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Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles, and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown, and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures-the food vacuole, the apicoplast, and the parasite plasma membrane-and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1; PF3D7_1135300), a unique Plasmodium -specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium . Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have diverse intracellular localizations and putative functions. IMPORTANCE Plasmodium falciparum-infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes not only facilitate movement of nutrients, metabolites, and other molecules between these compartments, but also are common therapeutic targets and can confer antimalarial drug resistance. Orphan membrane transporters in P. falciparum without sequence homology to transporters in other evolutionary lineages and divergent from host transporters may constitute attractive targets for novel intervention approaches. Here, we localized six of these putative transporters at different subcellular compartments and probed their importance during asexual parasite growth by using reverse genetic approaches. In total, only two candidates turned out to be dispensable for the parasite, highlighting four candidates as putative targets for therapeutic interventions. This study reveals the importance of several orphan transporters to blood stage P. falciparum development.

Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum. Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to a proportion of merozoites failing to invade and was associated with elongation of the rhoptry organelle during merozoite development and inhibition of rhoptry antigen processing. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion., (© 2022. The Author(s).)

During the symptomatic human blood phase, malaria parasites replicate within red blood cells. Parasite proliferation relies on the uptake of nutrients, such as amino acids, from the host cell and blood plasma, requiring transport across multiple membranes. Amino acids are delivered to the parasite through the parasite-surrounding vacuolar compartment by specialized nutrient-permeable channels of the erythrocyte membrane and the parasitophorous vacuole membrane (PVM). However, further transport of amino acids across the parasite plasma membrane (PPM) is currently not well characterized. In this study, we focused on a family of Apicomplexan amino acid transporters (ApiATs) that comprises five members in Plasmodium falciparum. First, we localized four of the P. falciparum ApiATs ( Pf ApiATs) at the PPM using endogenous green fluorescent protein (GFP) tagging. Next, we applied reverse genetic approaches to probe into their essentiality during asexual replication and gametocytogenesis. Upon inducible knockdown and targeted gene disruption, a reduced asexual parasite proliferation was detected for Pf ApiAT2 and Pf ApiAT4. Functional inactivation of individual Pf ApiATs targeted in this study had no effect on gametocyte development. Our data suggest that individual Pf ApiATs are partially redundant during asexual in vitro proliferation and fully redundant during gametocytogenesis of P. falciparum parasites. IMPORTANCE Malaria parasites live and multiply inside cells. To facilitate their extremely fast intracellular proliferation, they hijack and transform their host cells. This also requires the active uptake of nutrients, such as amino acids, from the host cell and the surrounding environment through various membranes that are the consequence of the parasite's intracellular lifestyle. In this paper, we focus on a family of putative amino acid transporters termed ApiAT. We show expression and localization of four transporters in the parasite plasma membrane of Plasmodium falciparum-infected erythrocytes that represent one interface of the pathogen to its host cell. We probed into the impact of functional inactivation of individual transporters on parasite growth in asexual and sexual blood stages of P. falciparum and reveal that only two of them show a modest but significant reduction in parasite proliferation but no impact on gametocytogenesis, pointing toward dispensability within this transporter family.

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single-celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites., (© 2021 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Parasites of the genus Plasmodium cause human and animal malaria, leading to significant health and economic impacts. A key aspect of the complex life cycle of Plasmodium parasites is the invasion of the parasite into its host cell, which is mediated by secretory organelles. The largest of these organelles, the rhoptry, undergoes rapid and profound physiological changes when it secretes its contents during merozoite and sporozoite invasion of the host erythrocyte and hepatocyte, respectively. Here we discuss recent advancements in our understanding of the dynamic rhoptry biology during the parasite's invasive stages, with a focus on the roles of cytosolically exposed rhoptry-interacting proteins (C-RIPs). We explore potential similarities between the molecular mechanisms driving merozoite and sporozoite rhoptry function., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Sequestration of Plasmodium falciparum ( P. falciparum )-infected erythrocytes to host endothelium through the parasite-derived P. falciparum erythrocyte membrane protein 1 ( Pf EMP1) adhesion proteins is central to the development of malaria pathogenesis. Pf EMP1 proteins have diversified and expanded to encompass many sequence variants, conferring each parasite a similar array of human endothelial receptor-binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum- infected adult travellers returning to Germany. Patients were categorized into either malaria naive (n = 15) or pre-exposed (n = 17), and into severe (n = 8) or non-severe (n = 24) cases. For differential expression analysis, Pf EMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var- expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with Pf EMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding Pf EMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic Pf EMP1 variants are more common in patients with a naive immune status, and/or adverse inflammatory host responses to first infections favor the growth of EPCR-binding parasites., Competing Interests: JW, GT, TT, RK, BK, JS, Hv, JS, HS, RW, LT, FL, AS, IB, ET, RF, TO, TL, TG, MD, AB No competing interests declared, (© 2021, Wichers et al.)

