant proteins we showed that PfSec22 forms direct binding interactions with six other PfSNAREs in vitro. These included the PfSyn5, PfBet1, PfGS27, PfSyn6, PfSyn16 and PfSyn18 PfSNAREs. By generating GFPexpressing parasites, we successfully localized the SNARE proteins PfSyn5, PfBet1 and PfGS27 to the parasite cis-Golgi compartment. We confirmed the association of PfSec22 with PfSyn5, PfBet1 and PfGS27 in vivo by immunoprecipitation analyses. Our data indicate a conserved ERto-Golgi SNARE assembly in P. falciparum, and suggest that the malaria Sec22 protein might form novel SNARE complexes required for vesicle traffic within P. falciparum-infected erythrocytes

Spirocyclic chromanes exhibit antiplasmodial activities and inhibit all intraerythrocytic life cycle stages

AbstractWe screened a collection of synthetic compounds consisting of natural-product-like substructural motifs to identify a spirocyclic chromane as a novel antiplasmodial pharmacophore using an unbiased cell-based assay. The most active spirocyclic compound UCF 201 exhibits a 50% effective concentration (EC50) of 350 nM against the chloroquine-resistant Dd2 strain and a selectivity over 50 using human liver HepG2 cells. Our analyses of physicochemical properties of UCF 201 showed that it is in compliance with Lipinski's parameters and has an acceptable physicochemical profile. We have performed a limited structure-activity-relationship study with commercially available chromanes preserving the spirocyclic motif. Our evaluation of stage specificities of UCF 201 indicated that the compound is early-acting in blocking parasite development at ring, trophozoite and schizont stages of development as well as merozoite invasion. SPC is an attractive lead candidate scaffold because of its ability to act on all stages of parasite's aexual life cycle unlike current antimalarials

A plastid two-pore channel essential for inter-organelle communication and growth of Toxoplasma gondii.

Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca2+-dependent events. Little is known about TPCs in unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca2+ indicator targeted to the apicoplast, we show that Ca2+ signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca2+ uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge

Computational Design and Preliminary Serological Analysis of a Novel Multi-Epitope Vaccine Candidate Against Onchocerciasis and Related Filarial Diseases

Onchocerciasis is a skin and eye disease that exerts a heavy socio-economic burden, particularly in sub-Saharan Africa, a region which harbours greater than 96% of either infected or at-risk populations. The elimination plan for the disease is currently challenged by many factors including amongst others; the potential emergence of resistance to the main chemotherapeutic agent, ivermectin (IVM). Novel tools, including preventative and therapeutic vaccines, could provide additional impetus to the disease elimination tool portfolio. Several observations in both humans and animals have provided evidence for the development of both natural and artificial acquired immunity. In this study, immuno-informatics tools were applied to design a filarial-conserved multi-epitope subunit vaccine candidate, (designated Ov-DKR-2) consisting of B-and T-lymphocyte epitopes of eight immunogenic antigens previously assessed in pre-clinical studies. The high-percentage conservation of the selected proteins and epitopes predicted in related nematode parasitic species hints that the generated chimera may be instrumental for cross-protection. Bioinformatics analyses were employed for the prediction, refinement, and validation of the 3D structure of the Ov-DKR-2 chimera. In-silico immune simulation projected significantly high levels of IgG1, T-helper, T-cytotoxic cells, INF-γ, and IL-2 responses. Preliminary immunological analyses revealed that the multi-epitope vaccine candidate reacted with antibodies in sera from both onchocerciasis-infected individuals, endemic normals as well as loiasis-infected persons but not with the control sera from European individuals. These results support the premise for further characterisation of the engineered protein as a vaccine candidate for onchocerciasis

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Identification Of Plasmodium Falciparum Family Of Snares

SNARE proteins function as specificity determinants in all eukaryotic vesicle-mediated transport pathways. Although the intra-erythrocytic parasite Plasmodium falciparum is known to target nuclear-encoded proteins via transport vesicles to several destinations within and beyond its plasma membrane, little is known about the role of SNARE proteins in these unusual trafficking pathways. In this study, we identified and compared the subunit structure of P. falciparum homologues of SNAREs (PfSNAREs) with their complements in mammals, and determined the subcellular localizations of some family members. A comprehensive bioinformatics analysis of the P. falciparum genome revealed 18 SNARE-like proteins that could be classified into five main phylogenetic groups: membrin-like, Bet1-like, VAMP-like, syntaxin5-like, and a P. falciparum-specific syntaxin-like subfamily. Unique to some PfSNARE proteins were presence of atypical amino acid residues at the 0 layer position, presence of up to two transmembrane segments, and frequent occurrence of low-complexity regions. Subcellular distribution of green fluorescence protein (GFP)-tagged P. falciparum SNARE orthologues indicates that PfSyn5p and PfSec22p are partly associated to ER and Golgi compartments, and to other punctuated structures within the parasite plasma membrane. Our data confirms of a conserved SNARE-mediated anterograde transport system in the parasite and argues against any involvement of these two SNAREs in vesicular trafficking within the host cell compartment. © 2007 Elsevier B.V. All rights reserved

Characterization Of A Prl Protein Tyrosine Phosphatase From Plasmodium Falciparum

