Biology subjects

Sigala, P. A.

Publications and source records attributed to Sigala, P. A..

12 recordsLinked to original sources

bioRxiv2026

Identification of a Putative Metal Transporter in the Apicoplast of Malaria Parasites

Plasmodium falciparum malaria parasites harbor an essential plastid organelle, called the apicoplast, which produces key metabolites required for organelle function and parasite viability. Apicoplast functions depend on iron and other metals, but the membrane transporters that mediate metal import into this organelle have been challenging to identify. Tetracycline antibiotics, including doxycycline, specifically target the apicoplast and can exhibit metal-dependent activity. Using tetracycline-affinity proteomics, we identified a doxycycline-interacting, uncharacterized transmembrane protein (UCT) targeted to the apicoplast periphery but not proteolytically processed. Although lacking sequence similarity to proteins of known function, UCT has a predicted structure with high similarity to pentameric CorA-family metal transporters that mediate metal uptake in other organisms. Functional tests revealed that UCT is dispensable for blood-stage asexual parasites, suggesting that the apicoplast has evolved redundant mechanisms for metal uptake. UCT knockdown in gametocytes, however, impairs the development of sexual parasites, which are critical for mosquito transmission. Our study identifies an apicoplast membrane protein with localization and structural properties that predict a role in metal transport into this key organelle. This discovery can provide a biochemical springboard to unravel broader apicoplast mechanisms of metal uptake across multiple stages of parasite development, including mosquito-stage parasites that display heightened UCT expression.

biochemistry↗

bioRxiv2026

Acyl Carrier Protein is Essential for Apicoplast Biogenesis in Malaria Parasites Independent of Fatty Acid Synthesis

Acyl carrier protein (ACP) and its 4-phosphopantetheine prosthetic group canonically function as the soluble scaffold for acyl chain assembly and elongation during type-II fatty acid synthesis (FASII). Plasmodium malaria parasites retain a FASII pathway in the apicoplast organelle that has been the subject of considerable scrutiny and confusion. Although apicoplast FASII is essential for P. falciparum growth within mosquitoes and the human liver, this pathway is dispensable and largely inactive in blood-stage parasites that can scavenge host fatty acids. In contrast to FASII enzymes that can be disrupted without fitness defect, we report that knockout or ligand-dependent knockdown of apicoplast ACP is lethal to blood-stage P. falciparum, indicating an essential FASII-independent function. Loss of ACP impairs the biosynthesis of essential isoprenoid precursors and blocks apicoplast biogenesis. Using proximity biotinylation and biochemical interaction studies, we identified a key role for ACP in binding and stabilizing apicoplast pyruvate kinase II (PKII). This critical enzyme is the only known source of nucleoside triphosphates (NTPs) in this organelle and is required for isoprenoid synthesis and apicoplast biogenesis. Our work reveals that ACP knockdown results in destabilization and loss of PKII, which is sufficient to explain ACP essentiality in this stage. This work unveils essential ACP function at a key biochemical hub controlling broad apicoplast metabolism in malaria parasites that is independent of the canonical ACP role in FASII.

biochemistry↗

bioRxiv2026

Evolutionary rate covariation across malaria parasite species enables inference of protein interactions

Despite publication of the Plasmodium falciparum reference genome over 20 years ago, one-third of its genes remain functionally unannotated, and information is limited for many others. Proteins that act in the same pathway or complex tend to experience similar shifts in evolutionary pressure, such that correlated constraints across species can indicate co-functional proteins. To investigate this connection, we calculated relative evolutionary rates across the genome on a phylogeny of 22 Plasmodium species and assigned each protein-protein pair a score representing the strength of evolutionary rate covariation (ERC). We show that known pathways and interacting proteins across lifecycle stages in Plasmodium have strong ERC signals. By scanning genome-wide for additional proteins showing high ERC with established interacting proteins, we find enrichment of stage expression and physically interacting protein pairs supporting new candidate functions for proteins. More generally, we demonstrate the utility of ERC to prioritize proteins for hypothesis-driven functional follow up by showing that a protein with little functional characterization (PF3D7_0811600) shows high ERC and co-localizes with high molecular weight rhoptry proteins 2 and 3 (RhopH2 and RhopH3), which form an ion channel that enhances parasite permeability of infected red blood cells. The ERC matrix can be queried to extract Plasmodium proteins showing high ERC with any protein of interest to prioritize candidate genes and accelerate discovery of novel functional connections.

