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Biochemistry Nov 2022, the causative agent of malaria, belongs to the phylum Apicomplexa. Most apicomplexans, including , contain an essential nonphotosynthetic plastid called the apicoplast...
, the causative agent of malaria, belongs to the phylum Apicomplexa. Most apicomplexans, including , contain an essential nonphotosynthetic plastid called the apicoplast that harbors its own genome that is replicated by a dedicated organellar replisome. This replisome employs a single DNA polymerase (apPol), which is expected to perform both replicative and translesion synthesis. Unlike other replicative polymerases, no processivity factor for apPol has been identified. While preliminary structural and biochemical studies have provided an overall characterization of apPol, the kinetic mechanism of apPol's activity remains unknown. We have used transient state methods to determine the kinetics of replicative and translesion synthesis by apPol and show that apPol has low processivity and efficiency while copying undamaged DNA. Moreover, while apPol can bypass oxidatively damaged lesions, the bypass is error-prone. Taken together, our results raise the following question─how does a polymerase with low processivity, efficiency, and fidelity (for translesion synthesis) faithfully replicate the apicoplast organellar DNA within the hostile environment of the human host? We hypothesize that interactions with putative components of the apicoplast replisome and/or an as-yet-undiscovered processivity factor transform apPol into an efficient and accurate enzyme.
Topics: Humans; Apicoplasts; Plasmodium falciparum; DNA Replication; DNA-Directed DNA Polymerase; DNA
PubMed: 36251801
DOI: 10.1021/acs.biochem.2c00446 -
The Journal of Eukaryotic Microbiology Nov 2022Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. T. gondii shares... (Review)
Review
Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. T. gondii shares several characteristics with plants including a nonphotosynthetic plastid termed apicoplast and a multivesicular organelle that was named the plant-like vacuole (PLV) or vacuolar compartment (VAC). The name plant-like vacuole was selected based on its resemblance in composition and function to plant vacuoles. The name VAC represents its general vacuolar characteristics. We will refer to the organelle as PLVAC in this review. New findings in recent years have revealed that the PLVAC represents the lysosomal compartment of T. gondii which has adapted peculiarities to fulfill specific Toxoplasma needs. In this review, we discuss the composition and functions of the PLVAC highlighting its roles in ion storage and homeostasis, endocytosis, exocytosis, and autophagy.
Topics: Humans; Toxoplasma; Vacuoles; Protozoan Proteins; Apicoplasts; Plants
PubMed: 36218001
DOI: 10.1111/jeu.12951 -
Protein Expression and Purification Feb 2023Recombinant expression and purification of proteins have become a staple of modern drug discovery as it enables more precise in vitro analyses of drug targets, which may...
Recombinant expression and purification of proteins have become a staple of modern drug discovery as it enables more precise in vitro analyses of drug targets, which may help obtain biochemical and biophysical parameters of a known enzyme and even uncover unknown characteristics indicative of novel enzymatic functions. Such information is often necessary to prepare adequate screening assays and drug-discovery experiments in general. Toxoplasma gondii is an obligate protozoan parasite that is a member of the phylum Apicomplexa, can develop several neuro-degenerative symptoms and, in specific cases, certain death for human hosts. Its relict non-photosynthetic plastid, the apicoplast, harbours a unique de novo long-chain fatty acid synthesis pathway of a prokaryotic character, FASII. The FASII pathway shows plasticity and, is essential for many intracellular and membranal components, along with fatty acid uptake via salvaging from the host, therefore, its disruption causes parasite death. TgFabG, a FASII enzyme responsible for a single reduction step in the pathway, was recombinantly expressed, purified and biochemically and biophysically characterised in this study. The bioengineering hurdle of expressing the recombinant gene of a eukaryotic, signal peptide-containing protein in a prokaryotic system was overcome for the apicomplexan enzyme TgFabG, by truncating the N-terminal signal peptide. TgFabG was ultimately recombinantly produced in a plasmid expression vector from its 1131 base pair gene, purified as 260 and 272 amino acid proteins using a hexahistidine (6 × Histag) affinity chromatography and its biochemical (enzyme activity and kinetics) and biophysical characteristics were analysed in vitro.
