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Now showing 1 - 10 of 20
  • Publication
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    SAP1 is a critical post-transcriptional regulator of infectivity in malaria parasite sporozoite stages
    (2011-02-01T00:00:00Z) Aly, Ahmed Sayed Ibrahım; Lindner, Scott E.; MacKellar, Drew C.; Peng, Xinxia; Kappe, Stefan H. I.; ALY, AHMED SAYED IBRAHıM
    Plasmodium salivary gland sporozoites upregulate expression of a unique subset of genes, collectively called the UIS (upregulated in infectious sporozoites). Many UIS were shown to be essential for early liver stage development, although little is known about their regulation. We previously identified a conserved sporozoite-specific protein, SAP1, which has an essential role in Plasmodium liver infection. Targeted deletion of SAP1 in Plasmodium yoelii caused the depletion of a number of selectively tested UIS transcripts in sporozoites, resulting in a complete early liver stage arrest. Here, we use a global gene expression survey to more comprehensively identify transcripts that are affected by SAP1 deletion. We find an effect upon both the transcript abundance of UIS genes, as well as of select genes previously not grouped as UIS. Importantly, we show that the lack of SAP1 causes the specific degradation of these transcripts. Collectively, our data suggest that SAP1 is involved in a selective post-transcriptional mechanism to regulate the abundance of transcripts critical to the infectivity of sporozoites. Although Pysap1(-) sporozoites are depleted of many of these important transcripts, they confer long-lasting sterile protection against wild-type sporozoite challenge in mice. SAP1 is therefore an appealing candidate locus for attenuation of Plasmodium falciparum.
  • Publication
    Metadata only
    Genetic Characterization of Coenzyme A Biosynthesis Reveals Essential Distinctive Functions during Malaria Parasite Development in Blood and Mosquito
    (2017-06-20T00:00:00Z) Hart, Robert J.; Abraham, Amanah; Aly, Ahmed Sayed Ibrahım; ALY, AHMED SAYED IBRAHıM
    Coenzyme A (CoA) is an essential universal cofactor for all prokaryotic and eukaryotic cells. In nearly all non-photosynthetic cells, CoA biosynthesis depends on the uptake and phosphorylation of vitamin B5 (pantothenic acid or pantothenate). Recently, putative pantothenate transporter (PAT) and pantothenate kinases (PanKs) were functionally characterized in P yoelii. PAT and PanKs were shown to be dispensable for blood stage development, but they were essential for mosquito stages development. Yet, little is known about the cellular functions of the other enzymes of the CoA biosynthesis pathway in malaria parasite life cycle stages. All enzymes of this pathway were targeted for deletion or deletion/complementation analyses by knockout/knock-in plasmid constructs to reveal their essential roles in P yoelii life cycle stages. The intermediate enzymes PPCS (Phosphopantothenylcysteine Synthase), PPCDC (Phosphopantothenylcysteine Decarboxylase) were shown to be dispensable for asexual and sexual blood stage development, but they were essential for oocyst development and the production of sporozoites. However, the last two enzymes of this pathway, PPAT (Phosphopantetheine Adenylyltransferase) and DPCK (Dephospho-CoA Kinase), were essential for blood stage development. These results indicate alternative first substrate requirement for the malaria parasite, other than the canonical pantothenate, for the synthesis of CoA in the blood but not inside the mosquito midgut. Collectively, our data shows that CoA de novo biosynthesis is essential for both blood and mosquito stages, and thus validates the enzymes of this pathway as potential antimalarial targets.
  • Publication
    Metadata only
    A systematic analysis of the early transcribed membrane protein family throughout the life cycle of Plasmodium yoelii
    (2011-11-01T00:00:00Z) MacKellar, Drew C.; Vaughan, Ashley M.; Aly, Ahmed Sayed Ibrahım; DeLeon, Sasha; Kappe, Stefan H. I.; ALY, AHMED SAYED IBRAHıM
    The early transcribed membrane proteins (ETRAMPs) are a family of small, highly charged transmembrane proteins unique to malaria parasites. Some members of the ETRAMP family have been localized to the parasitophorous vacuole membrane that separates the intracellular parasite from the host cell and thus presumably have a role in hostparasite interactions. Although it was previously shown that two ETRAMPs are critical for rodent malaria parasite liver-stage development, the importance of most ETRAMPs during the parasite life cycle remains unknown. Here, we comprehensively identify nine new etramps in the genome of the rodent malaria parasite Plasmodium yoelii, and elucidate their conservation in other malaria parasites. etramp expression profiles are diverse throughout the parasite life cycle as measured by RT-PCR. Epitope tagging of two ETRAMPs demonstrates protein expression in blood and liver stages, and reveals differences in both their timing of expression and their subcellular localization. Gene targeting studies of each of the nine uncharacterized etramps show that two are refractory to deletion and thus likely essential for blood-stage replication. Seven etramps are not essential for any life cycle stage. Systematic characterization of the members of the ETRAMP family reveals the diversity in importance of each family member at the interface between host and parasite throughout the developmental cycle of the malaria parasite.
