Optical diffraction tomography and image reconstruction to measure host cell alterations caused by divergent Plasmodium species.

Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium. Understanding the biological features of various parasite forms is important for the optical diagnosis and defining pathological states, which are often constrained by the lack of ambient visualization approaches. Here, we employ a label-free tomographic technique to visualize the host red blood cell (RBC) remodeling process and quantify changes in biochemical properties arising from parasitization. Through this, we provide a quantitative body of information pertaining to the influence of host cell environment on growth, survival, and replication of P. falciparum and P. vivax in their respective host cells: mature erythrocytes and young reticulocytes. These exquisite three-dimensional measurements of infected red cells demonstrats the potential of evolving 3D imaging to advance our understanding of Plasmodium biology and host-parasite interactions.

Plasmodium vivax binds host CD98hc (SLC3A2) to enter immature red blood cells.

More than one-third of the world's population is exposed to Plasmodium vivax malaria, mainly in Asia1. P. vivax preferentially invades reticulocytes (immature red blood cells)2-4. Previous work has identified 11 parasite proteins involved in reticulocyte invasion, including erythrocyte binding protein 2 (ref. 5) and the reticulocyte-binding proteins (PvRBPs)6-10. PvRBP2b binds to the transferrin receptor CD71 (ref. 11), which is selectively expressed on immature reticulocytes12. Here, we identified CD98 heavy chain (CD98), a heteromeric amino acid transporter from the SLC3 family (also known as SLCA2), as a reticulocyte-specific receptor for the PvRBP2a parasite ligand using mass spectrometry, flow cytometry, biochemical and parasite invasion assays. We characterized the expression level of CD98 at the surface of immature reticulocytes (CD71+) and identified an interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results identify CD98 as an additional host membrane protein, besides CD71, that is directly associated with P. vivax reticulocyte tropism. These findings highlight the potential of using PvRBP2a as a vaccine target against P. vivax malaria.

Comparing infrared spectroscopic methods for the characterization of Plasmodium falciparum-infected human erythrocytes.

Malaria, caused by parasites of the species Plasmodium, is among the major life-threatening diseases to afflict humanity. The infectious cycle of Plasmodium is very complex involving distinct life stages and transitions characterized by cellular and molecular alterations. Therefore, novel single-cell technologies are warranted to extract details pertinent to Plasmodium-host cell interactions and underpinning biological transformations. Herein, we tested two emerging spectroscopic approaches: (a) Optical Photothermal Infrared spectroscopy and (b) Atomic Force Microscopy combined with infrared spectroscopy in contrast to (c) Fourier Transform InfraRed microspectroscopy, to investigate Plasmodium-infected erythrocytes. Chemical spatial distributions of selected bands and spectra captured using the three modalities for major macromolecules together with advantages and limitations of each method is presented here. These results indicate that O-PTIR and AFM-IR techniques can be explored for extracting sub-micron resolution molecular signatures within heterogeneous and dynamic samples such as Plasmodium-infected human RBCs.

Targeted Phenotypic Screening in Plasmodium falciparum and Toxoplasma gondii Reveals Novel Modes of Action of Medicines for Malaria Venture Malaria Box Molecules.