Topics: Humans; Apicoplasts; Toxoplasma; Acyl Carrier Protein; Oxidoreductases; Fatty Acids; Protein Sorting Signals; Protozoan Proteins
PubMed: 36216219
DOI: 10.1016/j.pep.2022.106187 -
Microbiology Spectrum Oct 2022Toxoplasma gondii (T. gondii) is a pathogen belonging to the apicomplexan phylum, and it threatens human and animal health. Calcium ions, a critical second messenger in...
Toxoplasma gondii (T. gondii) is a pathogen belonging to the apicomplexan phylum, and it threatens human and animal health. Calcium ions, a critical second messenger in cells, can regulate important biological processes, including parasite invasion and egress. Calmodulin (CaM) is a small, highly conserved, Ca-binding protein found in all eukaryotic cells. After binding to Ca, CaM can be activated to interact with various proteins. However, little is known about CaM's function and its interacting proteins in T. gondii. In this study, we successfully knocked down CaM in the T. gondii parent strain TATI using a tetracycline-off system with the CaM promoter. The results indicated that CaM was required for tachyzoite proliferation, invasion, and egress and that CaM depletion resulted in apicoplast loss, thus threatening parasite survival in the next lytic cycle. In the tachyzoite stage, CaM loss caused significant anomalies in the parasite's basal constriction, motility, and parasite rosette-like arrangement in the parasitophorous vacuole (PV). These phenotypic defects caused by CaM depletion indicate the importance of CaM in T. gondii. Therefore, it is important to identify the CaM-interacting proteins in T. gondii. Applying BioID technology, more than 300 CaM's proximal interacting proteins were identified from T. gondii. These CaM partners were broadly distributed throughout the parasite. Furthermore, the protein interactome and transcriptome analyses indicated the potential role of CaM in ion binding, cation binding, metal ion binding, calcium ion binding, and oxidation-reduction. Our findings shed light on the CaM function and CaM-interactome in T. gondii and other eukaryotes. Toxoplasma gondii is an intracellular pathogen that threatens human and animal health. This unicellular parasite is active in many biological processes, such as egress and invasion. The implementation efficiency of T. gondii biological processes is dependent on signal transmission. Ca, as a second messenger, is essential for the parasite's life cycle. Calmodulin, a ubiquitous Ca receptor protein, is highly conserved and mediates numerous Ca-dependent events in eukaryotes. Few CaM functions or regulated partners have been characterized in T. gondii tachyzoites. Here, we reported the essential functions of calmodulin in T. gondii tachyzoite and the identification of its interacting partners using BioID technology, shedding light on the CaM function and CaM-interactome in Toxoplasma gondii and other eukaryotes.
Topics: Animals; Humans; Toxoplasma; Calmodulin; Parasites; Calcium; Technology; Tetracyclines; Cations; Protozoan Proteins
PubMed: 36214684
DOI: 10.1128/spectrum.01363-22 -
European Journal of Medicinal Chemistry Dec 2022Malaria is caused by the parasite Plasmodium falciparum, which contains an essential non-photosynthetic plastid called the apicoplast. A single DNA polymerase, apPOL, is...
Malaria is caused by the parasite Plasmodium falciparum, which contains an essential non-photosynthetic plastid called the apicoplast. A single DNA polymerase, apPOL, is targeted to the apicoplast, where it replicates and repairs the genome. apPOL has no direct orthologs in mammals and is considered a promising drug target for the treatment and/or prevention of malaria. We previously reported screening the Malaria Box to identify MMV666123 as an inhibitor of apPOL. Herein we extend our studies and report structure-activity relationships for MMV666123 and identify key structural motifs necessary for inhibition. Although attempts to crystallize apPOL with the inhibitor were not fruitful, kinetic analysis and crystal structure determinations of WT and mutant apo-enzymes, facilitated model building and provided insights into the putative inhibitor binding site. Our results validate apPOL as an antimalarial target and provide an avenue for the design of high potency, specific inhibitors of apPOL and other A-family DNA polymerases.