  • Publication
    Metadata only
    The antimalarial activity of the pantothenamide alpha-PanAm is via inhibition of pantothenate phosphorylation
    (2017-10-27T00:00:00Z) Chiu, Joy E.; Thekkiniath, Jose; Choi, Jae-Yeon; Perrin, Benjamin A.; Lawres, Lauren; Plummer, Mark; Virji, Azan Z.; Abraham, Amanah; Toh, Justin Y.; Van Zandt, Michael; Aly, Ahmed Sayed Ibrahım; Voelker, Dennis R.; Ben Mamoun, Choukri; ALY, AHMED SAYED IBRAHıM
    The biosynthesis of the major acyl carrier Coenzyme A from pantothenic acid (PA) is critical for survival of Plasmodium falciparum within human erythrocytes. Accordingly, a PA analog alpha-PanAm showed potent activity against blood stage parasites in vitro; however, its efficacy in vivo and its mode of action remain unknown. We developed a new synthesis route for alpha-PanAm and showed that the compound is highly effective against blood stages of drug-sensitive and -resistant P. falciparum strains, inhibits development of P. berghei in hepatocytes, and at doses up to 100 mg/kg also inhibits blood stage development of P. chabaudi in mice. We used yeast and its pantothenate kinase Cab1 as models to characterize mode of action of alpha-PanAm and found that alpha-PanAm inhibits yeast growth in a PA-dependent manner, and its potency increases dramatically in a yeast mutant with defective pantothenate kinase activity. Biochemical analyses using C-14-PA as a substrate demonstrated that alpha-PanAm is a competitive inhibitor of Cab1. Interestingly, biochemical and mass spectrometry analyses also showed that the compound is phosphorylated by Cab1. Together, these data suggest that alpha-PanAm exerts its antimicrobial activity by direct competition with the natural substrate PA for phosphorylation by the pantothenate kinase.
  • Publication
    Metadata only
    Plasmodium yoelii Vitamin B-5 Pantothenate Transporter Candidate is Essential for Parasite Transmission to the Mosquito
    (2014-07-11T00:00:00Z) Hart, Robert J.; Lawres, Lauren; Fritzen, Emma; Ben Mamoun, Choukri; Aly, Ahmed Sayed Ibrahım; ALY, AHMED SAYED IBRAHıM
    In nearly all non-photosynthetic cells, pantothenate (vitamin B-5) transport and utilization are prerequisites for the synthesis of the universal essential cofactor Coenzyme A (CoA). Early studies showed that human malaria parasites rely on the uptake of pantothenate across the parasite plasma membrane for survival within erythrocytes. Recently, a P. falciparum candidate pantothenate transporter (PAT) was characterized by functional complementation in yeast. These studies revealed that PfPAT mediated survival of yeast cells in low pantothenate concentrations and restored sensitivity of yeast cells lacking pantothenate uptake to fenpropimorph. In addition, PfPAT was refractory to deletion in P. falciparum in vitro, but nothing is known about the in vivo functions of PAT in Plasmodium life cycle stages. Herein, we used gene-targeting techniques to delete PAT in Plasmodium yoelii. Parasites lacking PAT displayed normal asexual and sexual blood stage development compared to wild-type (WT) and WT-like p230p(-) parasites. However, progression from the ookinete to the oocyst stage and sporozoite formation were completely abolished in pat(-) parasites. These studies provide the first evidence for an essential role of a candidate pantothenate transport in malaria transmission to Anopheles mosquitoes. This will set the stage for the development of PAT inhibitors against multiple parasite life cycle stages.
  • Publication
    Metadata only
    A Plasmodium /-hydrolase modulates the development of invasive stages
    (2015-12-01T00:00:00Z) Groat-Carmona, Anna M.; Kain, Heather; Brownell, Jessica; Douglass, Alyse N.; Aly, Ahmed Sayed Ibrahım; Kappe, Stefan H. I.; ALY, AHMED SAYED IBRAHıM
    The bud emergence (BEM)46 proteins are evolutionarily conserved members of the /-hydrolase superfamily, which includes enzymes with diverse functions and a wide range of substrates. Here, we identified a PlasmodiumBEM46-like protein (PBLP) and characterized it throughout the life cycle of the rodent malaria parasite Plasmodium yoelii. The PlasmodiumBEM46-like protein is shown to be closely associated with the parasite plasma membrane of asexual erythrocytic stage schizonts and exo-erythrocytic schizonts; however, PBLP localizes to unique intracellular structures in sporozoites. Generation and analysis of P.yoelii knockout (pblp) parasite lines showed that PBLP has an important role in erythrocytic stage merozoite development with pblp parasites forming fewer merozoites during schizogony, which results in decreased parasitemia when compared with wild-type (WT) parasites. pblp parasites showed no defects in gametogenesis or transmission to mosquitoes; however, because they formed fewer oocysts there was a reduction in the number of developed sporozoites in infected mosquitoes when compared with WT. Although pblp sporozoites showed no apparent defect in mosquito salivary gland infection, they showed decreased infectivity in hepatocytes in vitro. Similarly, mice infected with pblp sporozoites exhibited a delay in the onset of blood-stage patency, which is likely caused by reduced sporozoite infectivity and a discernible delay in exo-erythrocytic merozoite formation. These data are consistent with the model that PBLP has an important role in parasite invasive-stage morphogenesis throughout the parasite life cycle.
