Functional Characterisation Of Novel Plasmodium falciparum Proteins And Their Role In Erythrocyte Invasion

Malaria caused by Plasmodium falciparum is responsible for the deaths of hundreds of thousands of children every year, and imparts an overwhelming economic burden on the world’s poorest countries. All symptoms of malaria are caused during the asexual blood-stage of the P. falciparum lifecycle, which is reliant on merozoite invasion of host red blood cells (RBCs). Due to its essentiality for parasite survival, and its exposure to the host immune system, merozoite invasion is an attractive target for the development of antimalarial therapeutics. Merozoite invasion is coordinated by a series of secretory organelles, of which the largest and most well characterised are the rhoptries. Previous studies into rhoptry proteins have been strongly foccused on antigens that are secreted from the rhoptries during invasion; primarily due to their promise as vaccine candidates. As such, very little is known about proteins that coordinate rhoptry biogenesis, structure, or function. Prior to this study the P. falciparum proteins Pf3D7_0210600 and Pf3D7_0405200, hereafter referred to as P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting proteins (PfCERLI) 1 and 2, were largely uncharacterised proteins that previous studies had suggested may play a role in merozoite invasion. We hypothesised that PfCERLI1 and PfCERLI2 were rhoptry proteins that shared an evolutionary relationship and were both essential for merozoite invasion. We aimed to test these hypotheses through bioinformatic analyses, immunofluorescence microscopy, and gene disruption or inducible knockdown. Bioinformatic and phylogenetic analyses showed that cerli1 and cerli2 arose through an ancestral gene duplication of cerli1 that was present in the most recent common ancestor of haematozoa and coccidia. Analysis of the structure of CERLI proteins revealed they possess a conserved motif with the consensus sequence PHISE/DxxP that we have termed PHIS, along with conserved C2 and Pleckstrin homology (PH) domains that are likely ito have a role in membrane association. Using selection linked integration targeted gene disruption (SLI-TGD) we determined that both Pfcerli1 and Pfcerli2 were refractory to gene deletion and likely important for blood-stage growth. To assess their functions, we used an inducible protein knockdown system whereby the addition of glucosamine (GLCN) results in specific mRNA degradation prior to translation. Knockdown of either CERLI1 or CERLI2 resulted in growth inhibition caused by an inability of merozoites to invade RBCs. Immunofluorescnce microscopy and biochemical techniques revealed that both proteins are peripheral membrane proteins that localise to the cytosolic face of the rhoptry bulb. Rhoptry secretion assays showed that knockdown of PfCERLI1, but not PfCERLI2, leads to a defect in the secretion of key rhoptry antigens. By contrast, electron microscopy analysis of rhoptry size indicated a significant increase in rhoptry length following PfCERLI2 knockdown, but no change with PfCERLI1 knockdown. Semi-quantiative super-resolution microscopy analysis determined that knockdown of PfCERLI1 alters rhoptry antigen distribution, and it was shown that both PfCERLI1 and PfCERLI2 knockdown inhibit processing of key rhoptry antigens. The findings of these studies show that the previously uncharacterised proteins, PfCERLI1 and PfCERLI2, are related rhoptry proteins whose functions are essential for maintaining rhoptry morphology, rhoptry secretion, and rhoptry antigen processing.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

Expansion Microscopy Reveals Plasmodium falciparum Blood-Stage Parasites Undergo Anaphase with A Chromatin Bridge in the Absence of Mini-Chromosome Maintenance Complex Binding Protein

The malaria parasite Plasmodium falciparum undergoes closed mitosis, which occurs within an intact nuclear envelope, and differs significantly from its human host. Mitosis is underpinned by the dynamics of microtubules and the nuclear envelope. To date, our ability to study P. falciparum mitosis by microscopy has been hindered by the small size of the P. falciparum nuclei. Ultrastructure expansion microscopy (U-ExM) has recently been developed for P. falciparum, allowing the visualization of mitosis at the individual nucleus level. Using U-ExM, three intranuclear microtubule structures are observed: hemispindles, mitotic spindles, and interpolar spindles. A previous study demonstrated that the mini-chromosome maintenance complex binding-protein (MCMBP) depletion caused abnormal nuclear morphology and microtubule defects. To investigate the role of microtubules following MCMBP depletion and study the nuclear envelope in these parasites, we developed the first nuclear stain enabled by U-ExM in P. falciparum. MCMBP-deficient parasites show aberrant hemispindles and mitotic spindles. Moreover, anaphase chromatin bridges and individual nuclei containing multiple microtubule structures were observed following MCMBP knockdown. Collectively, this study refines our understanding of MCMBP-deficient parasites and highlights the utility of U-ExM coupled with a nuclear envelope stain for studying mitosis in P. falciparum

Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum

Highlights: • Crystal structure of the malaria parasite lipocalin • Comparative analysis of lipocalin superfamily members in alveolate genomes • Localization of PfLipocalin to the parasitophorous vacuole and food vacuole • Reverse genetics reveal PfLipocalin function in oxidative damage control Summary: Proteins of the lipocalin family are known to bind small hydrophobic ligands and are involved in various physiological processes ranging from lipid transport to oxidative stress responses. The genome of the malaria parasite Plasmodium falciparum contains a single protein PF3D7_0925900 with a lipocalin signature. Using crystallography and small-angle X-ray scattering, we show that the protein has a tetrameric structure of typical lipocalin monomers; hence we name it P. falciparum lipocalin (PfLCN). We show that PfLCN is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of PfLCN impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion. This suggests a key function of PfLCN in counteracting oxidative stress-induced cell damage during multiplication of parasites within erythrocytes

Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology

Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum

Proteins of the lipocalin family are known to bind small hydrophobic ligands and are involved in various physiological processes ranging from lipid transport to oxidative stress responses. The genome of the malaria parasite Plasmodium falciparum contains a single protein PF3D7_0925900 with a lipocalin signature. Using crystallography and small-angle X-ray scattering, we show that the protein has a tetrameric structure of typical lipocalin monomers; hence we name it P. falciparum lipocalin (PfLCN). We show that PfLCN is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of PfLCN impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion. This suggests a key function of PfLCN in counteracting oxidative stress-induced cell damage during multiplication of parasites within erythrocytes

PMRT1, a Plasmodium specific parasite plasma membrane transporter is essential for asexual and sexual blood stage development

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

Cell biological analysis reveals an essential role for Pfcerli2 in erythrocyte invasion by malaria parasites

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

Structural Insights Into PfARO and Characterization of its Interaction With PfAIP

Highlights: • We provide for the first time a crystal structure of a P. falciparum rhoptry protein. • We identify a PfARO interacting protein (PfAIP) and provide an in-depth phylogenetic analysis. • Structure-based mutagenesis, high-resolution microscopy and proximity-based protein identification. Abstract: 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

Dissecting the Gene Expression, Localization, Membrane Topology, and Function of the Plasmodium falciparum STEVOR Protein Family

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