Deletion of the Plasmodium falciparum exported protein PTP7 leads to Maurer's clefts vesiculation, host cell remodeling defects, and loss of surface presentation of EMP1.

Presentation of the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (EMP1), at knob-like protrusions on the surface of infected red blood cells, underpins the parasite's pathogenicity. Here we describe a protein PF3D7_0301700 (PTP7), that functions at the nexus between the intermediate trafficking organelle, the Maurer's cleft, and the infected red blood cell surface. Genetic disruption of PTP7 leads to accumulation of vesicles at the Maurer's clefts, grossly aberrant knob morphology, and failure to deliver EMP1 to the red blood cell surface. We show that an expanded low complexity sequence in the C-terminal region of PTP7, identified only in the Laverania clade of Plasmodium, is critical for efficient virulence protein trafficking.

Surface area-to-volume ratio, not cellular viscoelasticity, is the major determinant of red blood cell traversal through small channels.

The remarkable deformability of red blood cells (RBCs) depends on the viscoelasticity of the plasma membrane and cell contents and the surface area to volume (SA:V) ratio; however, it remains unclear which of these factors is the key determinant for passage through small capillaries. We used a microfluidic device to examine the traversal of normal, stiffened, swollen, parasitised and immature RBCs. We show that dramatic stiffening of RBCs had no measurable effect on their ability to traverse small channels. By contrast, a moderate decrease in the SA:V ratio had a marked effect on the equivalent cylinder diameter that is traversable by RBCs of similar cellular viscoelasticity. We developed a finite element model that provides a coherent rationale for the experimental observations, based on the nonlinear mechanical behaviour of the RBC membrane skeleton. We conclude that the SA:V ratio should be given more prominence in studies of RBC pathologies.

Multimodal analysis of Plasmodium knowlesi-infected erythrocytes reveals large invaginations, swelling of the host cell, and rheological defects.

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block-face scanning electron microscopy to explore the topography of P. knowlesi-infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi-infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python-based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi-infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.

Arabidopsis thaliana RHAMNOSE 1 condensate formation drives UDP-rhamnose synthesis.

Rhamnose is an essential component of the plant cell wall and is synthesized from uridine diphosphate (UDP)-glucose by the RHAMNOSE1 (RHM1) enzyme. RHM1 localizes to biomolecular condensates in plants, but their identity, formation, and function remain elusive. Combining live imaging, genetics, and biochemical approaches in Arabidopsis and heterologous systems, we show that RHM1 alone is sufficient to form enzymatically active condensates, which we name rhamnosomes. Rhamnosome formation is required for UDP-rhamnose synthesis and organ development. Overall, our study demonstrates a novel role for biomolecular condensation in metabolism and organismal development, and provides further support for how organisms have harnessed this biophysical process to regulate small molecule metabolism.

VAR2CSA Ectodomain Labeling in Plasmodium falciparum Infected Red Blood Cells and Analysis via Flow Cytometry.

Presentation of the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected red blood cells (RBCs) underpins the malaria parasite's pathogenicity. The transport of EMP1 to the RBC surface is facilitated by a parasite-derived trafficking system, in which over 500 parasite proteins are exported into the host cell cytoplasm. To understand how genetic ablation of selected exported proteins affects EMP1 transport, several EMP1 surface presentation assays have been developed, including: 1) trypsinization of surface-exposed EMP1 and analysis by SDS-PAGE and immunoblotting; and 2) infected RBC binding assays, to determine binding efficiency to immobilized ligand under physiological flow conditions. Here, we describe a third EMP1 surface presentation assay, where antibodies to the ectodomain of EMP1 and flow cytometry are used to quantify surface-exposed EMP1 in live cells. The advantages of this assay include higher throughput capacity and data better suited for robust quantitative analysis. This protocol can also be applied to other cellular contexts where an antibody can be developed for the ectodomain of the protein of interest.

Role of Plasmodium falciparum Protein GEXP07 in Maurer's Cleft Morphology, Knob Architecture, and P. falciparum EMP1 Trafficking.

The malaria parasite Plasmodium falciparum traffics the virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer's clefts. We developed a method for efficient enrichment of Maurer's clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterized or poorly characterized Maurer's cleft proteins. We generated transfectants expressing green fluorescent protein (GFP) fusions of 7 proteins and confirmed their Maurer's cleft location. Using co-immunoprecipitation and mass spectrometry, we generated an interaction map of proteins at the Maurer's clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurer's clefts. We focus on a putative PfEMP1 loading complex that includes the protein GEXP07/CX3CL1-binding protein 2 (CBP2). Disruption of GEXP07 causes Maurer's cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression, and loss of the PfEMP1-mediated adhesion. ΔGEXP07 parasites have a growth advantage compared to wild-type parasites, and the infected RBCs are more deformable and more osmotically fragile.IMPORTANCE The trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer's clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.

Genetic Pathways of Neuroregeneration in a Novel Mild Traumatic Brain Injury Model in Adult Zebrafish.

Mild traumatic brain injuries (mTBIs) are one of the most prevalent neurological disorders, and humans are severely limited in their ability to repair and regenerate central nervous system (CNS) tissue postinjury. However, zebrafish (Danio rerio) maintain the remarkable ability to undergo complete and functional neuroregeneration as an adult. We wish to extend knowledge of the known mechanisms of neuroregeneration by analyzing the differentially expressed genes (DEGs) in a novel adult zebrafish model of mTBI. In this study, a rodent weight drop model of mTBI was adapted to the adult zebrafish. A memory test showed significant deficits in spatial memory in the mTBI group. We identified DEGs at 3 and 21 days postinjury (dpi) through RNA-sequencing analysis. The resulting DEGs were categorized according to gene ontology (GO) categories. At 3 dpi, GO categories consisted of peak injury response pathways. Significantly, at 21 dpi, GO categories consisted of neuroregeneration pathways. Ultimately, these results validate a novel zebrafish model of mTBI and elucidate significant DEGs of interest in CNS injury and neuroregeneration.