scholarly journals AGIA Tag System for Ultrastructural Protein Localization Analysis in Blood-Stage Plasmodium falciparum

2021 ◽  
Vol 11 ◽  
Author(s):  
Masayuki Morita ◽  
Bernard N. Kanoi ◽  
Naoaki Shinzawa ◽  
Rie Kubota ◽  
Hiroyuki Takeda ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Subcellular Localization ◽  
Western Blotting ◽  
Protein Localization ◽  
Transmembrane Protein ◽  
Physiological Role ◽  
Immunofluorescence Assay ◽  
Blood Stage ◽  
Parasite Development ◽  
High Signal

Precise subcellular localization of proteins is the key to elucidating the physiological role of these molecules in malaria parasite development, understanding of pathogenesis, and protective immunity. In Plasmodium falciparum, however, detection of proteins in the blood-stage parasites is greatly hampered by the lack of versatile protein tags which can intrinsically label such molecules. Thus, in this study, to develop a novel system that can be used to evaluate subcellular localization of known and novel proteins, we assessed the application of AGIA tag, consisting of 9 amino acids (EEAAGIARP), in P. falciparum blood-stage parasites. Specifically, AGIA-tagged ring-infected erythrocyte surface antigen (RESA-AGIA) was episomally expressed in P. falciparum 3D7 strain. The RESA-AGIA protein was detected by Western blotting and immunofluorescence assay (IFA) using recombinant rabbit anti-AGIA tag monoclonal antibody (mAb) with a high signal/noise ratio. Similarly, AGIA-tagged multidrug resistance protein 1 (MDR1-AGIA), as an example of polyptic transmembrane protein, was endogenously expressed and detected by Western blotting and IFA with anti-AGIA tag mAb. Immunoelectron microscopy of the RESA-AGIA transfected merozoites revealed that mouse anti-RESA and the rabbit anti-AGIA mAb signals could definitively co-localize to the dense granules. Put together, this study demonstrates AGIA tag/anti-AGIA rabbit mAb system as a potentially useful tool for elucidating the subcellular localization of new and understudied proteins in blood-stage malaria parasites at the nanometer-level resolution.

scholarly journals Localization and function of a Plasmodium falciparum protein (PF3D7_1459400) during erythrocyte invasion

2020 ◽  
pp. 153537022096176
Author(s):  
Emmanuel Amlabu ◽  
Prince B Nyarko ◽  
Grace Opoku ◽  
Damata Ibrahim-Dey ◽  
Philip Ilani ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Subcellular Localization ◽  
Protein Expression ◽  
Functional Characterization ◽  
Blood Stage ◽  
Protein Subcellular Localization ◽  
Erythrocyte Invasion ◽  
Invasion Inhibition ◽  
Blood Stage Malaria ◽  
Peptide Antibodies

Nearly 60% of Plasmodium falciparum proteins are still uncharacterized and their functions are unknown. In this report, we carried out the functional characterization of a 45 kDa protein (PF3D7_1459400) and showed its potential as a target for blood stage malaria vaccine development. Analysis of protein subcellular localization, native protein expression profile, and erythrocyte invasion inhibition of both clinical and laboratory parasite strains by peptide antibodies suggest a functional role of PF3D7_1459400 protein during erythrocyte invasion. Also, immunoreactivity screens using synthetic peptides of the protein showed that adults resident in malaria endemic regions in Ghana have naturally acquired plasma antibodies against PF3D7_1459400 protein. Altogether, this study presents PF3D7_1459400 protein as a potential target for the development of peptide-based vaccine for blood-stage malaria. Impact statement Plasmodium falciparum malaria is a global health problem. Erythrocyte invasion by P. falciparum merozoites appears to be a promising target to curb malaria. We have identified and characterized a novel protein that is involved in erythrocyte invasion. Our data on protein subcellular localization, stage-specific protein expression pattern, and merozoite invasion inhibition by α-peptide antibodies suggest a role for PF3D7_1459400 protein during P. falciparum erythrocyte invasion. Even more, the human immunoepidemiology data present PF3D7_1459400 protein as an immunogenic antigen which could be further exploited for the development of new anti-infective therapy against malaria.

scholarly journals Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

2019 ◽  
Author(s):  
Yang Wu ◽  
Vesela Encheva ◽  
Judith L. Green ◽  
Edwin Lasonder ◽  
Adchara Prommaban ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Homology Model ◽  
Food Vacuole ◽  
Blood Stage ◽  
Parasite Development ◽  
Parasite Line ◽  
Schizont Stage ◽  
Post Translational Modification ◽  
Asexual Blood Stage ◽  
Erythrocyte Invasion

AbstractUbiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite’s elaboration of a new food vacuole.

