The protozoan parasite Plasmodium falciparum, which is responsible for the deadliest form of human malaria, accounts for over half a million deaths a year. These parasites proliferate in human red blood cells by consecutive rounds of closed mitoses called schizogony. Their virulence is attributed to their ability to modify the infected red cells to adhere to the vascular endothelium and to evade immunity through antigenic switches. Spatial dynamics at the nuclear periphery were associated with the regulation of processes that enable the parasites to establish long-term infection. However, our knowledge of components of the nuclear envelope (NE) in Plasmodium remains limited. One of the major protein complexes at the NE is the linker of nucleoskeleton and cytoskeleton (LINC) complex that forms a connecting bridge between the cytoplasm and the nucleus through the interaction of SUN and KASH domain proteins. Here, we have identified two SUN-domain proteins as possible components of the LINC complex of P. falciparum and show that their proper expression is essential for the parasite's proliferation in human red blood cells, and their depletion leads to the formation of membranous whorls and morphological changes of the NE. In addition, their differential expression highlights different functions at the nuclear periphery as PfSUN2 is specifically associated with heterochromatin, while PfSUN1 expression is essential for activation of the DNA damage response. Our data provide indications for the involvement of the LINC complex in crucial biological processes in the intraerythrocytic development cycle of malaria parasites. IMPORTANCE:Plasmodium falciparum, the parasite causing the deadliest form of malaria, is able to thrive in its human host by tight regulation of cellular processes, orchestrating nuclear dynamics with cytoplasmic machineries that are separated by the nuclear envelope. One of the major protein complexes that connect nuclear and cytoplasmic processes in eukaryotes is the linker of nucleoskeleton and cytoskeleton (LINC) complex. However, while the nuclear periphery of P. falciparum was implicated in several important functions, the role of the LINC complex in Plasmodium biology is unknown. Here, we identify two components of P. falciparum LINC complex and demonstrate that they are essential for the parasites' proliferation in human blood, and their depletion leads to the formation of morphological changes in the cell. In addition, the two components have different functions in activating the DNA damage response and in their association with heterochromatin. Our data provide evidence for their essential roles in the parasites' cell cycle.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. The crystalline structure of hemozoin was established by X-ray powder diffraction using a synthetic analog, β-hematin. Here, we apply emerging methods of in situ cryo-electron tomography and 3D electron diffraction to obtain a definitive structure of hemozoin directly from ruptured parasite cells. Biogenic hemozoin crystals take a striking polar morphology. Like β-hematin, the unit cell contains a heme dimer, which may form four distinct stereoisomers: two centrosymmetric and two chiral enantiomers. Diffraction analysis, supported by density functional theory analysis, reveals a selective mixture in the hemozoin lattice of one centrosymmetric and one chiral dimer. Absolute configuration has been determined by morphological analysis and confirmed by a novel method of exit-wave reconstruction from a focal series. Atomic disorder appears on specific facets asymmetrically, and the polar morphology can be understood in light of water binding. Structural modeling of the heme detoxification protein suggests a function as a chiral agent to bias the dimer formation in favor of rapid growth of a single crystalline phase. The refined structure of hemozoin should serve as a guide to new drug development.

Long noncoding RNAs (lncRNA) are emerging as important regulators of gene expression in eukaryotes. In recent years, a large repertoire of lncRNA were discovered in Apicomplexan parasites and were implicated in several mechanisms of gene expression, including marking genes for activation, contributing to the formation of subnuclear compartments and organization, regulating the deposition of epigenetic modifications, influencing chromatin and chromosomal structure and manipulating host gene expression. Here, we aim to update recent knowledge on the role of lncRNAs as regulators in Apicomplexan parasites and highlight the possible molecular mechanisms by which they function. We hope that some of the hypotheses raised here will contribute to further investigation and lead to new mechanistic insight and better understanding of the role of lncRNA in parasite’s biology.