Topics: Animals; Apicoplasts; Plasmodium falciparum; Antimalarials; Kinetics; DNA-Directed DNA Polymerase; Malaria; Protozoan Proteins; Mammals
PubMed: 36191407
DOI: 10.1016/j.ejmech.2022.114751 -
Microbiology Spectrum Oct 2022Most commercial products cannot be used for clearance of contamination from cultures of apicomplexan parasites due to the parasites' dependence on the apicoplast, an...
Most commercial products cannot be used for clearance of contamination from cultures of apicomplexan parasites due to the parasites' dependence on the apicoplast, an essential organelle with DNA replication and translation machinery of cyanobacterial origin. The lone exception, mycoplasma removal agent (MRA), is relatively expensive, and some mycoplasma strains have shown resistance to clearance with MRA. Here, we report that the fluoroquinolone antibiotic sparfloxacin is a safe, effective, and inexpensive alternative for treatment of mycoplasma contamination in cultures of apicomplexan parasites. Sparfloxacin cleared both MRA-sensitive and MRA-resistant mycoplasma species from P. falciparum cultures at 1 and 4 μg/mL, respectively. We show that cultures of three different apicomplexan parasites can be maintained at concentrations of sparfloxacin required to clear mycoplasma without resulting in substantial deleterious effects on parasite growth. We also describe an alternative low-cost, in-house PCR assay for detecting mycoplasma. These findings will be useful to laboratories maintaining apicomplexan parasites , especially in low-resource environments, where the high cost of commercial products creates an economic barrier for detecting and eliminating mycoplasma from culture. These findings will be useful to laboratories maintaining apicomplexan parasites , especially in low-resource environments, where the high cost of commercial products creates an economic barrier for detecting and eliminating from culture.
Topics: Animals; Parasites; Mycoplasma; Fluoroquinolones; Anti-Bacterial Agents
PubMed: 36190416
DOI: 10.1128/spectrum.03497-22 -
Foodborne Pathogens and Disease Oct 2022infections are common in humans and animals worldwide. Ingestion of raw or undercooked meat containing tissue cysts of is one major source of transmission of this...
infections are common in humans and animals worldwide. Ingestion of raw or undercooked meat containing tissue cysts of is one major source of transmission of this parasite. It is important to guarantee the meat quality of China since our pork industry produces about half of the world's pork. In this study, a total of 746 pig samples were collected from Zhejiang and Jiangsu provinces in eastern China, and examined for infection by PCR amplification targeting gene. In this study, we found that 57 of 746 (7.6%) pigs were positive for gene, with 8.5% (48/562) in Zhejiang province and 4.9% (9/184) in Jiangsu province, respectively. The positive DNA samples were further genotyped at 11 genetic markers, including SAG1, 5'-and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico through PCR-restriction fragment length polymorphism (PCR-RFLP) technology. Two genotypes (ToxoDB 9 and ToxoDB 10) of were identified by PCR-RFLP in Zhejiang province. However, both genotypes were not determined from Jiangsu province, which is speculated on the low DNA concentration and the small number of samples. These results indicate that infection is endemic in pigs in eastern China and may raise public food safety concerns, suggesting more interventions for -related risks are needed in the future.
Topics: Humans; Swine; Animals; Toxoplasma; Toxoplasmosis, Animal; Genotype; Genetic Markers; Polymorphism, Restriction Fragment Length
PubMed: 36179062
DOI: 10.1089/fpd.2022.0036 -
Autophagy Apr 2023In apicomplexan parasites, the macroautophagy/autophagy machinery is repurposed to maintain the plastid-like organelle apicoplast. Previously, we showed that in and ,...
