Protein Sorting in Plasmodium Falciparum.

Plasmodium falciparum is a unicellular eukaryote with a very polarized secretory system composed of micronemes rhoptries and dense granules that are required for host cell invasion. P. falciparum, like its relative T. gondii, uses the endolysosomal system to produce the secretory organelles and to ingest host cell proteins. The parasite also has an apicoplast, a secondary endosymbiotic organelle, which depends on vesicular trafficking for appropriate incorporation of nuclear-encoded proteins into the apicoplast. Recently, the central molecules responsible for sorting and trafficking in P. falciparum and T. gondii have been characterized. From these studies, it is now evident that P. falciparum has repurposed the molecules of the endosomal system to the secretory pathway. Additionally, the sorting and vesicular trafficking mechanism seem to be conserved among apicomplexans. This review described the most recent findings on the molecular mechanisms of protein sorting and vesicular trafficking in P. falciparum and revealed that P. falciparum has an amazing secretory machinery that has been cleverly modified to its intracellular lifestyle.

Introduction to effective mentorship for early-career research scientists.

Diversifying the scientific workforce remains a national priority due to the continued lack of representation from underrepresented individuals in STEM fields. Quality mentoring has been identified as a stimulus to enhance not only research success, but also recruitment and retention of underrepresented groups pursuing STEM careers. Utilizing the Entering Mentoring training curriculum framework, this report provides a brief synopsis and key takeaways from the 2019 NIH-ASCB Accomplishing Career Transition (ACT) workshop, "Introduction to Effective Mentorship for Scientists" for 30 senior postdoctoral and early-career faculty researchers from historically underrepresented racial and ethnicity backgrounds. In addition, effective strategies and best practices to enhance STEM mentoring for early-career researchers are provided, which have practical applications for diverse mentoring relationships across disciplines, career stages, and mentee types.

Human erythrocyte band 3 is a host receptor for Plasmodium falciparum glutamic acid-rich protein.

Malaria remains a major global threat to human health and economic development. Microvascular lesions caused by Plasmodium falciparum-infected human erythrocytes/red blood cells are hallmarks of severe pathogenesis contributing to high mortality, particularly in children from sub-Saharan Africa. In this study, we used a phage display complementary DNA library screening strategy to identify P falciparum glutamic acid-rich protein (PfGARP) as a secreted ligand that recognizes an ectodomain of human erythrocyte anion-exchanger, band 3/AE1, as a host receptor. Domain mapping of PfGARP revealed distinct nonoverlapping repeats encoding the immune response epitopes and core erythrocyte-binding activity. Synthetic peptides derived from the erythrocyte-binding repeats of PfGARP induced erythrocyte aggregation reminiscent of the rosetting phenomenon. Using peptides derived from the immunogenic repeats, a quantitative immunoassay was developed to detect a selective immune response against PfGARP in human plasma samples obtained from patients in rural Mali, suggesting the feasibility of PfGARP as a potential biomarker of disease progression. Collectively, our results suggest that PfGARP may play a functional role in enhancing the adhesive properties of human erythrocytes by engaging band 3 as a host receptor. We propose that immunological and pharmacological inhibition of PfGARP may unveil new therapeutic options for mitigating lesions in cerebral and pregnancy-associated malaria.

Assessment and Molecular Characterization of Human Intestinal Parasites in Bivalves from Orchard Beach, NY, USA.

Bivalves have been shown to be carriers of the human intestinal parasites Cryptosporidium parvum and Toxoplasma gondii. The goal of this study is to determine the prevalence of protozoan parasites in mollusks of New York City using a polymerase chain reaction (PCR)-based assay. Four species of mollusks, Mya arenaria, Geukensia demissa, Crassostrea virginica, and Mytilis edulis, were collected from Orchard Beach, NY in the fall of 2014, totaling 159 specimens. Each individual mollusk was dissected to harvest the digestive gland, the mantle, the gills, the foot and the siphon. The tissues were assayed for the presence of Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii DNA by using primers that target parasite-specific genes. C. parvum was found at a prevalence of 50%, 11.3%, and 1%, respectively, in Mya arenaria, G. demissa, and Mytilis edulis. C. parvum DNA was detected in all the tissues of these bivalve species, except the gills. Furthermore, G. lamblia was detected in Mya arenaria, G. demissa, Crassostrea virginica and Mytilis edulis at a prevalence of 37.5%, 4.5%, 60%, and 20.6%, respectively, while T. gondii DNA was not detected.