Background Artemisinin-based combination therapies (ACTs) are recommended as first-line treatment against uncomplicated Plasmodium falciparum infection. Mutations in the PfKelch13 (PF3D7_1343700) gene led to resistance to artemisinin in Southeast Asia. Mutations in the Pfcoronin (PF3D7_1251200) gene confer reduced artemisinin susceptibility in vitro to an African Plasmodium strain, but their role in clinical resistance has not been established.Methods We conducted a retrospective observational study of Israeli travellers returning from sub-Saharan Africa with P. falciparum malaria, including patients with artemether-lumefantrine (AL) failure. Blood samples from all malaria-positive patients are delivered to the national Parasitology Reference Laboratory along with personal information. Confirmation of malaria, species identification and comparative parasite load analysis were performed using real-time PCR. DNA extractions from stored leftover samples were analysed for the presence of mutations in Pfkelch13 and Pfcoronin. Age, weight, initial parasitaemia level and Pfcoronin status were compared in patients who failed treatment vs responders.Results During 2009-2020, 338 patients had P. falciparum malaria acquired in Africa. Of those, 15 (24-69 years old, 14 males) failed treatment with AL. Four were still parasitemic at the end of treatment, and 11 had malaria recrudescence. Treatment failure rates were 0% during 2009-2012, 9.1% during 2013-2016 and 17.4% during 2017-2020. In all patients, the Pfkelch13 propeller domain had a wild-type sequence. We did find the P76S mutation in the propeller domain of Pfcoronin in 4/15 (28.6%) of the treatment-failure cases compared to only 3/56 (5.5%) in the successfully treated patients (P = 0.027).Conclusion AL treatment failure emergence was not associated with mutations in Pfkelch13. However, P76S mutation in the Pfcoronin gene was more frequently present in the treatment-failure group and merits further investigation. The increase of malaria incidence in sub-Saharan-Africa partly attributed to the COVID-19 pandemic might also reflect a wider spread of ACT resistance.

Neutrophils play critical roles in a broad spectrum of clinical conditions. Accordingly, manipulation of neutrophil function may provide a powerful immunotherapeutic approach. However, due to neutrophils characteristic short half-life and their large population number, this possibility was considered impractical. Here we describe the identification of peptides which specifically bind either murine or human neutrophils. Although the murine and human neutrophil-specific peptides are not cross-reactive, we identified CD177 as the neutrophil-expressed binding partner in both species. Decorating nanoparticles with a neutrophil-specific peptide confers neutrophil specificity and these neutrophil-specific nanoparticles accumulate in sites of inflammation. Significantly, we demonstrate that encapsulating neutrophil modifying small molecules within these nanoparticles yields specific modulation of neutrophil function (ROS production, degranulation, polarization), intracellular signaling and longevity both in vitro and in vivo. Collectively, our findings demonstrate that neutrophil specific targeting may serve as a novel mode of immunotherapy in disease.

In eukaryotic organisms, noncoding RNAs (ncRNAs) have been implicated as important regulators of multifaceted biological processes, including transcriptional, posttranscriptional, and epigenetic regulation of gene expression. In recent years, it is becoming clear that protozoan parasites encode diverse ncRNA transcripts; however, little is known about their cellular functions. Recent advances in high-throughput "omic" studies identified many novel long ncRNAs (lncRNAs) in apicomplexan parasites, some of which undergo splicing, polyadenylation, and encode small proteins. To date, only a few of them are characterized, leaving a big gap in our understanding regarding their origin, mode of action, and functions in parasite biology. In this review, we focus on lncRNAs of the human malaria parasite Plasmodium falciparum and highlight their cellular functions and possible mechanisms of action.

The aim of this paper was to evaluate the effect of vehicle, chemical enhancer and iontophoresis on the skin accumulation of clobetasol propionate (CP) and mometasone furoate (MF). In vitro permeation experiments were performed using pig ear skin as barrier and HPLC as quantification method. The formulations tested were chitosan gels, sodium-deoxycholate gels and commercial creams of CP and MF. The results obtained indicate that Na-DOC gel had an enhancing effect on the skin accumulation of both active agents. This effect was more evident with CP especially in the stratum corneum and epidermis which are the target sites of topical steroidal treatment. Two terpene derivatives (d-limonene and nerolidol) and Transcutol® P were evaluated as chemical penetration enhancers. Nerolidol produced considerable increase in the amount of CP and MF accumulated without any permeation across the skin. The application of electric current (anodal iontophoresis) to the gels improved the accumulation of MF while it did not effect the accumulation of CP. Due to the best accumulation results of nerolidol, the enhancement effect in combination with iontophoresis was also investigated. It was shown that, the combination of anodal iontophoresis and chemical enhancer (nerolidol) produced no further enhancement for both active agents.