evolutionary biology↗

bioRxiv2025

A Divergent Cytochrome c in Malaria Parasites with an Anomalously Low Redox Potential

Eukaryotic cytochrome (cyt) c is a highly conserved mitochondrial protein central to cellular respiration, featuring a covalently attached hexacoordinate heme whose redox potential is tuned by axial His/Met ligands and surrounding residues to support electron transport chain (ETC) function. We have identified an unrecognized lineage of eukaryotic cyt c homologs in Apicomplexa, a phylum of intracellular pathogens that includes Plasmodium falciparum malaria parasites. P. falciparum cyt c-2 (Pfcyt c-2) exemplifies this divergent lineage and has an unusual pentacoordinate heme despite conservation of His/Met ligands. We determined that Pfcyt c-2 has a redox potential of -278 mV that is over 500 mV lower than canonical cyt c homologs (+250 mV) and contradicts a conserved ETC role. This anomalous redox potential is lower than any natural monoheme c-type cyt. Nevertheless, Pfcyt c-2 displays canonical thermostability and low-level peroxidase activity, while showing signs of elevated structural heterogeneity. These results reveal a new clade of eukaryotic cyt c variants with divergent biochemical properties and biological roles, opening new scaffolds for mechanistic discovery and redox engineering.

biochemistry↗

bioRxiv2025

Chemical propulsion of hemozoin crystal motion in malaria parasites

Malaria parasites infect red blood cells where they digest host hemoglobin and release free heme inside a lysosome-like organelle called the food vacuole. To detoxify excess heme, parasites form hemozoin crystals that rapidly tumble inside this compartment. Hemozoin formation is critical for parasite survival and central to antimalarial drug activity. Although the static structural properties of hemozoin have been extensively investigated, crystal motion and its underlying mechanism have remained puzzling. We used quantitative image analysis to determine the timescale of motion, which requires the intact vacuole but does not require the parasite itself. Using single particle tracking and Brownian dynamics simulations with experimentally derived interaction potentials, we found that hemozoin motion exhibits unexpectedly tight confinement but is much faster than thermal diffusion. Hydrogen peroxide, which is generated at high levels in the food vacuole, has been shown to stimulate the motion of synthetic metallic nanoparticles via surface-catalyzed peroxide decomposition that generates propulsive kinetic energy. We observed that peroxide stimulated the motion of isolated crystals in solution and that conditions that suppress peroxide formation slowed hemozoin motion inside parasites. These data suggest that surface-exposed metals on hemozoin catalyze peroxide decomposition to drive crystal motion. This work reveals hemozoin motion in malaria parasites as a biological example of an endogenous self-propelled nanoparticle. This mechanism of propulsion likely serves a physiological role to reduce oxidative stress to parasites from hydrogen peroxide produced by large-scale hemoglobin digestion during blood-stage infection. Significance statementHemozoin crystal formation is a major antimalarial drug target as it is essential for survival of Plasmodium parasites that cause malaria, one of the worlds most devastating infectious diseases. Hemozoin crystals rapidly tumble inside the parasite food vacuole, and the mechanism and significance of this motion have been mysterious. Using quantitative live-cell imaging and computational modeling, we discovered that hemozoin motion is driven by catalytic decomposition of hydrogen peroxide on the crystal surface, a mechanism analogous to synthetic nanomotors. Hemozoin crystals have been viewed as inert detoxification products. Our work reframes the physiological role of these crystals as catalytically active nanoparticles whose mechanism of propulsion helps to neutralize toxic hydrogen peroxide generated by parasite digestion of hemoglobin during blood-stage infection.

biophysics↗

bioRxiv2024

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis

Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 [A]-resolution X-ray structure that revealed a highly -helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.