Toxoplasma gondii and Giardia duodenalis infections in domestic dogs in New York City public parks.

Toxoplasma gondii and Giardia duodenalis have been widely reported to produce major diseases in humans and domestic animals. Little is known about the occurrence of these protozoan parasites in domestic dogs in the United States. The goal of this study was to determine the prevalence and genotypes of T. gondii and G. duodenalis among dogs in New York City. Fecal samples from domestic dogs were analyzed by polymerase chain reaction (PCR). Overall, 3% of the samples tested positive for T. gondii, while 15% were positive for G. duodenalis. PCR-RFLP of T. gondii-positive samples revealed genotypes I and III, while sequence analysis of the G. duodenalis-positive samples indicated that 94.1% of the dogs were infected with the zoonotic assemblage A. Further studies are needed to determine the prevalence of zoonotic protozoan parasites in domestic dogs.

Plasmodium falciparum double C2 domain protein, PfDOC2, binds to calcium when associated with membranes.

The pathogenesis of malaria is strongly correlated with secretion of the micronemes, the apical organelles which contain the adhesins required for invasion of Plasmodium falciparum into human erythrocytes. A critical event in P. falciparum erythrocyte invasion is the production of calcium transients. After entering the cell, Ca(2+) binds to soluble Ca(2+)-binding proteins, such as the double C2 domains (DOC2). Recently, deletion of a P. falciparum DOC2 protein, PfDOC2, was shown to cause impairment in microneme secretion. However, PfDOC2 remains poorly characterized. Here, we report that PfDOC2 is expressed throughout the erythrocytic cycle and demonstrate that it is associated with membrane fractions and binds to calcium when it is part of these membranous structures. In summary, we show that PfDOC2 is a calcium lipid-binding protein of the protein kinase C type of DOC2 proteins.

Presence of Plasmodium and Haemoproteus in breeding prothonotary warblers (Protonotaria citrea: Parulidae): temporal and spatial trends in infection prevalence.

Prothonotary warbler (Protonotaria citrea) has shown a long-term decline in abundance in the United States. As a long-range migrant, these warblers are exposed to parasites in both tropical and temperate regions. The focus of this study was to use molecular techniques to examine the temporal prevalence patterns of heamosopridian parasites Plasmodium and Haemoproteus in breeding prothonotary warblers. The prevalence (presence or absence) of Plasmodium and Haemoproteus species was assayed using primer sets for the cytochrome b gene of the mitochondrial DNA. Blood samples were obtained from 187 adult prothonotary warblers collected at 3 central Virginia, U.S.A., breeding sites. The relationship between haemosporidian parasite infections and reproductive success also was examined. We found that 71% of captured prothonotary warblers were infected with haemosporidian parasites, specifically, with 36% prevalence for Haemoproteus spp. and 44% prevalence for Plasmodium spp., during the 2008 breeding season; for both parasites, prevalence increased throughout the season. We found significant variation in haemosporidian parasite prevalence across the breeding season that was strongly site specific. Conversely, we found no significant effects of haemosporidian parasite infections on the reproductive success of prothonotary warblers. This is in sharp contrast to recent reports suggesting considerable effects of these parasites on the reproductive success of wild birds.

Antimalarial activity of a cis-terpenone.

BACKGROUND: Malaria is the third most prevalent cause of infectious disease in the world. Resistance of the parasite to classical drugs makes the discovery of new and effective drugs more urgent. The oxidized derivative of hydroxy-cis terpenone (OHCT) is a synthetic molecule that is not toxic to cultured human liver cells at concentrations as high as 60 microM and inhibits activity of cytochrome P450s that metabolize many drugs. METHODS: OHCT activity against chloroquine-sensitive and -resistant strains of Plasmodium falciparum, and a P. falciparum clone that is partially resistant to artemisinin was assayed in vitro. RESULTS: OHCT at nanomolar concentrations was effective against all intraerythrocytic stages of P. falciparum and exhibited activity in vitro against both chloroquine-sensitive and -resistant strains of P. falciparum as well as a P. falciparum clone that is partially resistant to artemisinin. Moreover, OHCT exhibited potent activity against gametocytes, the form that is transmitted by mosquitoes and essential for the spread of malaria. CONCLUSION: OHCT displays strong growth inhibitory activity against all stages of P. falciparum and no evidence of toxicity to human cells in culture. It is easily synthesized and has the potential for inhibiting metabolism of drugs used in combination therapies.