Apicomplexan parasites, such as human malaria parasites, have complex lifecycles encompassing multiple and diverse environmental niches. Invading, replicating, and escaping from different cell types, along with exploiting each intracellular niche, necessitate large and dynamic changes in parasite morphology and cellular architecture. The inner membrane complex (IMC) is a unique structural element that is intricately involved with these distinct morphological changes. The IMC is a double membrane organelle that forms de novo and is located beneath the plasma membrane of these single-celled organisms. In Plasmodium spp. parasites it has three major purposes: it confers stability and shape to the cell, functions as an important scaffolding compartment during the formation of daughter cells, and plays a major role in motility and invasion. Recent years have revealed greater insights into the architecture, protein composition and function of the IMC. Here, we discuss the multiple roles of the IMC in each parasite lifecycle stage as well as insights into its sub-compartmentalization, biogenesis, disassembly and regulation during stage conversion of P. falciparum ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RT declared providing some of the pictures used in this review article, and confirms the absence of any other collaboration with the authors to the handling editor., (Copyright © 2021 Ferreira, Heincke, Wichers, Liffner, Wilson and Gilberger.)

Characterizing the adhesive dynamics of Plasmodium falciparum infected erythrocytes (IEs) to different endothelial cell receptors (ECRs) in flow is a big challenge considering available methods. This study investigated the adhesive dynamics of IEs to five ECRs (CD36, ICAM-1, P-selectin, CD9, CSA) using simulations of in vivo-like flow and febrile conditions. To characterize the interactions between ECRs and knobby and knobless IEs of two laboratory-adapted P. falciplarum isolates, cytoadhesion analysis over time was performed using a new tracking bioinformatics method. The results revealed that IEs performed rolling adhesion exclusively over CD36, but exhibited stationary binding to the other four ECRs. The absence of knobs affected rolling adhesion both with respect to the distance travelled by IEs and their velocity. Knobs played a critical role at febrile temperatures by stabilizing the binding interaction. Our results clearly underline the complexity of the IE-receptor interaction and the importance of knobs for the survival of the parasite at fever temperatures, and lead us to propose a new hypothesis that could open up new strategies for the treatment of malaria.

Apicomplexan parasites contain rhoptries, which are specialized secretory organelles that coordinate host cell invasion. During the process of invasion, rhoptries secrete their contents to facilitate interaction with, and entry into, the host cell. Here, we report the crystal structure of the rhoptry protein Armadillo Repeats-Only (ARO) from the human malaria parasite, Plasmodium falciparum (PfARO). The structure of PfARO comprises five tandem Armadillo-like (ARM) repeats, with adjacent ARM repeats stacked in a head-to-tail orientation resulting in PfARO adopting an elongated curved shape. Interestingly, the concave face of PfARO contains two distinct patches of highly conserved residues that appear to play an important role in protein-protein interaction. We functionally characterized the P. falciparum homolog of ARO interacting protein (PfAIP) and demonstrate that it localizes to the rhoptries. We show that conditional mislocalization of PfAIP leads to deficient red blood cell invasion. Guided by the structure, we identified mutations of PfARO that lead to mislocalization of PfAIP. Using proximity-based biotinylation we probe into PfAIP interacting proteins., (Copyright © 2019. Published by Elsevier Ltd.)

During its intraerythrocytic development, the malaria parasite Plasmodium falciparum exposes variant surface antigens (VSAs) on infected erythrocytes to establish and maintain an infection. One family of small VSAs is the polymorphic STEVOR proteins, which are marked for export to the host cell surface through their PEXEL signal peptide. Interestingly, some STEVORs have also been reported to localize to the parasite plasma membrane and apical organelles, pointing toward a putative function in host cell egress or invasion. Using deep RNA sequencing analysis, we characterized P. falciparum stevor gene expression across the intraerythrocytic development cycle, including free merozoites, in detail and used the resulting stevor expression profiles for hierarchical clustering. We found that most stevor genes show biphasic expression oscillation, with maximum expression during trophozoite stages and a second peak in late schizonts. We selected four STEVOR variants, confirmed the expected export of these proteins to the host cell membrane, and tracked them to a secondary location, either to the parasite plasma membrane or the secretory organelles of merozoites in late schizont stages. We investigated the function of a particular STEVOR that showed rhoptry localization and demonstrated its role at the parasite-host interface during host cell invasion by specific antisera and targeted gene disruption. Experimentally determined membrane topology of this STEVOR revealed a single transmembrane domain exposing the semiconserved as well as variable protein regions to the cell surface. IMPORTANCE Malaria claims about half a million lives each year. Plasmodium falciparum , the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for individual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation., (Copyright © 2019 Wichers et al.)

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host's immune system, thus promoting overall parasite survival in the face of host immunity., Competing Interests: BKLS (Executive Vice President Process Development and Manufacturing) and SLH (Chief Executive and Scientific Officer) are employees of Sanaria Inc. This affiliation does not alter our adherence to all PLOS policies on sharing data and materials. All other authors declare that no competing interests exist.