Biology subjects

Nofal, S. D.

Publications and source records attributed to Nofal, S. D..

9 recordsLinked to original sources

bioRxiv2026

Dynamic phosphorylation of the Plasmodium falciparum Myosin A motor regulates motor speed and is important for asexual replication

Malaria parasite invasion of red blood cells is powered by Myosin A (MyoA), the actomyosin motor which is a core component of the glideosome. Our previous phosphoproteomic study identified a cyclic GMP-dependent phosphorylation site on MyoA at Ser19, suggesting that cyclic nucleotide signalling may directly regulate motor activity during invasion. However, the functional significance of this phosphorylation site remains unclear. Here, we show that Plasmodium falciparum MyoA phosphorylation at Ser19 is dynamically regulated during the intraerythrocytic cycle, peaking in extracellular merozoites and rapidly declining following invasion. Using conditional depletion of MyoA, we show that MyoA is essential for red blood cell invasion but dispensable for gametocyte development. Conditional complementation with mutant versions of MyoA that either mimic (S19D) or ablate phosphorylation (S19A) reveal markedly impaired asexual parasite proliferation, highlighting the importance of precise regulation of MyoA phosphorylation at Ser19. In vitro motility assays using parasite-derived MyoA showed that ablating phosphorylation either pharmacologically or by introducing an S19A mutation, reduces motor speed. Conversely, introducing a phosphomimetic S19D mutation restores motility levels comparable to phosphorylated wild type MyoA. Collectively, these results establish MyoA Ser19 phosphorylation as a regulatory switch for actomyosin motor activity.

cell biology↗

bioRxiv2025

Physiological febrile heat stress increases cytoadhesion through increased protein trafficking of Plasmodium falciparum surface proteins into the red blood cell

Fever is a hallmark of malaria. Several studies have linked febrile temperatures to reduced parasite viability, but also to increased cytoadhesion, a key driver of pathology. However, different mechanisms have been proposed to cause changes in cytoadhesion and parasite sensitivity to heat. Here, we demonstrate that exposure of Plasmodium falciparum-infected red blood cells (iRBCs) to physiologically relevant febrile heat stress (39 {degrees}C), derived from patient data, enhances cytoadhesion through increased trafficking of the major virulence factor PfEMP1 to the iRBC surface. This phenomenon is not limited to PfEMP1 and common laboratory strains, as it extends to the surface nutrient channel PSAC in four clinical isolates of diverse geographic origin. The increased surface protein display occurs without changes in overall protein expression or parasite developmental progression. Using phosphoproteomics and proximity labelling, we find that elevated temperature also increases trafficking and phosphorylation of exported proteins into the RBC. Enhanced export is likely reliant on the presence of a transmembrane domain as shown by NanoLuc reporter assays. Collectively, our results indicate that febrile temperatures commonly experienced during infection can accelerate protein export, likely at the parasitophorous vacuole. This enhanced export following heat stress is relevant because increased cytoadhesion could influence disease severity through earlier iRBC sequestration and elevated bound parasite mass.

cell biology↗

bioRxiv2024

Beta-hairpin Mechanism of Autoinhibition and Activation in the Kinesin-2 Family

Members of the kinesin-2 family coordinate with other motors to power diverse physiological processes, but the structural mechanisms regulating kinesin-2 activity have been unknown. Distinctively, kinesin-2s canonically function as heterotrimers of two different motor subunits (e.g. Kif3A and Kif3B) and Kap3, but the role of heterotrimerization has yet to fully emerge. Here, we combine structural, cell biological, and single-molecule approaches to dissect kinesin-2 regulation as a heterodimer, heterotrimer, and quaternary complex with a cargo adaptor (APC). We identify a conserved motif in the tail of kinesin-2s (the beta-hairpin motif) that controls kinesin-2 motility by sequestering the motor domains away from their microtubule track, using striking molecular mimicry. Our data reveal how Kap3 binds via a multipartite interface with Kif3A and Kif3B, and - rather than activating motility directly - provides a platform on which cargo adaptors can engage and occlude the beta-hairpin motif. Together, these data articulate a structural framework for kinesin-2 activation, coordination with dynein, and adaptation for different biological functions.

cell biology↗

bioRxiv2024

Evolution and inhibition of the FIKK effector kinase family in P. falciparum

Among the [~]200 Plasmodium species that infect vertebrates, six infect humans. Of these, P. falciparum causes >95% of all [~]500,000 annual fatalities. Phylogenetically, P. falciparum belongs to the Laverania subgenus, a group of Plasmodium species that infect great apes. Common to Laverania species is the family of FIKK kinases. One million years ago, a single FIKK kinase conserved in all Plasmodium species gained an export element in the Laverania subgenus and expanded into the family of [~]20 atypical FIKK kinases, most of which are exported into the host cell. The fikk genes are conserved in syntenic loci across the Laverania, arguing for a rapid expansion controlling important functions in host cell remodelling and pathogenesis. We provide evidence that the FIKK paralogues evolved specific and mutually exclusive phosphorylation motif preferences, conserved across their Laverania orthologues, in a short evolutionary timeframe. Surprisingly, we find that FIKK13 has evolved exclusive tyrosine-phosphorylation preference, which was thought to be absent in Plasmodium species. Combining a crystal structure with AlphaFold2 predictions, we identify residues that determine kinase-specificity within the FIKK family in a fast-evolving flexible loop. Finally, we show that all expressed members of the FIKK kinase family can be chemically inhibited in vitro using a single compound. Such a pan-specific inhibitor of this kinase family important for virulence could reduce the ability of the parasite to gain escape-mutations and resistance.

