Biology subjects

Fierro, M. A.

Publications and source records attributed to Fierro, M. A..

8 recordsLinked to original sources

bioRxiv2025

The inner membrane complex protein, IMC55, is dispensable for intra-erythrocytic development of Plasmodium falciparum

Plasmodium falciparum, an obligate intracellular Apicomplexan parasite and the causative agent of malaria must reside and replicate within a host cell during the intra-erythrocytic stage. The parasite utilizes a specialized double-membraned organelle, called the inner membrane complex for schizont-stage segmentation and merozoite motility during subsequent re-invasion. In this study, we investigate the function of IMC55, a multi-transmembrane inner membrane complex protein, which we identified as a putative interacting partner of the P. falciparum ER-resident calcium binding protein, PfERC. We have previously shown that PfERC is essential for parasite egress and our data shows that PfERC may also function in parasite invasion into the RBC. The whole genome transposon mutagenesis screen predicts that IMC55 is essential for the intra-erythrocytic asexual cycle. In this work, we show that IMC55 is not required for asexual replication. Following inducible expression of this gene using two conditional systems, we find there is no defect in replicative fitness during the asexual blood stage. Taken together, these data demonstrate that the function of IMC55 is either dispensable or redundant for the Plasmodium falciparum asexual blood stage.

cell biology↗

bioRxiv2025

Spatiotemporal dynamics of signal dependent exocytosis and parasitophorous vacuolar membrane rupture during Plasmodium falciparum egress.

Malaria, caused by intracellular Plasmodium parasites, remains a major global health concern. These parasites reside and replicate within a vacuole in host red blood cells. Egress of daughter parasites out of the vacuolar and host membranes is tightly regulated via a complex mechanism. Prior studies have suggested that a cyclic-GMP driven calcium signaling pathway leads to the signal-dependent exocytosis of egress-specific vesicles that discharge several proteases into the parasitophorous vacuole. However, signal-dependent exocytosis during egress has not yet been observed in live parasitized RBCs. We targeted the exocytosis reporter, superecliptic pHlourin or SEP, to these egress-specific vesicles and utilized time-lapse imaging to observe exocytosis in live P. falciparum parasites. The spatiotemporal relationship between exocytosis and the breakdown of the parasitophorous vacuolar membrane (PVM) as well as parasite egress was also determined using a fluorescent reporter targeted to the PVM. Our data showed that exocytosis is triggered as early as 3 hours prior to merozoite egress. These data suggest that the PVM rupture occurs at a single site and rapidly expands from that initial site of rupture, releasing the merozoites into the RBC. This is followed by RBC membrane rupture and egress of merozoites. Using conditional mutants of the P. falciparum endoplasmic reticulum calcium-binding protein (PfERC), we demonstrate that knockdown of PfERC inhibits signal-dependent exocytosis of egress-specific vesicles. Together, these data demonstrate that signal-dependent exocytosis of egress-specific vesicles starts well before merozoites are formed via cytokinesis, PVM ruptures at a single site, and that PfERC is required for exocytosis of egress-specific vesicles.

cell biology↗

bioRxiv2024

Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBC) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs, begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from two specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains seven transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only one rhoptry each. The single rhoptry in RON11 deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11 deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11 deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.

cell biology↗

bioRxiv2023

PEXEL is a proteolytic maturation site for both exported and non-exported Plasmodium proteins

Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric Plasmodium export element (PEXEL)/Host Targeting Motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N-terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here we report that the non-exported parasitophorous vacuole protein UIS2 contains a bona fide PEXEL motif that is processed in the P. falciparum blood-stage. While the N-termini of exported proteins containing the PEXEL and immediately downstream [~]10 residues is sufficient to mediate translocation into the RBC, the equivalent UIS2 N-terminus does not promote export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position which constitutes the extreme N-terminal residue following PEXEL cleavage (P1, RIL{tau}DE). Using a series of chimeric reporter fusions, we show that Asp at P1 is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N-terminus mediates export, not PEXEL processing per se. Prompted by this observation, we further show that PEXEL sequences in the N-termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export. ImportanceHost erythrocyte remodeling by malaria parasite exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite Plasmodium falciparum, most exported proteins undergo proteolytic maturation via recognition of the pentameric Plasmodium export element (PEXEL)/Host Targeting motif by the aspartic protease Plasmepsin V (PMV) which exposes a mature N-terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing by PMV, reinforcing that features of the mature N-terminus, and not PEXEL cleavage, identify cargo for export cargo. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

microbiology↗

bioRxiv2022

The PTEX pore component EXP2 is important for intrahepatic development during the Plasmodium liver stage

During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To transcend this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are indicated by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3UTR enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Post-invasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid to late liver stage marker LISP2. As an orthogonal approach to monitor development, EXP2-glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding with merosome detachment that marks the culmination of liver stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal EXP2 is important for intrahepatic parasite development, indicating PTEX components also function at the hepatocyte-parasite interface.

microbiology↗

bioRxiv2022

A novel knock sideways approach for Plasmodium secreted proteins reveals a surprising insensitivity to vacuolar depletion of PTEX component HSP101

Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting Plasmodium secreted protein functions are limited. We report knockER, a novel DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiple Plasmodium species to interrogate the trafficking, topology and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a novel tool for dissecting secreted protein function with sub-compartmental resolution that should be widely amenable to genetically tractable eukaryotes. SignificanceProtein trafficking and secretion through the endomembrane system is a defining feature of eukaryotes. The secretory pathway is central to the unique biology and pathology of the obligate intracellular malaria parasite, however tools for studying secreted protein function are limited. Knock-sideways is a powerful mutagenesis strategy that conditionally sequesters a protein away from its site of function but is generally not applicable to secreted proteins. We developed a simple approach to conditionally sequester Plasmodium secreted proteins in the ER by inducible C-terminal fusion with a KDEL ER-retrieval sequence that can be used for trafficking, topology and loss-of-function studies. The knockER strategy is broadly applicable to functional dissection of proteins that traverse the eukaryotic secretory pathway.

microbiology↗

bioRxiv2018

An ER-resident calcium binding protein modulates egress of malaria parasites from erythrocytes

The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be the calcium (Ca2+) signaling hub and required for biogenesis of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum Endoplasmic Reticulum-resident Calcium-binding protein (PfERC), a member of the CREC family. Knockdown of PfERC shows that this gene is essential for asexual growth of P. falciparum. Analysis of the intraerythrocytic lifecycle revealed that PfERC is essential for parasite egress but not required for protein trafficking or Ca2+ storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease, SUB1. Using double mutant parasites, we show that PfERC is required for the proteolytic maturation of the essential aspartic protease, Plasmepsin X, which cleaves SUB1. Further, we show that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish the ER-resident CREC family protein, PfERC, as a key early regulator of the egress proteolytic cascade of malaria parasites.

cell biology↗

bioRxiv2018

The ER chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites.

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterized ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localizes to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2 kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

cell biology↗