Glycophorin B is the erythrocyte receptor of Plasmodium falciparum erythrocyte-binding ligand, EBL-1.

In the war against Plasmodium, humans have evolved to eliminate or modify proteins on the erythrocyte surface that serve as receptors for parasite invasion, such as the Duffy blood group, a receptor for Plasmodium vivax, and the Gerbich-negative modification of glycophorin C for Plasmodium falciparum. In turn, the parasite counters with expansion and diversification of ligand families. The high degree of polymorphism in glycophorin B found in malaria-endemic regions suggests that it also may be a receptor for Plasmodium, but, to date, none has been identified. We provide evidence from erythrocyte-binding that glycophorin B is a receptor for the P. falciparum protein EBL-1, a member of the Duffy-binding-like erythrocyte-binding protein (DBL-EBP) receptor family. The erythrocyte-binding domain, region 2 of EBL-1, expressed on CHO-K1 cells, bound glycophorin B(+) but not glycophorin B-null erythrocytes. In addition, glycophorin B(+) but not glycophorin B-null erythrocytes adsorbed native EBL-1 from the P. falciparum culture supernatants. Interestingly, the Efe pygmies of the Ituri forest in the Democratic Republic of the Congo have the highest gene frequency of glycophorin B-null in the world, raising the possibility that the DBL-EBP family may have expanded in response to the high frequency of glycophorin B-null in the population.

The glycophorin C N-linked glycan is a critical component of the ligand for the Plasmodium falciparum erythrocyte receptor BAEBL.

Plasmodium vivax uses a single member of the Duffy binding-like (DBL) receptor family to invade erythrocytes and is not found in West Africa where its erythrocyte ligand, the Duffy blood group antigen, is missing. In contrast, Plasmodium falciparum expresses four members of the DBL family, and remarkably, single-point mutations of two of these receptors (BAEBL and JESEBL) bind to entirely different erythrocyte ligands, greatly expanding the range of erythrocytes that P. falciparum can invade. In this article, we describe the molecular basis of the binding specificity for one BAEBL variant (VSTK) that binds to glycophorin C. We demonstrate that soluble glycophorin C completely blocks the binding of BAEBL (VSTK) to human erythrocytes, requiring 0.7 microM for 50% inhibition, a concentration similar to that required by glycophorin A to block the binding of erythrocyte-binding antigen 175 to erythrocytes. BAEBL (VSTK) does not bind to Gerbich-negative erythrocytes that express a truncated form of glycophorin C because it lacks exon 3. The N-linked oligosaccharide of Gerbich-negative glycophorin C has a markedly different composition than the wild-type glycophorin C. Moreover, removal of the N-linked oligosaccharide from the wild-type glycophorin C eliminates its ability to inhibit binding of BAEBL (VSTK) to erythrocytes. These findings are consistent with the ligand for BAEBL (VSTK) being, in part, the N-linked oligosaccharide and suggest that single-point mutations in BAEBL allow P. falciparum to recognize oligosaccharides on different erythrocyte surface glycoproteins or glycolipids, greatly increasing its invasion range.

Domain III of Plasmodium falciparum apical membrane antigen 1 binds to the erythrocyte membrane protein Kx.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is located in the merozoite micronemes, an organelle that contains receptors for invasion, suggesting that AMA1 may play a role in this process. However, direct evidence that P. falciparum AMA1 binds to human erythrocytes is lacking. In this study, we determined that domain III of AMA1 binds to the erythrocyte membrane protein, Kx, and that the rate of invasion of Kx(null) erythrocytes is reduced, indicating a significant but not unique role of AMA1 and Kx in parasite invasion of erythrocytes. Domains I/II/III, domains I/II and domain III of AMA1 were expressed on the surface of CHO-K1 cells, and their ability to bind erythrocytes was determined. We observed that each of these domains failed to bind untreated human erythrocytes. In contrast, domain III, but not the other domains of AMA1, bound to trypsin-treated human erythrocytes. We tested the binding of AMA1 to trypsin-treated genetically mutant human erythrocytes, missing various erythrocyte membrane proteins. AMA1 failed to bind trypsin-treated Kx(null) (McLeod) erythrocytes, which lack the Kx protein. Furthermore, treatment of human erythrocytes with trypsin, followed by alpha-chymotrypsin, cleaved Kx and destroyed the binding of AMA1 to human erythrocytes. Lastly, the rate of invasion of Kx null erythrocytes by P. falciparum was significantly lower than Kx-expressing erythrocytes. Taken together, our data suggest that AMA1 plays an important, but not exclusive, role in invasion of human erythrocytes through a process that involves exposure or modification of the erythrocyte surface protein, Kx, by a trypsin-like enzyme.

Parasite ligand-host receptor interactions during invasion of erythrocytes by Plasmodium merozoites.

Malaria parasites must recognise and invade different cells during their life cycle. The efficiency with which Plasmodium falciparum invades erythrocytes of all ages is an important virulence factor, since the ability of the parasite to reach high levels of parasitemia is often associated with severe pathology and morbidity. The merozoite invasion of erythrocytes is a highly complex, multi-step process that is dependent on a cascade of specific molecular interactions. Although many proteins are known to play an important role in invasion, their functional characteristics remain unclear. Therefore, a complete understanding of the molecular interactions that are the basis of the invasion process is absolutely crucial, not only in improving our knowledge about the basic biology of the malarial parasite, but also for the development of intervention strategies to counter the disease. Here we review the current state of knowledge about the receptor-ligand interactions that mediate merozoite invasion of erythrocytes.

Polymorphism in the Plasmodium falciparum erythrocyte-binding ligand JESEBL/EBA-181 alters its receptor specificity.

The malaria parasite lives within erythrocytes and depends on the binding of parasite ligands to host cell surface receptors for invasion. The most virulent human malaria parasite, Plasmodium falciparum, uses multiple ligands, including EBA-175, BAEBL, and JESEBL of the Duffy-binding-like (DBL) family of erythrocyte-binding proteins, for invasion of human erythrocytes. Region II of these parasite ligands is the erythrocyte-binding domain. Previously, we had shown that polymorphism in region II of BAEBL leads to different erythrocyte-binding specificities. We have now identified and characterized the binding specificity of six JESEBL variants. We sequenced region II of JESEBL from 20 P. falciparum clones collected from various parts of the world where malaria is endemic. We observed eight JESEBL variants that contained amino acid polymorphisms at five positions among all clones. Seven of the eight variants could be connected by a single base change that led to an amino acid change. We investigated the functional significance of these polymorphisms by transiently expressing region II from six of JESEBL variants on the surface of Chinese hamster ovary cells. We observed four erythrocyte-binding patterns to enzyme-treated erythrocytes. Thus, P. falciparum DBL ligands JESEBL and BAEBL can recognize multiple receptors on the erythrocyte surface. In contrast to Plasmodium vivax, which has disappeared from West Africa because of the Duffy-negative blood group, P. falciparum may have been successful in endemic areas because it has mutated the ligands of the DBL family to create multiple pathways of invasion, thus making selection of refractory erythrocytes unlikely.

Polymorphism in a Plasmodium falciparum erythrocyte-binding ligand changes its receptor specificity.

Recognition of human erythrocytes by Plasmodium species depends in part on Region II of the Duffy binding-like family of parasite ligands, which includes BA erythrocyte binding ligand (BAEBL) of P. falciparum. In previous studies of BAEBL from two clones, Dd2/Nm from Vietnam and E12 from Papua New Guinea (PNG), it was found that BAEBL bound different erythrocyte receptors. Because of variation in binding specificity, we studied the sequence and erythrocyte binding specificity of Region II of BAEBL in P. falciparum clones from different parts of the world. We observed five nucleotide substitutions leading to five amino acid changes and five polymorphisms in Region II of BAEBL in parasites from both PNG and other parts of the world. We expressed four of the polymorphisms on COS cells and determined their binding to enzyme-treated erythrocytes and to Gerbich-negative erythrocytes. We also performed erythrocyte-binding assay using the native protein from radiolabeled culture supernatant. Both assays demonstrated that each of the four polymorphisms in the parasite ligand, BAEBL, bound to a different receptor on erythrocytes. These results suggest that P. falciparum has evolved multiple invasion pathways dependent on polymorphisms in the BAEBL ligand.