One of the key mechanisms contributing to the virulence of Plasmodium falciparum is its ability to undergo antigenic switching among antigenically distinct variants of the PfEMP1 adhesive proteins, encoded by the var gene family. To avoid premature exposure of its antigenic repertoire, the parasite transcribes its var genes in a mutually exclusive manner, and switch expression at a very slow rate. This process is epigenetically regulated and it relies on "epigenetic memory," which imprints the single active var gene to remain active for multiple replication cycles. Erasing this epigenetic memory in parasites grown in culture resembles parasites, which egress from the liver. It could therefore be of interest for investigating var switching patterns at the onset of malaria infections. In addition, this procedure could be used for creating heterogeneity of var expression among parasite populations. The methodology described here for resetting of var gene expression is based on promoter titration, also known as molecular sponging.

The virulence ofPlasmodium falciparum, which causes the deadliest form of human malaria, is attributed to its ability to evade the human immune response. These parasites “choose” to express a single variant from a repertoire of surface antigens called PfEMP1, which are placed on the surface of the infected red cell. Immune evasion is achieved by switches in expression betweenvargenes, each encoding a differentPfEMP1 variant. While the mechanisms that regulate mutually exclusive expression ofvargenes are still elusive, antisense long-noncoding RNAs (lncRNAs) transcribed from the intron of the activevargene were implicated in the “choice” of the single activevargene. Here, we show that this lncRNA colocalizes with the site ofvarmRNA transcription and is anchored to thevarlocus via DNA:RNA interactions. We define thevarlncRNA interactome and identify a redox sensor,P. falciparumthioredoxin peroxidase I (PfTPx-1), as one of the proteins associated with thevarantisense lncRNA. We show thatPfTPx-1 localizes to a nuclear subcompartment associated with active transcription on the nuclear periphery, in ring-stage parasite, whenvartranscription occurs. In addition,PfTPx-1 colocalizes with S-adenosylmethionine synthetase (PfSAMS) in the nucleus, and its overexpression leads to activation ofvar2csa,similar to overexpression ofPfSAMS. Furthermore, we show thatPfTPx-1 knockdown alters thevarswitch rate as well as activation of additional gene subsets. Taken together, our data indicate that nuclearPfTPx-1 plays a role in gene activation possibly by providing a redox-controlled nuclear microenvironment ideal for active transcription.

The serine-arginine-rich (SR) proteins play an exceptionally important role in eukaryotic gene expression, primarily by regulating constitutive and alternative splicing events. In addition to their primary role as splicing factors, SR proteins have emerged as multifunctional RNA-binding proteins that act as key regulators of almost every step of RNA metabolism. As in higher eukaryotes, Plasmodium parasites encode several SR proteins, which were implicated in pre-mRNA splicing. However, only a few have been characterized and their biological roles remain understudied. Intriguingly, in addition to splicing regulation, unexpected functions of particular SR proteins have been reported in Plasmodium in recent years. Here, we highlight the key characteristics and different noncanonical splicing functions of SR proteins and discuss potential mechanisms, which might be involved in their multifaceted functionality in Plasmodium.

Plasmodium falciparum, the deadliest form of human malaria, remains one of the major threats to human health in endemic regions. Its virulence is attributed to its ability to modify infected red blood cells (iRBC) to adhere to endothelial receptors by placing variable antigens known as PfEMP1 on the surface of the red cell. PfEMP1 expression on the red cell surface determines the cytoadhesive properties of the iRBCs and is implicated in severe manifestations of malaria. To evade antibody mediated responses the parasite undergoes continuous switches of expression between different PfEMP1 variants. Recently it became clear that in addition to antibody mediated responses, PfEMP1 triggers an innate immune response, however, the role of neutrophils, the most abundant white blood cells in the human circulation, in malaria remains elusive. Here we show that neutrophils recognize and kill blood stages of several P. falciparum isolates, and we identify neutrophil ICAM-1 and specific PfEMP1s implicated in cerebral malaria as the key molecules involved in this killing. Our data provide mechanistic insight into the interactions between neutrophils and iRBCs and demonstrate the important influence of PfEMP1 on the selective innate response to cerebral malaria.

