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Nucleic Acids Research Jun 2024The human malaria parasite Plasmodium falciparum genome is among the most A + T rich, with low complexity regions (LCRs) inserted in coding sequences including those...
The human malaria parasite Plasmodium falciparum genome is among the most A + T rich, with low complexity regions (LCRs) inserted in coding sequences including those for proteins targeted to its essential relict plastid (apicoplast). Replication of the apicoplast genome (plDNA), mediated by the atypical multifunctional DNA polymerase PfPrex, would require additional enzymatic functions for lagging strand processing. We identified an apicoplast-targeted, [4Fe-4S]-containing, FEN/Exo (PfExo) with a long LCR insertion and detected its interaction with PfPrex. Distinct from other known exonucleases across organisms, PfExo recognized a wide substrate range; it hydrolyzed 5'-flaps, processed dsDNA as a 5'-3' exonuclease, and was a bipolar nuclease on ssDNA and RNA-DNA hybrids. Comparison with the rodent P. berghei ortholog PbExo, which lacked the insertion and [4Fe-4S], revealed interspecies functional differences. The insertion-deleted PfExoΔins behaved like PbExo with a limited substrate repertoire because of compromised DNA binding. Introduction of the PfExo insertion into PbExo led to gain of activities that the latter initially lacked. Knockout of PbExo indicated essentiality of the enzyme for survival. Our results demonstrate the presence of a novel apicoplast exonuclease with a functional LCR that diversifies substrate recognition, and identify it as the candidate flap-endonuclease and RNaseH required for plDNA replication and maintenance.
PubMed: 38888125
DOI: 10.1093/nar/gkae512 -
Proceedings of the National Academy of... Jun 2024Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its interorganelle shuttling...
Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its interorganelle shuttling is crucial for cell fitness. Many apicomplexan parasites harbor a unique organelle called the apicoplast that houses metabolic pathways like fatty acid and isoprenoid precursor biosyntheses, requiring pyruvate as a substrate. However, how pyruvate is supplied in the apicoplast remains enigmatic. Here, deploying the zoonotic parasite as a model apicomplexan, we identified two proteins residing in the apicoplast membranes that together constitute a functional apicoplast pyruvate carrier (APC) to mediate the import of cytosolic pyruvate. Depletion of APC results in reduced activities of metabolic pathways in the apicoplast and impaired integrity of this organelle, leading to parasite growth arrest. APC is a pyruvate transporter in diverse apicomplexan parasites, suggesting a common strategy for pyruvate acquisition by the apicoplast in these clinically relevant intracellular pathogens.
Topics: Apicoplasts; Toxoplasma; Pyruvic Acid; Protozoan Proteins; Animals; Membrane Transport Proteins; Biological Transport; Metabolic Networks and Pathways
PubMed: 38865262
DOI: 10.1073/pnas.2314314121 -
BioRxiv : the Preprint Server For... May 2024Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection,...
Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, 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 Å-resolution X-ray structure that revealed a highly α-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 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.
PubMed: 38853871
DOI: 10.1101/2024.05.30.596652 -
Advances in Microbial Physiology 2024Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the... (Review)
Review
Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the diversity and function of protistan organelles has increased in recent years, providing a complex panoply of structure/function relationships. This is particularly noticeable with the description of hydrogenosomes, and the diverse array of structures that followed, having hybrid hydrogenosome/mitochondria attributes. These diverse organelles have lost the major, at one time, definitive components of the mitochondrion (tricarboxylic cycle enzymes and cytochromes), however they all contain the machinery for the assembly of Fe-S clusters, which is the single unifying feature they share. The plasticity of organelles, like the mitochondrion, is therefore evident from its ability to lose its identity as an aerobic energy generating powerhouse while retaining key ancestral functions common to both aerobes and anaerobes. It is interesting to note that the apicoplast, a non-photosynthetic plastid that is present in all apicomplexan protozoa, apart from Cryptosporidium and possibly the gregarines, is also the site of Fe-S cluster assembly proteins. It turns out that in Cryptosporidium proteins involved in Fe-S cluster biosynthesis are localized in the mitochondrial remnant organelle termed the mitosome. Hence, different organisms have solved the same problem of packaging a life-requiring set of reactions in different ways, using different ancestral organelles, discarding what is not needed and keeping what is essential. Don't judge an organelle by its cover, more by the things it does, and always be prepared for surprises.
Topics: Organelles; Mitochondria; Eukaryota; Iron-Sulfur Proteins
PubMed: 38821633
DOI: 10.1016/bs.ampbs.2024.02.001 -
Current Biology : CB Jun 2024Apicomplexans are obligate intracellular parasites that have evolved from a free-living, phototrophic ancestor. They have been reported from marine environmental samples...
Apicomplexans are obligate intracellular parasites that have evolved from a free-living, phototrophic ancestor. They have been reported from marine environmental samples in high numbers, with several clades of apicomplexan-related lineages (ARLs) having been described from environmental sequencing data (16S rRNA gene metabarcoding). The most notable of these are the corallicolids (previously ARL-V), which possess chlorophyll-biosynthesis genes in their relic chloroplast (apicoplast) and are geographically widespread and abundant symbionts of anthozoans. Corallicolids are related to the Eimeriorina, a suborder of apicomplexan coccidians that include other notable members such as Toxoplasma gondii.Ophioblennius macclurei, the redlip blenny, along with other tropical reef fishes, is known to be infected by Haemogregarina-like and Haemohormidium-like parasites supposedly belonging to the Adeleorina; however, phylogenetics shows that these parasites are instead related to the Eimeriorina. Hybrid genomic sequencing of apicomplexan-infected O. macclurei blood recovered the entire rRNA operon of this apicomplexan parasite along with the complete mitochondrion and apicoplast genomes. Phylogenetic analyses using this new genomic information consistently place these fish-infecting apicomplexans, hereby informally named ichthyocolids, sister to the corallicolids within Coccidia. The apicoplast genome did not contain chlorophyll biosynthesis genes, providing evidence for another independent loss of this pathway within Apicomplexa. Based on the 16S rRNA gene found in the apicoplast, this group corresponds to the previously described ARL-VI. Screening of fish microbiome studies using the plastid 16S rRNA gene shows these parasites to be geographically and taxonomically widespread in fish species across the globe with implications for commercial fisheries and oceanic food webs.