The Malaria Box collection includes 400 chemically diverse small molecules with documented potency against malaria parasite growth, but the underlying modes of action are largely unknown. Using complementary phenotypic screens against Plasmodium falciparum and Toxoplasma gondii, we report phenotype-specific hits based on inhibition of overall parasite growth, apicoplast segregation, and egress or host invasion, providing hitherto unavailable insights into the possible mechanisms affected. First, the Malaria Box library was screened against tachyzoite stage T. gondii and the half-maximal effective concentrations (EC50s) of molecules showing ≥80% growth inhibition at 10 µM were determined. Comparison of the EC50s for T. gondii and P. falciparum identified a subset of 24 molecules with nanomolar potency against both parasites. Thirty molecules that failed to induce acute growth inhibition in T. gondii tachyzoites in a 2-day assay caused delayed parasite death upon extended exposure, with at least three molecules interfering with apicoplast segregation during daughter cell formation. Using flow cytometry and microscopy-based examinations, we prioritized 26 molecules with the potential to inhibit host cell egress/invasion during asexual developmental stages of P. falciparum. None of the inhibitors affected digestive vacuole integrity, ruling out a mechanism mediated by broadly specific protease inhibitor activity. Interestingly, five of the plasmodial egress inhibitors inhibited ionophore-induced egress of T. gondii tachyzoites. These findings highlight the advantage of comparative and targeted phenotypic screens in related species as a means to identify lead molecules with a conserved mode of action. Further work on target identification and mechanism analysis will facilitate the development of antiparasitic compounds with cross-species efficacy. IMPORTANCE The phylum Apicomplexa includes many human and animal pathogens, such as Plasmodium falciparum (human malaria) and Toxoplasma gondii (human and animal toxoplasmosis). Widespread resistance to current antimalarials and the lack of a commercial vaccine necessitate novel pharmacological interventions with distinct modes of action against malaria. For toxoplasmosis, new drugs to effectively eliminate tissue-dwelling latent cysts of the parasite are needed. The Malaria Box antimalarial collection, managed and distributed by the Medicines for Malaria Venture, includes molecules of novel chemical classes with proven antimalarial efficacy. Using targeted phenotypic assays of P. falciparum and T. gondii, we have identified a subset of the Malaria Box molecules as potent inhibitors of plastid segregation and parasite invasion and egress, thereby providing early insights into their probable mode of action. Five molecules that inhibit the egress of both parasites have been identified for further mechanistic studies. Thus, the approach we have used to identify novel molecules with defined modes of action in multiple parasites can expedite the development of pan-active antiparasitic agents.

Quantitative mass spectrometry of human reticulocytes reveal proteome-wide modifications during maturation.

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.

Synthesis and in vitro evaluation of hydrazinyl phthalazines against malaria parasite, Plasmodium falciparum.

In this report, we describe the synthesis of 1-(Phthalazin-4-yl)-hydrazine using bronsted acidic ionic liquids and demonstrate their ability to inhibit asexual stage development of human malaria parasite, Plasmodium falciparum. Through computational studies, we short-listed chemical scaffolds with potential binding affinity to an essential parasite protein, dihydroorotate dehydrogenase (DHODH). Further, these compounds were synthesized in the lab and tested against P. falciparum. Several compounds from our library showed inhibitory activity at low micro-molar concentrations with minimal cytotoxic effects. These results indicate the potential of hydralazine derivatives as reference scaffolds to develop novel antimalarials.

Synthesis, characterization and in vitro evaluation of novel enantiomerically-pure sulphonamide antimalarials.

Malaria parasites are currently gaining drug-resistance rapidly, across countries and continents. Hence, the discovery and development of novel chemical scaffolds, with superior antimalarial activity remain an important priority, for the developing world. Our report describes the development, characterization and evaluation of novel bepotastine-based sulphonamide antimalarials inhibiting asexual stage development of Plasmodium falciparum parasites in vitro. The screening results showed potent inhibitory activity of a number of novel sulphonamides against P. falciparum at low micromolar concentrations, in particular in late-stage parasite development. Based on computational studies we hypothesize N-myristoyltransferase as the target of the compounds developed here. Our results demonstrate the value of novel bepotastine-based sulphonamide compounds for targeting the asexual developmental stages of P. falciparum.

Single-cell evaluation of red blood cell bio-mechanical and nano-structural alterations upon chemically induced oxidative stress.

Erythroid cells, specifically red blood cells (RBCs), are constantly exposed to highly reactive radicals during cellular gaseous exchange. Such exposure often exceeds the cells' innate anti-oxidant defense systems, leading to progressive damage and eventual senescence. One of the contributing factors to this process are alterations to hemoglobin conformation and globin binding to red cell cytoskeleton. However, in addition to the aforementioned changes, it is possible that oxidative damage induces critical changes to the erythrocyte cytoskeleton and corresponding bio-mechanical and nano-structural properties of the red cell membrane. To quantitatively characterize how oxidative damage accounts for such changes, we employed single-cell manipulation techniques such as micropipette aspiration and atomic force microscopy (AFM) on RBCs. These investigations demonstrated visible morphological changes upon chemically induced oxidative damage (using hydrogen peroxide, diamide, primaquine bisphosphate and cumene hydroperoxide). Our results provide previously unavailable observations on remarkable changes in red cell cytoskeletal architecture and membrane stiffness due to oxidative damage. Furthermore, we also demonstrate that a pathogen that infects human blood cells, Plasmodium falciparum was unable to penetrate through the oxidant-exposed RBCs that have damaged cytoskeleton and stiffer membranes. This indicates the importance of bio-physical factors pertinent to aged RBCs and it's relevance to malaria infectivity.