We conducted a randomized single-blinded trial comparing the efficacy and safety of artesunate (AS) + amodiaquine (AQ, 3 days) versus AS (3 days) + sulfadoxine-pyrimethamine (SP, single dose) versus AS monotherapy (5 days) in Southern Mali. Uncomplicated malaria cases were followed for 28 days. Molecular markers of drug resistance were determined. After identification of recrudescences by genotyping, both artemisinin-based combination therapies (ACTs) reached nearly 100% efficacy at Day 14 and Day 28 versus 98.3% and 96.5% for AS, respectively (P > 0.05). AS + SP significantly selected DHFR and DHPS mutations associated with sulfadoxine and pyrimethamine resistance (P < 0.001), and AS + AQ equally selected PfCRT and PfMDR1 point mutations associated with chloroquine and AQ resistance (P < 0.001). No significant adverse event attributable to any of the study drugs was found. The ACTs were efficacious and safe, but the selection of markers for resistance to the partner drugs raises concerns over their lifespan in areas of intense malaria transmission.
Toxoplasma gondii is an intracellular parasite that causes disseminated infections in fetuses and immunocompromised individuals. Although gene regulation is important for parasite differentiation and pathogenesis, little is known about protein organization in the nucleus. Here we show that the fucose-binding Aleuria aurantia lectin (AAL) binds to numerous punctate structures in the nuclei of tachyzoites, bradyzoites, and sporozoites but not oocysts. AAL also binds to Hammondia and Neospora nuclei but not to more distantly related apicomplexans. Analyses of the AAL-enriched fraction indicate that AAL binds O-linked fucose added to Ser/Thr residues present in or adjacent to Ser-rich domains (SRDs). Sixty-nine Ser-rich proteins were reproducibly enriched with AAL, including nucleoporins, mRNA-processing enzymes, and cell-signaling proteins. Two endogenous SRDs-containing proteins and an SRD-YFP fusion localize with AAL to the nuclear membrane. Superresolution microscopy showed that the majority of the AAL signal localizes in proximity to nuclear pore complexes. Host cells modify secreted proteins with O-fucose; here we describe the O-fucosylation pathway in the nucleocytosol of a eukaryote. Furthermore, these results suggest O-fucosylation is a mechanism by which proteins involved in gene expression accumulate near the NPC.toxoplasma | fucose | nuclear glycosylation | nuclear pore complex T he apicomplexan parasite Toxoplasma gondii causes disseminated infections in humans, and these infections can lead to severe damage in immunocompromised individuals and fetuses (1, 2). There is no human vaccine against T. gondii, and recently the price of pyrimethamine, the drug used to treat toxoplasmosis in the United States, has increased more than 50-fold (2).T. gondii has a complex life cycle, and the parasite's ability to differentiate through its life stages in response to stresses and environmental conditions is fundamental for its pathogenicity and transmission (3). Transcriptome analyses have revealed that a large percentage of mRNAs show life stage-specific expression (4) and/or cell cycle regulation (5). Recent studies have increased our understanding of gene expression in T. gondii by identifying the AP2 family of transcription factors (6-8) and by describing posttranslational modifications (PTMs) of histones and some of the enzymes responsible for them (9-11). However, little is known about protein organization at the nuclear periphery, a subnuclear compartment that plays a critical role in transcriptional regulation in many eukaryotes. In particular, the gene-gating model (12) suggests that the nuclear pore complex (NPC) has a role in transcriptional regulation and chromatin organization as well as in protein and mRNA transport (13,14).In T. gondii chromodomain protein 1 localizes with heterochromatin at the nuclear periphery (15), and centromeres sequester to an apical nuclear region (16). Although the nuclear localization signal (NLS) and importin-α system are present, key nuclear import and export molecules are...
Unlike most cells, protozoa in the phylum Apicomplexa divide by a distinctive process in which multiple daughters are assembled within the mother (schizogony or endodyogeny), using scaffolding known as the inner membrane complex (IMC). The IMC underlies the plasma membrane during interphase, but new daughters develop in the cytoplasm, as cytoskeletal filaments associate with flattened membrane cisternae (alveolae), which elongate rapidly to encapsulate subcellular organelles. Newly assembled daughters acquire their plasma membrane as they emerge from the mother, leaving behind vestiges of the maternal cell. Although the maternal plasma membrane remains intact throughout this process, the maternal IMC disappears – is it degraded, or recycled to form the daughter IMC? Exploiting fluorescently tagged IMC markers, we have used live-cell imaging, fluorescence recovery after photobleaching (FRAP) and mEos2 photoactivation to monitor the dynamics of IMC biogenesis and turnover during the replication of Toxoplasma gondii tachyzoites. These studies reveal that the formation of the T. gondii IMC involves two distinct steps – de novo assembly during daughter IMC elongation within the mother cell, followed by recycling of maternal IMC membranes after the emergence of daughters from the mother cell.
Sulfadoxine–pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.
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