Malaria is a potentially fatal infectious disease caused by the obligate intracellular parasite Plasmodium falciparum . The parasite infects human red blood cells (RBC) and derives nutrition by catabolism of hemoglobin. As amino acids are assimilated from the protein component, the toxic heme is released. Molecular heme is detoxified by rapid sequestration to physiologically insoluble hemozoin crystals within the parasite’s digestive vacuole (DV). Common antimalarial drugs interfere with this crystallization process, leaving the parasites vulnerable to the by-product of their own metabolism. A fundamental debate with important implications on drug mechanism regards the chemical environment of crystallization in vivo , whether aqueous or lipid. This issue had been addressed previously by cryogenic soft X-ray tomography. We employ cryo-scanning transmission electron tomography (CSTET) to probe parasite cells throughout the life cycle in a fully hydrated, vitrified state at higher resolution. During the acquisition of CSTET data, Bragg diffraction from the hemozoin provides a uniquely clear view of the crystal boundary at nanometer resolution. No intermediate medium, such as a lipid coating or shroud, could be detected surrounding the crystals. The present study describes a unique application of CSTET in the study of malaria. The findings can be extended to evaluate new drug candidates affecting hemozoin crystal growth.

Plasmodium falciparum, which causes the deadliest form of human malaria, is able to evade antibody-mediated immune responses through switches in expression of surface antigens. Thus, over the years, the focus of most research has been on the role of the adaptive immune response in the course of malaria. However, in recent years there is mounting evidence for the role of the innate immune response to Plasmodium infections. In this context, very little is known on the protective role of neutrophils against blood-stage parasites and the mechanisms by which they recognize and eliminate infected red blood cells. Here we describe several useful methodologies that enable the study and quantification of the interactions between human neutrophils and P. falciparum-infected red blood cells.

The virulence of Plasmodium falciparum has been attributed in large part to the expression on the surface of infected red blood cells of the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Different forms of this protein are encoded by individual members of the multicopy gene family called var. Two attributes of the var gene family are key to the pathogenesis of malaria caused by P. falciparum; the hyperrecombinogenic nature of the var gene family that continuously generates antigenic diversity within parasite populations, and the ability of parasites to express only a single var gene at a time and to switch which gene is expressed over the course of an infection. The unique attributes of CRISPR-Cas9 have been applied to help decipher the molecular mechanisms underlying these unusual properties of the var gene family, both as a source of the DNA double strand breaks that initiate var gene recombination and as a way to recruit molecular probes to specific regions of the genome. In this chapter, we describe these somewhat unusual applications of the CRISPR-Cas9 system.

Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. X-ray powder diffraction has established that the unit cell contains a cyclic hematin dimer, yet the pro-chiral nature of heme supports formation of four distinct stereoisomers, two centrosymmetric and two chiral enantiomers. Here we apply emerging methods of in situ cryo-electron tomography and diffraction to obtain a definitive structure of biogenic hemozoin. Individual crystals take a striking polar morphology. Diffraction analysis, supported by density functional theory, indicates a compositional mixture of one centrosymmetric and one chiral dimer, whose absolute configuration has been determined on the basis of crystal morphology and interaction with the aqueous medium. Structural modeling of the heme detoxification protein suggests a mechanism for dimer selection. The refined structure of hemozoin should serve as a guide to new drug development.

Plasmodium falciparum is the deadliest human parasite that causes malaria when it reaches the bloodstream and begins proliferating inside red blood cells, where the parasites are particularly prone to DNA damage. The molecular mechanisms that allow these pathogens to maintain their genome integrity under such conditions are also the driving force for acquiring genome plasticity that enables them to create antigenic variation and become resistant to essentially all available drugs.

Plasmodium falciparum , the parasite responsible for the deadliest form of human malaria, maintains a complex life cycle with a relatively small number of genes. Pf SR1 is an alternative splicing factor that regulates expansion of the P. falciparum protein repertoire. To further investigate Pf SR1 functions, we set to unveil its interactome. We found that Pf SR1 interacts with proteins, which are linked to various processes of RNA metabolism in a stage-dependent manner. These include: chromatin re-modeling, transcription, splicing and translation. Intriguingly, some of the Pf SR1 interacting proteins are orthologues of proteins implicated in the DNA damage response. We demonstrate that Pf SR1 expression is important for preventing the accumulation of DNA damage in proliferating parasites. In addition, following parasites’ exposure to a source of DNA damage, Pf SR1 is recruited to damaged foci where it interacts with the phosphorylated core histone Pf H2A, which marks damaged chromatin. Furthermore, Pf SR1 expression was found to be essential for the ability of the parasite to activate the DNA repair machinery and recover from DNA damage caused by either irradiation or exposure to artemisinin, the first line anti-malarial drug. These findings unveil a novel role of Pf SR1 in protecting P. falciparum from DNA damage and artemisinin exposure.

Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. In mouse development, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a unique perspective of BBB TJs and open new directions for understanding TJ functionality in biological barriers, ultimately enabling restoration in disease or modulation for drug delivery.