Subcellular Localization of Quinate: Oxidoreductase from Phaseolus mungo L. Sprouts

1994 ◽  
Vol 49 (7-8) ◽  
pp. 415-420 ◽  
Author(s):  
Xiangbo Kang ◽  
H. Ekkehard Neuhaus ◽  
Renate Scheibe
Keyword(s):  
Subcellular Localization ◽  
Western Blotting ◽  
Mung Bean ◽  
Plant Material ◽  
Physiological Role ◽  
Single Band ◽  
Phaseolus Mungo ◽  
Monospecific Antiserum

Quinate:oxidoreductase (QORase, EC 1.1.1.24) was isolated and purified from etiolated mung bean (Phaseolus mungo L.) sprouts and a monospecific antiserum was raised in rabbit to the homogeneous protein. Highly intact etioplasts were isolated from the same plant material. The stroma of the purified etioplasts was enzymatically characterized. Contamination by cytosol, mitochondria and vacuole was estimated from activities of marker en­zymes. QORase activity was localized in the stroma (about 91% for both NAD+ and NADP+ as a cofactor). Western blotting and immunoprinting of the stroma proteins revealed a single band that migrated identically with the purified QORase. The results suggest that the QOR-ase is localized predominantly, if not exclusively, in the etioplast stroma. The physiological role of the enzyme is discussed

scholarly journals Evidence for a Common Role for the Serine-Type Plasmodium falciparum Serine Repeat Antigen Proteases: Implications for Vaccine and Drug Design

2007 ◽  
Vol 75 (12) ◽  
pp. 5565-5574 ◽  
Author(s):  
Joanne E. McCoubrie ◽  
Susanne K. Miller ◽  
Tobias Sargeant ◽  
Robert T. Good ◽  
Anthony N. Hodder ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Functional Redundancy ◽  
Blood Stage ◽  
Parasite Development ◽  
Serine Residue ◽  
Human Malaria Parasite ◽  
Knock Out ◽  
Genetic Deletion ◽  
Stage Parasite ◽  
Blood Stage Parasite

ABSTRACT Serine repeat antigens (SERAs) are a family of secreted “cysteine-like” proteases of Plasmodium parasites. Several SERAs possess an atypical active-site serine residue in place of the canonical cysteine. The human malaria parasite Plasmodium falciparum possesses six “serine-type” (SERA1 to SERA5 and SERA9) and three “cysteine-type” (SERA6 to SERA8) SERAs. Here, we investigate the importance of the serine-type SERAs to blood-stage parasite development and examine the extent of functional redundancy among this group. We attempted to knock out the four P. falciparum serine-type SERA genes that have not been disrupted previously. SERA1, SERA4, and SERA9 knockout lines were generated, while only SERA5, the most strongly expressed member of the SERA family, remained refractory to genetic deletion. Interestingly, we discovered that while SERA4-null parasites completed the blood-stage cycle normally, they exhibited a twofold increase in the level of SERA5 mRNA. The inability to disrupt SERA5 and the apparent compensatory increase in SERA5 expression in response to the deletion of SERA4 provides evidence for an important blood-stage function for the serine-type SERAs and supports the notion of functional redundancy among this group. Such redundancy is consistent with our phylogenetic analysis, which reveals a monophyletic grouping of the serine-type SERAs across the genus Plasmodium and a predominance of postspeciation expansion. While SERA5 is to some extent further validated as a target for vaccine and drug development, our data suggest that the expression level of other serine-type SERAs is the only barrier to escape from anti-SERA5-specific interventions.

scholarly journals Stage-dependent effect of deferoxamine on growth of Plasmodium falciparum in vitro

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1250-1255 ◽  
Author(s):  
S Whitehead ◽  
TE Peto
Keyword(s):  
Plasmodium Falciparum ◽  
Growth Inhibition ◽  
Antimalarial Activity ◽  
Clinical Malaria ◽  
Inhibitory Effect ◽  
Parasite Development ◽  
And Control ◽  
Speed Of Onset

Abstract Deferoxamine (DF) has antimalarial activity that can be demonstrated in vitro and in vivo. This study is designed to examine the speed of onset and stage dependency of growth inhibition by DF and to determine whether its antimalarial activity is cytostatic or cytocidal. Growth inhibition was assessed by suppression of hypoxanthine incorporation and differences in morphologic appearance between treated and control parasites. Using synchronized in vitro cultures of Plasmodium falciparum, growth inhibition by DF was detected within a single parasite cycle. Ring and nonpigmented trophozoite stages were sensitive to the inhibitory effect of DF but cytostatic antimalarial activity was suggested by evidence of parasite recovery in later cycles. However, profound growth inhibition, with no evidence of subsequent recovery, occurred when pigmented trophozoites and early schizonts were exposed to DF. At this stage in parasite development, the activity of DF was cytocidal and furthermore, the critical period of exposure may be as short as 6 hours. These observations suggest that iron chelators may have a role in the treatment of clinical malaria.

scholarly journals The Structure of the Cysteine-Rich Domain of Plasmodium falciparum P113 Identifies the Location of the RH5 Binding Site

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Ivan Campeotto ◽  
Francis Galaway ◽  
Shahid Mehmood ◽  
Lea K. Barfod ◽  
Doris Quinkert ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Binding Site ◽  
Structural Information ◽  
Blood Stage ◽  
Site Directed Mutagenesis ◽  
Effective Vaccine ◽  
Binding Region ◽  
Fab Fragment ◽  
Content Type ◽  
Blood Stage Malaria

ABSTRACT Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5. IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum. It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it—and the other parasite proteins it interacts with—promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

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