biochemistry↗

bioRxiv2024

Unraveling mechanisms of iron acquisition in malaria parasites

Plasmodium falciparum malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, P. falciparum requires nutritional iron to support essential metabolic pathways, but the critical mechanisms of iron acquisition and trafficking during RBC infection have remained obscure. Parasites internalize and liberate massive amounts of heme during large-scale digestion of RBC hemoglobin within an acidic food vacuole (FV) but lack a heme oxygenase to release porphyrin-bound iron. Although most FV heme is sequestered into inert hemozoin crystals, prior studies indicate that trace heme escapes biomineralization and is susceptible to non-enzymatic degradation within the oxidizing FV environment to release labile iron. Parasites retain a homolog of divalent metal transporter 1 (DMT1), a known mammalian iron transporter, but its role in P. falciparum iron acquisition has not been tested. Our phylogenetic studies indicate that P. falciparum DMT1 (PfDMT1) retains conserved molecular features critical for metal transport. We localized this protein to the FV membrane and defined its orientation in an export-competent topology. Conditional knockdown of PfDMT1 expression is lethal to parasites, which display broad cellular defects in iron-dependent functions, including impaired apicoplast biogenesis and mitochondrial polarization. Parasites are selectively rescued from partial PfDMT1 knockdown by supplementation with exogenous iron, but not other metals. These results support a cellular paradigm whereby PfDMT1 is the molecular gatekeeper to essential iron acquisition by blood-stage malaria parasites and suggest that therapeutic targeting of PfDMT1 may be a potent antimalarial strategy.

cell biology↗

bioRxiv2024

Biogenesis of cytochromes c and c1 in the electron transport chain of malaria parasites

Plasmodium malaria parasites retain an essential mitochondrional electron transport chain (ETC) that is critical for growth within humans and mosquitoes and a key antimalarial drug target. ETC function requires cytochromes c and c1 that are unusual among heme proteins due to their covalent binding to heme via conserved CXXCH sequence motifs. Heme attachment to these proteins in most eukaryotes requires the mitochondrial enzyme holocytochrome c synthase (HCCS) that binds heme and the apo cytochrome to facilitate biogenesis of the mature cytochrome c or c1. Although humans encode a single bifunctional HCCS that attaches heme to both proteins, Plasmodium parasites are like yeast and encode two separate HCCS homologs thought to be specific for heme attachment to cyt c (HCCS) or cyt c1 (HCC1S). To test the function and specificity of P. falciparum HCCS and HCC1S, we used CRISPR/Cas9 to tag both genes for conditional expression. HCC1S knockdown selectively impaired cyt c1 biogenesis and caused lethal ETC dysfunction that was not reversed by over-expression of HCCS. Knockdown of HCCS caused a more modest growth defect but strongly sensitized parasites to mitochondrial depolarization by proguanil, revealing key defects in ETC function. These results and prior heterologous studies in E. coli of cyt c hemylation by P. falciparum HCCS and HCC1S strongly suggest that both homologs are essential for mitochondrial ETC function and have distinct specificities for biogenesis of cyt c and c1, respectively, in parasites. This study lays a foundation to develop novel strategies to selectively block ETC function in malaria parasites.

microbiology↗

bioRxiv2023

Direct Tests of Cytochrome Function in the Electron Transport Chain of Malaria Parasites

The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c-2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knock-out had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c-2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino-acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution. SIGNIFICANCE STATEMENTMitochondria are critical organelles in eukaryotic cells that drive oxidative metabolism. The mitochondrion of Plasmodium malaria parasites is a major drug target that has many differences from human cells and remains poorly studied. One key difference from humans is that malaria parasites express two cytochrome c proteins that differ significantly from each other and play untested and uncertain roles in the mitochondrial electron transport chain (ETC). Our study revealed that one cyt c is essential for ETC function and parasite viability while the second, more divergent protein has unusual structural and biochemical properties and is not required for growth of blood-stage parasites. This work elucidates key biochemical properties and evolutionary differences in the mitochondrial ETC of malaria parasites.

