Identification of Plasmodium Falciparum proteins interacting with the erythrocyte membrane skeleton protein spectrin

Malaria, which is caused by Plasmodium parasites, is responsible for the death of millions of humans every year in the tropical and subtropical regions of the world. Specifically P. falciparum, one of four malaria species infecting humans, is responsible for the greatest morbidity and mortality burden in African populations. The Anopheles mosquito transmits the parasite to the human host, where it infects and destroys human erythrocytes. The erythrocyte membrane therefore plays a vital role in all aspects of the pathogenic phase of the P. falciparum life cycle and protein-protein interactions between host and parasite are thus a key focus of research. The human erythrocyte maintains its shape with a structural network of proteins underneath the plasma membrane and the main protein component of this erythrocyte membrane skeleton is spectrin. To investigate host-parasite protein interactions, a novel application of phage display technology was developed, whereby purified human erythrocyte spectrin was biopanned against a P. falciparum phage-display library. The P. falciparum DNA inserts of interacting phage were compared to the PlasmoDB database and five interacting proteins were identified: a putative aminopeptidase (PfM18AAP); a putative Ebl-1 like protein, which is proposed to participate in erythrocyte invasion; and three hypothetical proteins. The interaction of the hypothetical proteins with spectrin is the first information available on the function of these proteins. The five gene sequences were cloned into the pET-15b or pGEX-4T-2 expression vectors for purification of the recombinant proteins from Escherichia coli. Only the 6His-PfM18AAP fusion protein was expressed in soluble form and purified by affinity selection. PfM18AAP migrated as a 67 kDa peptide on SDS-PAGE and native gel analysis revealed multiple subunits of the enzyme, predominantly a tetramer and higher oligomers. Cleavage of the 6His-tag and subsequent IEF SDS-PAGE revealed three 65 kDa entities with pI ~6.6, ~6.7 and ~6.9. An in vitro coupled enzyme assay showed that PfM18AAP cleaved an N-terminal aspartate from a peptide substrate with a maximum activity at pH 7.5 and 37 ºC. Inhibitor studies confirmed that the enzyme is a metalloprotease. Blot overlay assays with PfM18AAP against spectrin and erythrocyte membrane proteins verified that PfM18AAP binds strongly to β-spectrin, as well as protein 4.1, protein 4.2, actin and glyceraldehyde-3-phosphate dehydrogenase. Comparison of the PfM18AAP protein sequence to ten other M18 aminopeptidase sequences, including human and three other Plasmodium species, revealed that all the critical amino acids responsible for the binding of two catalytic metal ions, enzymatic catalysis and quaternary structure stabilisation are conserved. The peptide fragment, which initially bound to spectrin during phage display, is not found in other M18 aminopeptidases, suggesting that the presence of this fragment is an evolutionary development of P. falciparum that allows the protease to bind to human spectrin. Analysis of four M18 aminopeptidase crystal structures revealed that the spectrin-binding region forms an external loop on the protein and would thus be accessible to spectrin. Results from this study suggest that, apart from haemoglobin digestion, PfM18AAP performs additional functions in the parasite and infected erythrocyte by cleaving spectrin and other erythrocyte membrane proteins. This would destabilise and disrupt the erythrocyte membrane skeleton to facilitate entry or exit from the host cell, or the insertion of parasite proteins into the host cell membrane. Further analysis and characterisation of PfM18AAP and its interactions with the erythrocyte membrane proteins will shed more light on the multifunctional role of this parasite enzyme. Studies of this enzyme and the hypothetical proteins may also aid in the quest to discover new therapeutics to combat this killer disease

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BACKGROUND: The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. METHODS: P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. RESULTS: Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. CONCLUSION: The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane

Nowhere to hide : interrogating different metabolic parameters of Plasmodium falciparum gametocytes in a transmission blocking drug discovery pipeline towards malaria elimination

BACKGROUND : The discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions. METHODS : Gametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable. RESULTS : A highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4 %. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident. CONCLUSIONS : With this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broaddiversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.South African Medical Research Council Strategic Health Initiatives Partnerships with the Medicines for Malaria Venture as well as the Council for Scientific and Industrial Research, and the 3R Foundation (project 118–10).http://www.malariajournal.comhb201

The M18 aspartyl aminopeptidase of Plasmodium falciparum binds to human erythrocyte spectrin in vitro