microbiology↗

bioRxiv2022

CDC50 orthologues in Plasmodium falciparum have distinct roles in merozoite egress and trophozoite maturation

In model organisms P4-ATPases require cell division control protein 50 (CDC50) chaperones for their phospholipid flipping activity. In the malaria parasite, P. falciparum, guanylyl cyclase alpha (GC) is an integral membrane protein that is essential for release (egress) of merozoites from their host erythrocytes. GC is unusual in that it contains both a C-terminal cyclase domain and an N-terminal P4-ATPase domain of unknown function. We sought to investigate whether any of the three CDC50 orthologues (denoted A, B and C) encoded by P. falciparum are required for GC function. Using gene tagging and conditional gene disruption, we demonstrate that both CDC50B and CDC50C are expressed in the clinically important asexual blood stages and that CDC50B is a binding partner of GC whereas CDC50C is the binding partner of another putative P4-ATPase, ATP2. Our findings indicate that CDC50B has no essential role for intraerythrocytic parasite maturation but modulates the rate of parasite egress by interacting with GC for optimal cGMP synthesis. In contrast, CDC50C is essential for blood stage trophozoite maturation. Additionally, we find that the CDC50C-ATP2 complex may influence parasite endocytosis of host cell haemoglobin and consequently hemozoin formation.

molecular biology↗

bioRxiv2021

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in Toxoplasma gondii

Fundamental processes of obligate intracellular parasites, such as Toxoplasma gondii and Plasmodium falciparum, are controlled by a set of plant-like calcium dependent kinases (CDPKs), the conserved cAMP- and cGMP-dependent protein kinases (PKA and PKG), secondary messengers and lipid signalling. While some major components of the signalling networks have been identified, how these are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels to activate PKG or CDPKs, respectively. We show that both these inducers trigger near identical signalling pathways and provide evidence for a positive feedback loop involving CDPK3. We measure phospho- and lipid signalling in parasites treated with the Ca2+ ionophore A23187 in a sub-minute timecourse and show CDPK3-dependent regulation of diacylglycerol levels and increased phosphorylation of four phosphodiesterases (PDEs), suggesting their function in the feedback loop. Disruption of CDPK3 leads to elevated cAMP levels and inhibition of PKA signalling rescues the egress defect of {Delta}CDPK3 parasites treated with A23187. Biochemical analysis of the four PDEs identifies PDE2 as the only cAMP-specific PDE among these candidates, while the other PDEs are cGMP specific, two of which are inhibited by the predicted PDE inhibitor BIPPO. Conditional deletion of the four PDEs supports an important, but non-essential role for PDE1 and PDE2 in growth, with PDE2 controlling A23187-mediated egress. In summary we uncover a positive feedback loop that enhances signalling during egress and links several signalling pathways together.

microbiology↗

bioRxiv2020

Plasmodium falciparum guanylyl cyclase-alpha and the activity of its appended P4-ATPase domain are essential for cGMP synthesis and blood stage egress

In malaria parasites, guanylyl cyclases (GCs), which synthesise cyclic GMP (cGMP), are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here we investigate the role of Plasmodium falciparum guanylyl cyclase alpha (GC) and its associated P4-ATPase module, showing that asexual blood stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GC-null parasites cannot synthesise cGMP, or mobilise calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that ATPase activity is up stream of and linked to cGMP synthesis. Collectively, our results demonstrate that GC is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress.

molecular biology↗

bioRxiv2020

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization of Plasmodium falciparum and Plasmodium knowlesi asexual stage parasites

During the course of the asexual erythrocytic stage of development, Plasmodium spp. parasites undergo a series of morphological changes and induce alterations in the host cell. At the end of this stage, the parasites exit the host cell, after which the progeny invade a new host cell. These processes are rapid and occur in a time-dependent manner. Of particular importance, egress and invasion of erythrocytes by the parasite are difficult to capture in an unsynchronized culture, or even a culture that has been synchronized to within hours. Therefore, precise synchronization of parasite cultures is of paramount importance for the investigation of these processes. Here we describe a method for synchronizing Plasmodium falciparum and Plasmodium knowlesi asexual blood stage parasites with ML10, a highly specific inhibitor of the cGMP-dependent protein kinase (PKG) that arrests parasite growth approximately 15 minutes prior to egress. This inhibitor allows parasite cultures to be synchronized to within minutes, with a simple wash step. Furthermore, we show that parasites remain viable for several hours after becoming arrested by the compound and that ML10 has advantages over the previously used PKG inhibitor Compound 2. Here, we demonstrate that ML10 is an invaluable tool for the study of Plasmodium spp. asexual blood stage biology and for the routine synchronization of P. falciparum and P. knowlesi cultures.

microbiology↗

bioRxiv2019

Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the -subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2 I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to <2% for the 3D7 parental line (P = 0.0011 for each clone). In immunoprecipitation and localisation studies of HA-tagged AP-2, we identified interacting partners including AP-2, AP-1/2{beta}, AP-2{sigma} and a kelch-domain protein encoded on chromosome 10 of P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

microbiology↗