  • Publication
    Metadata only
    Gene Targeting Studies of the Malaria Parasite DNA Photolyase gene using CRISPR-Cas9 Genome Editing Technology
    (2021-01-08T00:00:00Z) Yılmaz, İlknur; Palabıyık, Bedia; Temel, Binnur; Aly, Ahmed Sayed Ibrahım; TEMEL, BİNNUR; ALY, AHMED SAYED IBRAHıM
  • Publication
    Open Access
    Automated wide-field malaria parasite infection detection using Fourier ptychography on stain-free thin-smears
    (2022-07-01T00:00:00Z) AKÇAKIR, OSMAN; Celebi, Lutfi Kadir; KAMIL, MOHD; ALY, Ahmed Sayed Ibrahım; AKÇAKIR, OSMAN; KAMIL, MOHD; ALY, AHMED SAYED IBRAHıM
    Diagnosis of malaria in endemic areas is hampered by the lack of a rapid, stain-free and sensitive method to directly identify parasites in peripheral blood. Herein, we report the use of Fourier ptychography to generate wide-field high-resolution quantitative phase images of erythrocytes infected with malaria parasites, from a whole blood sample. We are able to image thousands of erythrocytes (red blood cells) in a single field of view and make a determination of infection status of the quantitative phase image of each segmented cell based on machine learning (random forest) and deep learning (VGG16) models. Our random forest model makes use of morphology and texture based features of the quantitative phase images. In order to label the quantitative images of the cells as either infected or uninfected before training the models, we make use of a Plasmodium berghei strain expressing GFP (green fluorescent protein) in all life cycle stages. By overlaying the fluorescence image with the quantitative phase image we could identify the infected subpopulation of erythrocytes for labelling purposes. Our machine learning model (random forest) achieved 91% specificity and 72% sensitivity while our deep learning model (VGG16) achieved 98% specificity and 57% sensitivity. These results highlight the potential for quantitative phase imaging coupled with artificial intelligence to develop an easy to use platform for the rapid and sensitive diagnosis of malaria.
  • Publication
    Metadata only
    Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages
    (2019-05-01T00:00:00Z) Aly, Ahmed Sayed Ibrahım; Deveci, Gozde; Yilmaz, Ilknur; Abraham, Amanah; Golshan, Aneesa; Hart, Robert J.; ALY, AHMED SAYED IBRAHıM; DEVECİ, GÖZDE
    Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P. falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites. Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.
  • Publication
    Metadata only
    Mitochondrial Spermidine Synthase is Essential for Blood-stage growth of the Malaria Parasite
    (2022-12-01T00:00:00Z) KAMIL, MOHD; KINA, ÜMİT YAŞAR; DEVECİ, GÖZDE; Akyuz, Sevim N.; Yilmaz, Ilknur; ALY, Ahmed Sayed Ibrahım; KAMIL, MOHD; KINA, ÜMİT YAŞAR; DEVECİ, GÖZDE; ALY, AHMED SAYED IBRAHıM
    © 2022Positively-charged polyamines are essential molecules for the replication of eukaryotic cells and are particularly important for the rapid proliferation of parasitic protozoa and cancer cells. Unlike in Trypanosoma brucei, the inhibition of the synthesis of intermediate polyamine Putrescine caused only partial defect in malaria parasite blood-stage growth. In contrast, reducing the intracellular concentrations of Spermidine and Spermine by polyamine analogs caused significant defects in blood-stage growth in Plasmodium yoelii and P. falciparum. However, little is known about the synthesizing enzyme of Spermidine and Spermine in the malaria parasite. Herein, malaria parasite conserved Spermidine Synthase (SpdS) gene was targeted for deletion/complementation analyses by knockout/knock-in constructs in P. yoelii. SpdS was found to be essential for blood-stage growth. Live fluorescence imaging in blood-stages and sporozoites confirmed a specific mitochondrial localization, which is not known for any polyamine-synthesizing enzyme so far. This study identifies SpdS as an excellent drug targeting candidate against the malaria parasite, which is localized to the parasite mitochondrion.