In apicomplexan parasites, the macroautophagy/autophagy machinery is repurposed to maintain the plastid-like organelle apicoplast. Previously, we showed that in and , ATG12 interacts with ATG5 in a non-covalent manner, in contrast to the covalent interaction in most organisms. However, it remained unknown whether apicomplexan parasites have functional orthologs of ATG16L1, a protein that is essential for the function of the covalent ATG12-ATG5 complex in other organisms. Furthermore, the mechanism used by the autophagy machinery to maintain the apicoplast is unclear. We report that the ATG12-ATG5-ATG16L complex exists in (Tg). This complex is localized on isolated structures at the periphery of the apicoplast dependent on TgATG16L. Inducible depletion of TgATG12, TgATG5, or TgATG16L caused loss of the apicoplast and affected parasite growth. We found that a putative soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein, synaptosomal-associated protein 29 (TgSNAP29, Qbc SNARE), is required to maintain the apicoplast in . TgSNAP29 depletion disrupted TgATG8 localization at the apicoplast. Additionally, we identified a putative ubiquitin-interacting motif-docking site (UDS) of TgATG8. Mutation of the UDS site abolished TgATG8 localization on the apicoplast but not lipidation. These findings suggest that the TgATG12-TgATG5-TgATG16L complex is required for biogenesis of the apicoplast, in which TgATG8 is translocated to the apicoplast via vesicles in a SNARE -dependent manner in .: AID: auxin-inducible degron; CCD: coiled-coil domain; HFF: human foreskin fibroblast; IAA: indole-3-acetic acid; LAP: LC3-associated phagocytosis; NAA: 1-naphthaleneacetic acid; PtdIns3P: phosphatidylinositol-3-phosphate; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; UDS: ubiquitin-interacting motif-docking site; UIM: ubiquitin-interacting motif.
Topics: Animals; Humans; Toxoplasma; Apicoplasts; Ethylmaleimide; Autophagy; Parasites; Ubiquitins; Protozoan Proteins; Autophagy-Related Protein 12; Qb-SNARE Proteins; Qc-SNARE Proteins; Autophagy-Related Protein 5
PubMed: 36095096
DOI: 10.1080/15548627.2022.2123639 -
The Journal of Eukaryotic Microbiology Nov 2022Malaria parasites are diheteroxenous, requiring two hosts-a vertebrate and a mosquito-to complete their life cycle. Mosquitoes are the definitive host where malaria... (Review)
Review
Malaria parasites are diheteroxenous, requiring two hosts-a vertebrate and a mosquito-to complete their life cycle. Mosquitoes are the definitive host where malaria parasite sex occurs, and vertebrates are the intermediate host, supporting asexual amplification and more significant geographic spread. In this review, we examine the roles of a single malaria parasite compartment, the relict plastid known as the apicoplast, at each life cycle stage. We focus mainly on two malaria parasite species-Plasmodium falciparum and P. berghei-comparing the changing, yet ever crucial, roles of their apicoplasts.
Topics: Humans; Animals; Apicoplasts; Parasites; Rodentia; Plasmodium falciparum; Life Cycle Stages; Malaria; Protozoan Proteins
PubMed: 36070203
DOI: 10.1111/jeu.12947 -
Frontiers in Cellular and Infection... 2022Toxoplasmosis caused by the protozoan is one of the most common parasitic diseases in humans and almost all warm-blooded animals. Lys, Glu, and Gln-specific tRNAs...
Toxoplasmosis caused by the protozoan is one of the most common parasitic diseases in humans and almost all warm-blooded animals. Lys, Glu, and Gln-specific tRNAs contain a super-modified 2-thiourea (sU) derivatives at the position 34, which is essential for all living organisms by maintaining the structural stability and aminoacylation of tRNA, and the precision and efficiency of codon recognition during protein translation. However, the enzyme(s) involved in this modification in remains elusive. In this report, three putative tRNA-specific 2-thiolation enzymes were identified, of which two were involved in the sU34 modification of tRNA, tRNA, and tRNA. One was named MnmA, an apicoplast-located tRNA-specific 2-thiolation enzyme in . Knockout of showed that this enzyme is important for the lytic cycle of tachyzoites. Loss of MnmA also led to abnormities in apicoplast biogenesis and severely disturbed apicoplast genomic transcription. Notably, mice survived from the infection with 10 MnmA-KO RH tachyzoites. These findings provide new insights into sU34 tRNA modification in Apicomplexa, and suggest MnmA, the first apicoplast tRNA thiouridylase identified in all apicomplexans, as a potential drug target.
Topics: Animals; Apicoplasts; Humans; Mice; Mice, Knockout; Protozoan Proteins; RNA, Transfer; Toxoplasma; Toxoplasmosis
PubMed: 36046743
DOI: 10.3389/fcimb.2022.947039