Isoprenylated proteins have important functions in cell growth and differentiation of eukaryotic cells. Inhibitors of protein prenylation in malaria have recently shown strong promise as effective antimalarials. In studying protein prenylation in the malaria protozoan parasite Plasmodium falciparum, we have shown earlier that the incubation of P. falciparum cells with 3H-prenol precursors resulted in various size classes of labeled proteins. To understand the physiological function of prenylated proteins of malaria parasites, that are targets of prenyltransferase inhibitors, we searched the PlasmoDB database for proteins containing the C-terminus prenylation motif. We have identified, among other potentially prenylated proteins, an orthologue of a PRL (protein of regenerating liver) subgroup protein tyrosine phosphatases, termed PfPRL. Here, we show that PfPRL is expressed in the parasite\u27s intraerythrocytic stages, where it partially associates with endoplasmic reticulum and within a subcompartment of the food vacuole. Additionally, PfPRL targeting parallels that of apical membrane antigen-1 in developing merozoites. Recombinant PfPRL shows phosphatase activity that is preferentially inhibited by a tyrosine phosphatase inhibitor suggesting that PfPRL functions as a tyrosine phosphatase. Recombinant PfPRL can also be farnesylated in vitro. Inhibition of malarial farnesyltransferase activity can be achieved with the heptapetide RKCHFM, which corresponds to the C-terminus of PfPRL. This study provides the first evidence for expression of enzymatically active PRL-related protein tyrosine phosphatases in malarial parasites, and demonstrates the potential of peptides derived from Plasmodium prenylated proteins as malarial farnesyltransferase inhibitors. © 2007 Elsevier B.V. All rights reserved

Evidence For Prenylation-Dependent Targeting Of A Ykt6 Snare In Plasmodium Falciparum

Ykt6 proteins are the most versatile fusogens in eukaryotic cells, and the only SNAREs that can be both prenylated and acylated at a C-terminal CAAX motif. Unlike yeast and mammalian cells where a single Ykt6 gene is expressed, the Plasmodium falciparum genome encodes two Ykt6 proteins. We have investigated the expression and prenylation of the Ykt6 orthologue, PfYkt6.1 in intra-erythrocytic stages of P. falciparum. PfYkt6.1 localized to the parasite Golgi and other unidentified cytoplasmic compartments, and was partly cytosolic (∼50% in early trophozoites). The membrane-association of PfYkt6.1 was dependent on the presence of a conserved C-terminal CAAX motif (CCSIM). By expressing full-length and mutant proteins in Escherichia coli, we have shown that PfYkt6.1 indeed serves as substrate for prenylation by P. falciparum farnesyltransferases. Surprisingly, PfYkt6.1 could also be geranylgeranylated by parasite extracts independent of the C-terminal amino acid residue. Deletion of the CAAX motif inhibited both farnesylation and geranylgeranylation activities. Additionally, the PfYkt6.1 heptapeptide KQCCSIM, corresponding to the C-terminal CAAX sequence, inhibited the parasite farnesyltransferase activity with an IC50 of 1 μM. Our findings underscore the importance of CAAX motif-derived peptidomimetics for antimalarial drug development. © 2010 Elsevier B.V. All rights reserved

Spirocyclic Chromanes Exhibit Antiplasmodial Activities And Inhibit All Intraerythrocytic Life Cycle Stages

We screened a collection of synthetic compounds consisting of natural-product-like substructural motifs to identify a spirocyclic chromane as a novel antiplasmodial pharmacophore using an unbiased cell-based assay. The most active spirocyclic compound UCF 201 exhibits a 50% effective concentration (EC50) of 350 nM against the chloroquine-resistant Dd2 strain and a selectivity over 50 using human liver HepG2 cells. Our analyses of physicochemical properties of UCF 201 showed that it is in compliance with Lipinski\u27s parameters and has an acceptable physicochemical profile. We have performed a limited structure-activity-relationship study with commercially available chromanes preserving the spirocyclic motif. Our evaluation of stage specificities of UCF 201 indicated that the compound is early-acting in blocking parasite development at ring, trophozoite and schizont stages of development as well as merozoite invasion. SPC is an attractive lead candidate scaffold because of its ability to act on all stages of parasite\u27s aexual life cycle unlike current antimalarials

Nutritional Status and Humoral Immune Response to Plasmodium falciparum in Children Aged 6–59 Months

Malaria is a leading cause of morbidity and mortality in Africa. Children are mostly exposed to this disease; numerous studies have investigated the relationship between child malnutrition and either malaria morbidity or infection. Few studies demonstrated the interaction between child malnutrition and specific anti-Plasmodium falciparum immune responses. The purpose of this study was to investigate the impact of nutritional status and iron on total anti-Plasmodium falciparum IgG levels in children living in the Gado-Badzéré refugee camp. We carried out a cross-sectional study during August–November 2017 in the Gado-Badzéré refugee camp in the East region of Cameroon. Children aged from 6 to 59 months with fever were recruited from the medical center. The data were recorded using a standardized data collection sheet and were analyzed using SPSS and WHO Anthro software. The total anti-Pf 3D7 total IgG level was determined using an ELISA technique while a colorimetric method was used to measure the total iron level. A total of 83 patients aged 6–59 months were enrolled in this study. The prevalence of malaria and malnutrition was 47% and 31%, respectively. Acute malnutrition was statistically less recurrent in noninfected children compared with that in the infected children. The infection tended to have significant influence on the level of anti-Plasmodium falciparum antibodies in children. In addition, nutritional status and serum iron levels had no significant influence on children’s anti-Pf IgG T levels. Malaria and malnutrition remain real public health problems in the Gado-badzéré refugee camp. Knowledge of the nutritional profile of the population would be of great benefit in setting up an appropriate health program. We therefore suggest that more standardized studies be conducted to highlight the effect of nutrition and micronutrients on immunological status