Topics: Animals; Apicomplexa; Phylogeny; Fishes; Fish Diseases
PubMed: 38821048
DOI: 10.1016/j.cub.2024.04.084 -
BioRxiv : the Preprint Server For... Jun 2024Plasmodium falciparum malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, P. falciparum...
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.
PubMed: 38798484
DOI: 10.1101/2024.05.10.587216 -
Advances in Pharmacological and... 2024The discovery of a relict plastid, also known as an apicoplast (apicomplexan plastid), that houses housekeeping processes and metabolic pathways critical to parasites'... (Review)
Review
The discovery of a relict plastid, also known as an apicoplast (apicomplexan plastid), that houses housekeeping processes and metabolic pathways critical to parasites' survival has prompted increased research on identifying potent inhibitors that can impinge on apicoplast-localised processes. The apicoplast is absent in humans, yet it is proposed to originate from the eukaryote's secondary endosymbiosis of a primary symbiont. This symbiotic relationship provides a favourable microenvironment for metabolic processes such as haem biosynthesis, Fe-S cluster synthesis, isoprenoid biosynthesis, fatty acid synthesis, and housekeeping processes such as DNA replication, transcription, and translation, distinct from analogous mammalian processes. Recent advancements in comprehending the biology of the apicoplast reveal it as a vulnerable organelle for malaria parasites, offering numerous potential targets for effective antimalarial therapies. We provide an overview of the metabolic processes occurring in the apicoplast and discuss the organelle as a viable antimalarial target in light of current advances in drug discovery. We further highlighted the relevance of these metabolic processes to during the different stages of the lifecycle.
PubMed: 38765186
DOI: 10.1155/2024/9940468 -
Scientific Reports May 2024Plasmodium falciparum, the causative agent of malaria, poses a significant global health challenge, yet much of its biology remains elusive. A third of the genes in the...
Plasmodium falciparum, the causative agent of malaria, poses a significant global health challenge, yet much of its biology remains elusive. A third of the genes in the P. falciparum genome lack annotations regarding their function, impeding our understanding of the parasite's biology. In this study, we employ structure predictions and the DALI search algorithm to analyse proteins encoded by uncharacterized genes in the reference strain 3D7 of P. falciparum. By comparing AlphaFold predictions to experimentally determined protein structures in the Protein Data Bank, we found similarities to known domains in 353 proteins of unknown function, shedding light on their potential functions. The lowest-scoring 5% of similarities were additionally validated using the size-independent TM-align algorithm, confirming the detected similarities in 88% of the cases. Notably, in over 70 P. falciparum proteins the presence of domains resembling heptatricopeptide repeats, which are typically involvement in RNA binding and processing, was detected. This suggests this family, which is important in transcription in mitochondria and apicoplasts, is much larger in Plasmodium parasites than previously thought. The results of this domain search provide a resource to the malaria research community that is expected to inform and enable experimental studies.
Topics: Plasmodium falciparum; Protozoan Proteins; Algorithms; Protein Domains; Databases, Protein; Models, Molecular
PubMed: 38719885
DOI: 10.1038/s41598-024-60058-x -
Biology Mar 2024DNA polymerases replicate cellular genomes and/or participate in the maintenance of genome integrity. DNA polymerases sharing high sequence homology with DNA polymerase... (Review)
Review
DNA polymerases replicate cellular genomes and/or participate in the maintenance of genome integrity. DNA polymerases sharing high sequence homology with DNA polymerase I (pol I) have been grouped in Family A. Pol I participates in Okazaki fragment maturation and in bacterial genome repair. Since its discovery in 1956, pol I has been extensively studied, primarily to gain deeper insights into the mechanism of DNA replication. As research on DNA polymerases advances, many novel functions of this group of polymerases are being uncovered. For example, human DNA polymerase θ (a Family A DNA pol) has been shown to synthesize DNA using RNA as a template, a function typically attributed to retroviral reverse transcriptase. Increased interest in drug discovery against pol θ has emerged due to its roles in cancer. Likewise, Pol I family enzymes also appear attractive as drug-development targets against microbial infections. Development of antimalarial compounds targeting apicoplast apPOL, an ortholog of Pol I, further extends the targeting of this family of enzymes. Here, we summarize reported drug-development efforts against Family A polymerases and future perspective regarding these enzymes as antibiotic targets. Recently developed techniques, such as artificial intelligence, can be used to facilitate the development of new drugs.
PubMed: 38666816
DOI: 10.3390/biology13040204 -
Microbiology Resource Announcements Jun 2024We are reporting a genome containing four nuclear chromosomes, a mitochondrial genome, and an apicoplast from reference isolate NVSL348. This report includes a gapless...
We are reporting a genome containing four nuclear chromosomes, a mitochondrial genome, and an apicoplast from reference isolate NVSL348. This report includes a gapless assembly consisting of all six genetic molecules.
PubMed: 38651914
DOI: 10.1128/mra.00039-24