microbiology↗

bioRxiv2021

Critical Role for Isoprenoids in Apicoplast Biogenesis by Malaria Parasites

Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in Plasmodium falciparum malaria parasites and is required for prenylation-dependent vesicular trafficking and other cellular processes. We have elucidated a critical and previously uncharacterized role for IPP in apicoplast biogenesis. Inhibiting IPP synthesis blocks apicoplast elongation and inheritance by daughter merozoites, and apicoplast biogenesis is rescued by exogenous IPP and polyprenols. Knockout of the only known isoprenoid-dependent apicoplast pathway, tRNA prenylation by MiaA, has no effect on blood-stage parasites and thus cannot explain apicoplast reliance on IPP. However, we have localized an annotated polyprenyl synthase (PPS) to the apicoplast lumen. PPS knockdown is lethal to parasites, rescued by IPP, and blocks apicoplast biogenesis, thus explaining apicoplast dependence on isoprenoid synthesis. We hypothesize that PPS synthesizes long-chain polyprenols critical for apicoplast membrane fluidity and biogenesis. This work critically expands the paradigm for isoprenoid utilization in malaria parasites and identifies a novel essential branch of apicoplast metabolism suitable for therapeutic targeting.

microbiology↗

bioRxiv2021

Divergent Acyl Carrier Protein Mediates Mitochondrial Fe-S Cluster Biosynthesis in Malaria Parasites

Plasmodium falciparum malaria parasites are early-diverging eukaryotes with many unusual metabolic adaptations. Understanding these adaptations will give insight into parasite evolution and unveil new parasite-specific drug targets. Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis. In yeast and humans, mACP also functions to biochemically couple FASII activity to electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast to most eukaryotes, the Plasmodium mitochondrion lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC followed by broader cellular defects. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of both Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other Apicomplexan pathogens, including Toxoplasma and Babesia. This discovery also highlights the ancient, fundamental role of ACP in mitochondrial Fe-S cluster biogenesis and unveils an evolutionary driving force to retain this interaction with ACP independent of its eponymous function in FASII. Significance StatementPlasmodium malaria parasites are single-celled eukaryotes that evolved unusual metabolic adaptations. Parasites require a mitochondrion for blood-stage viability, but essential functions beyond the electron transport chain are sparsely understood. Unlike yeast and human cells, the Plasmodium mitochondrion lacks fatty acid synthesis enzymes but retains a divergent acyl carrier protein (mACP) incapable of tethering acyl groups. Nevertheless, mACP is essential for parasite viability by binding and stabilizing the core mitochondrial Fe-S cluster biogenesis complex via a divergent molecular interface lacking an acyl-pantetheine group that contrasts with other eukaryotes. This discovery unveils an essential metabolic adaptation in Plasmodium and other human parasites that decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis and evolved at or near the emergence of Apicomplexan parasitism.

microbiology↗

bioRxiv2020

Doxycycline has Distinct Apicoplast-Specific Mechanisms of Antimalarial Activity

Doxycycline (DOX) is a key antimalarial drug thought to kill Plasmodium parasites by blocking protein translation in the essential apicoplast organelle. Clinical use is primarily limited to prophylaxis due to delayed second-cycle parasite death at 1-3 M serum concentrations. DOX concentrations >5 M kill parasites with first-cycle activity but have been ascribed to off-target mechanisms outside the apicoplast. We report that 10 M DOX blocks apicoplast biogenesis in the first cycle and is rescued by isopentenyl pyrophosphate, an essential apicoplast product, confirming an apicoplast-specific mechanism. Exogenous iron rescues parasites and apicoplast biogenesis from first-but not second-cycle effects of 10 M DOX, revealing that first-cycle activity involves a metal-dependent mechanism distinct from the delayed-death mechanism. These results critically expand the paradigm for understanding the fundamental antiparasitic mechanisms of DOX and suggest repurposing DOX as a faster-acting antimalarial at higher dosing whose multiple mechanisms would be expected to limit parasite resistance.

microbiology↗