<p>Abstract</p> <p>Background</p> <p>During erythrocytic schizogony, <it>Plasmodium falciparum </it>interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the <it>P. falciparum </it>M18 aspartyl aminopeptidase (<it>Pf</it>M18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) <it>Pf</it>M18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.</p> <p>Methods</p> <p>r<it>Pf</it>M18AAP was produced as a hexahistidine-fusion protein in <it>Escherichia coli </it>and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin-<it>Pf</it>M18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization.</p> <p>Results</p> <p>r<it>Pf</it>M18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that r<it>Pf</it>M18AAP aggregated into oligomers. An <it>in vitro </it>coupled enzyme assay showed that r<it>Pf</it>M18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37°C. The spectrin-binding region of <it>Pf</it>M18AAP is not found in <it>Homo sapiens, Saccharomyces cerevisiae </it>and other<it>Plasmodium </it>species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with r<it>Pf</it>M18AAP against spectrin and erythrocyte membrane proteins indicated that r<it>Pf</it>M18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.</p> <p>Conclusion</p> <p>Studies characterizing r<it>Pf</it>M18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, <it>Pf</it>M18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.</p

Potent Plasmodium falciparum gametocytocidal compounds identified by exploring the kinase inhibitor chemical space for dual active antimalarials

OBJECTIVES : Novel chemical tools to eliminate malaria should ideally target both the asexual parasites and transmissible gametocytes. Several imidazopyridazines (IMPs) and 2-aminopyridines (2-APs) have been described as potent antimalarial candidates targeting lipid kinases. However, these have not been extensively explored for stage-specific inhibition of gametocytes in Plasmodium falciparum parasites. Here we provide an in-depth evaluation of the gametocytocidal activity of compounds from these chemotypes and identify novel starting points for dual-acting antimalarials. METHODS : We evaluated compounds against P. falciparum gametocytes using several assay platforms for cross-validation and stringently identified hits that were further profiled for stage specificity, speed of action and ex vivo efficacy. Physicochemical feature extraction and chemogenomic fingerprinting were applied to explore the kinase inhibition susceptibility profile. RESULTS : We identified 34 compounds with submicromolar activity against late stage gametocytes, validated across several assay platforms. Of these, 12 were potent at 1000-fold selectivity towards the parasite over mammalian cells. Front-runner compounds targeted mature gametocytes within 48 h and blocked transmission to mosquitoes. The resultant chemogenomic fingerprint of parasites treated with the lead compounds revealed the importance of targeting kinases in asexual parasites and gametocytes. CONCLUSIONS : This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.The South African Medical Research Council (SAMRC) Self-initiated Research (to JN) and Strategic Health Initiatives Partnerships (MRC-SHIP) programmes to L.B., T.C., D.M. K.C. further acknowledges the SAMRC for funding of the extramural Drug Discovery and Development Research Unit at UCT. The SAMRC is acknowledged for funding of the UP ISMC (LMB) and WRIM (TLC) as Collaborating Centres for Malaria Research. The Council for Scientific and Industrial Research and the 3R Foundation (project 118–10) to D.M. We thank the Medicines for Malaria Venture and South African Technology Innovation Agency (TIA) for funding to K.C. (Project MMV09/0002). The University of Cape Town, University of Pretoria, and South African Research Chairs Initiative of the Department of Science and Technology, administered through the South African National Research Foundation are gratefully acknowledged for support to K.C. and L.B. (UID84627). JN was supported through an International Society for Infectious Diseases grant.https://academic.oup.com/jac2019-05-01hj2018Biochemistr

Defining the clinical and cognitive phenotype of child savants with autism spectrum disorder

Objective: Whilst savant syndrome is most commonly observed in individuals with Autism Spectrum Disorder (ASD), it has historically been associated with intellectual impairment, and little is known about the clinical and cognitive characteristics of intellectually able individuals with ASD and savant skills. Methods: Participants with ASD and validated savant skills were compared with age and intelligence matched non-savants with ASD using a range of diagnostic and standardised tests. Results: Although the analysis of the clinical data revealed few differences between the groups, striking differences emerged during cognitive testing. Children with savant skills exhibited highly superior working memory and their scores on tests of analytic skills were also superior to those of non-savants. Conclusion: We propose that obsessionality, focused attention, superior working memory and analytic skills facilitate veridical mapping and pattern perception abilities characteristic in savant syndrome

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.Supplementary Data 1: Data of the supra-hexagonal plot in Figure 2ASupplementary Data 2: Complete dataset of all MMV PRB compounds’ activity on Plasmodium life cycle stagesSupplementary Data 3: Full SMFA dataset to support Figure 5CSupplementary Data 4: Transcriptome analysis of MMV1580488 (ML324) treated parasites to support Figure 6C.The Medicines for Malaria Venture and South African Technology Innovation Agency (TIA). This project was in part supported by the South African Medical Research Council with funds received from the South African Department of Science and Innovation, in partnership with the Medicines for Malaria Venture; and the DST/NRF South African Research Chairs Initiative Grant; and CSIR Parliamentary Grant funding as well as the Bill and Melinda Gates Foundation and the Australian NHMRC (APP1072217).http://www.nature.com/ncommshj2021BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC