scholarly journals Structure of the cysteine-rich domain of Plasmodium falciparum P113 identifies the location of the RH5 binding site

2020 ◽  
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 ◽  
Parasite Protein ◽  
Blood Stage Malaria

AbstractPlasmodium 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 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 non-inhibitory for RH5 binding. Using a Fab fragment as a crystallisation 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 the development of blood-stage malaria vaccines that target RH5.ImportanceMalaria 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 anti-malarial vaccines to ultimately prevent this disease which affects some of the poorest people on the planet.

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.

scholarly journals Bacterially Expressed Full-Length Recombinant Plasmodium falciparum RH5 Protein Binds Erythrocytes and Elicits Potent Strain-Transcending Parasite-Neutralizing Antibodies

2013 ◽  
Vol 82 (1) ◽  
pp. 152-164 ◽  
Author(s):  
K. Sony Reddy ◽  
Alok K. Pandey ◽  
Hina Singh ◽  
Tajali Sahar ◽  
Amlabu Emmanuel ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Neutralizing Antibodies ◽  
Malaria Vaccine ◽  
Expression System ◽  
Full Length ◽  
Blood Stage ◽  
Parasite Protein ◽  
Content Type ◽  
Erythrocyte Binding ◽  
Blood Stage Malaria

ABSTRACTPlasmodium falciparumreticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, basigin. PfRH5 is an attractive malaria vaccine candidate, as it is expressed by a wide number ofP. falciparumstrains, cannot be genetically disrupted, and exhibits limited sequence polymorphisms. Viral vector-induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial-cell-based expression system. In this study, we have produced full-length recombinant PfRH5 inEscherichia colithat exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also, importantly, elicits potent invasion-inhibitory antibodies against a number ofP. falciparumstrains. Antibasigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5, further confirming that they bind with basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics that of the native parasite protein and elicits potent strain-transcending parasite-neutralizing antibodies.P. falciparumhas the capability to develop immune escape mechanisms, and thus, blood-stage malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced potent additive inhibition against multiple worldwideP. falciparumstrains. PfRH5 was immunogenic when immunized with other antigens, eliciting potent invasion-inhibitory antibody responses with no immune interference. Our results strongly support the development of PfRH5 as a component of a combination blood-stage malaria vaccine.

scholarly journals IgM in human immunity to Plasmodium falciparum malaria

2019 ◽  
Vol 5 (9) ◽  
pp. eaax4489 ◽  
Author(s):  
M. J. Boyle ◽  
J. A. Chan ◽  
I. Handayuni ◽  
L. Reiling ◽  
G. Feng ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Blood Cells ◽  
Plasmodium Falciparum Malaria ◽  
Clinical Malaria ◽  
Blood Stage ◽  
Dependent Manner ◽  
Human Immunity ◽  
Lifetime Exposure ◽  
Blood Stage Malaria ◽  
Reduced Risk

Most studies on human immunity to malaria have focused on the roles of immunoglobulin G (IgG), whereas the roles of IgM remain undefined. Analyzing multiple human cohorts to assess the dynamics of malaria-specific IgM during experimentally induced and naturally acquired malaria, we identified IgM activity against blood-stage parasites. We found that merozoite-specific IgM appears rapidly in Plasmodium falciparum infection and is prominent during malaria in children and adults with lifetime exposure, together with IgG. Unexpectedly, IgM persisted for extended periods of time; we found no difference in decay of merozoite-specific IgM over time compared to that of IgG. IgM blocked merozoite invasion of red blood cells in a complement-dependent manner. IgM was also associated with significantly reduced risk of clinical malaria in a longitudinal cohort of children. These findings suggest that merozoite-specific IgM is an important functional and long-lived antibody response targeting blood-stage malaria parasites that contributes to malaria immunity.

Characterization of a Plasmodium falciparum PHISTc protein, PF3D7_0801000, in blood- stage malaria parasites

2021 ◽  
Vol 80 ◽  
pp. 102240
Author(s):  
Hikaru Nagaoka ◽  
Bernard N. Kanoi ◽  
Masayuki Morita ◽  
Takahiro Nakata ◽  
Nirianne M.Q. Palacpac ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Blood Stage ◽  
Malaria Parasites ◽  
Blood Stage Malaria

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.

Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1559-1567 ◽  
Author(s):  
Eloísa Andújar ◽  
Eduardo Santero
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Mutational Analysis ◽  
Structural Information ◽  
Substrate Inhibition ◽  
Site Directed Mutagenesis ◽  
Functional Importance ◽  
Extradiol Dioxygenase ◽  
Polycyclic Compounds ◽  
Extradiol Dioxygenases

The sequence of the extradiol dioxygenase ThnC, involved in tetralin biodegradation, was aligned with other extradiol dioxygenases involved in biodegradation of polycyclic compounds, and a three-dimensional model of ThnC, based on the structure of the previously crystallized 2,3-dihydroxybiphenyl dioxygenase from Burkholderia fungorum LB400, was built. In order to assess the functional importance of some non-active-site residues whose relevance could not be established by structural information, a number of positions surrounding the substrate-binding site were mutated in ThnC. Ten mutant proteins were purified and their activity towards 1,2-dihydroxytetralin, 1,2-dihydroxynaphthalene and 2,3-dihydroxybiphenyl was characterized. N213H, Q198H, G206M, A282R and A282G mutants increased k cat/K m at least twofold using 1,2-dihydroxytetralin as the substrate, thus showing that activity of ThnC is not maximized for this substrate. N213H and Q198H mutants increased k cat/K m using any of the substrates tested, thus showing the relevance for activity of these two histidines, which are highly conserved in dihydroxybiphenyl dioxygenases, but not present in dihydroxynaphthalene dioxygenases. Different substitutions in position 282 had different effects on general activity or substrate specificity, thus showing the functional importance of the most C-terminal β-sheet of the protein. A251M and G206M mutants showed increased activity specifically for a particular substrate. N213H, G206M, A282R, A282G and Y177I substitutions resulted in enzymes more tolerant to acidic pH, the most striking effect being observed in mutant Y177I, which showed maximal activity at pH 5·5. In addition, Q198D and V175D mutants, which had altered K m, also showed altered sensitivity to substrate inhibition, thus indicating that inhibition is exerted through the same binding site. This mutational analysis, therefore, identified conserved residues important for activity or substrate specificity, and also shed some light on the mechanism of substrate inhibition exhibited by extradiol dioxygenases.

scholarly journals High-throughput Screening for Potent and Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase

2005 ◽  
Vol 280 (23) ◽  
pp. 21847-21853 ◽  
Author(s):  
Jeffrey Baldwin ◽  
Carolyn H. Michnoff ◽  
Nicholas A. Malmquist ◽  
John White ◽  
Michael G. Roth ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Binding Site ◽  
High Throughput ◽  
High Throughput Screening ◽  
De Novo ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Dihydroorotate Dehydrogenase ◽  
Selective Inhibitors ◽  
Diverse Range

Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC50 values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC50 against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.

scholarly journals Identification of a Potent Combination of Key Plasmodium falciparum Merozoite Antigens That Elicit Strain-Transcending Parasite-Neutralizing Antibodies

2012 ◽  
Vol 81 (2) ◽  
pp. 441-451 ◽  
Author(s):  
Alok K. Pandey ◽  
K. Sony Reddy ◽  
Tajali Sahar ◽  
Sonal Gupta ◽  
Hina Singh ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Neutralizing Antibodies ◽  
Malaria Vaccine ◽  
Vaccine Development ◽  
Blood Stage ◽  
Content Type ◽  
Receptor Blocking ◽  
Erythrocyte Invasion ◽  
Invasion Inhibition ◽  
Blood Stage Malaria

ABSTRACTBlood-stage malaria vaccines that target singlePlasmodium falciparumantigens involved in erythrocyte invasion have not induced optimal protection in field trials. Blood-stage malaria vaccine development has faced two major hurdles, antigenic polymorphisms and molecular redundancy, which have led to an inability to demonstrate potent, strain-transcending, invasion-inhibitory antibodies. Vaccines that target multiple invasion-related parasite proteins may inhibit erythrocyte invasion more efficiently. Our approach is to develop a receptor-blocking blood-stage vaccine againstP. falciparumthat targets the erythrocyte binding domains of multiple parasite adhesins, blocking their interaction with their receptors and thus inhibiting erythrocyte invasion. However, with numerous invasion ligands, the challenge is to identify combinations that elicit potent strain-transcending invasion inhibition. We evaluated the invasion-inhibitory activities of 20 different triple combinations of antibodies mixedin vitroagainst a diverse set of six key merozoite ligands, including the novel ligandsP. falciparumapical asparagine-rich protein (PfAARP), EBA-175 (PfF2),P. falciparumreticulocyte binding-like homologous protein 1 (PfRH1), PfRH2, PfRH4, andPlasmodiumthrombospondin apical merozoite protein (PTRAMP), which are localized in different apical organelles and are translocated to the merozoite surface at different time points during invasion. They bind erythrocytes with different specificities and are thus involved in distinct invasion pathways. The antibody combination of EBA-175 (PfF2), PfRH2, and PfAARP produced the most efficacious strain-transcending inhibition of erythrocyte invasion against diverseP. falciparumclones. This potent antigen combination was selected for coimmunization as a mixture that induced balanced antibody responses against each antigen and inhibited erythrocyte invasion efficiently. We have thus demonstrated a novel two-step screening approach to identify a potent antigen combination that elicits strong strain-transcending invasion inhibition, supporting its development as a receptor-blocking malaria vaccine.

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