1,109 results on '"Protozoan Proteins physiology"'
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2. Essential role of a Plasmodium berghei heat shock protein (PBANKA_0938300) in gametocyte development.
- Author
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Kashif M, Quadiri A, and Singh AP
- Subjects
- Animals, Female, Heat-Shock Proteins genetics, Hot Temperature, Life Cycle Stages, Male, Gametogenesis physiology, Heat-Shock Proteins physiology, Plasmodium berghei physiology, Protozoan Proteins physiology
- Abstract
The continued existence of Plasmodium parasites in physiologically distinct environments during their transmission in mosquitoes and vertebrate hosts requires effector proteins encoded by parasite genes to provide adaptability. Parasites utilize their robust stress response system involving heat shock proteins for their survival. Molecular chaperones are involved in maintaining protein homeostasis within a cell during stress, protein biogenesis and the formation of protein complexes. Due to their critical role in parasite virulence, they are considered targets for therapeutic interventions. Our results identified a putative P. berghei heat shock protein (HSP) belonging to the HSP40 family (HspJ62), which is abundantly induced upon heat stress and expressed during all parasite stages. To determine the role HspJ62, a gene-disrupted P. berghei transgenic line was developed (ΔHspJ62), which resulted in disruption of gametocyte formation. Such parasites were unable to form subsequent sexual stages because of disrupted gametogenesis, indicating the essential role of HspJ62 in gametocyte formation. Transcriptomic analysis of the transgenic line showed downregulation of a number of genes, most of which were specific to male or female gametocytes. The transcription factor ApiAP2 was also downregulated in ΔHspJ62 parasites. Our findings suggest that the downregulation of ApiAP2 likely disrupts the transcriptional regulation of sexual stage genes, leading to impaired gametogenesis. This finding also highlights the critical role that HspJ62 indirectly plays in the development of P. berghei sexual stages and in facilitating the conversion from the asexual blood stage to the sexual stage. This study characterizes the HspJ62 protein as a fertility factor because parasites lacking it are unable to transmit to mosquitoes. This study adds an important contribution to ongoing research aimed at understanding gametocyte differentiation and formation in parasites. The molecule adds to the list of potential drug targets that can be targeted to inhibit parasite sexual development and consequently parasite transmission., (© 2021. The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
3. Plasmodium berghei -Released Factor, Pb TIP, Modulates the Host Innate Immune Responses.
- Author
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Kalia I, Anand R, Quadiri A, Bhattacharya S, Sahoo B, and Singh AP
- Subjects
- Amino Acid Sequence, Animals, Conserved Sequence, Cytokines biosynthesis, Cytokines genetics, Erythrocyte Membrane chemistry, Erythrocytes parasitology, Humans, Lipopolysaccharides pharmacology, Macrophages metabolism, Malaria parasitology, Mice, Mice, Inbred C57BL, Molecular Mimicry, Peptide Fragments blood, Peptide Fragments immunology, Protozoan Proteins immunology, RAW 264.7 Cells, Recombinant Proteins pharmacology, Sequence Alignment, Sequence Homology, Amino Acid, Transcriptome, Host-Pathogen Interactions immunology, Immune Evasion immunology, Immunity, Innate, Macrophages parasitology, Malaria immunology, Plasmodium berghei immunology, Protozoan Proteins physiology
- Abstract
The Plasmodium parasite has to cross various immunological barriers for successful infection. Parasites have evolved mechanisms to evade host immune responses, which hugely contributes to the successful infection and transmission by parasites. One way in which a parasite evades immune surveillance is by expressing molecular mimics of the host molecules in order to manipulate the host responses. In this study, we report a Plasmodium berghei hypothetical protein, Pb TIP (PbANKA_124360.0), which is a Plasmodium homolog of the human T-cell immunomodulatory protein (TIP). The latter possesses immunomodulatory activities and suppressed the host immune responses in a mouse acute graft- versus -host disease (GvHD) model. The Plasmodium berghei protein, Pb TIP, is expressed on the merozoite surface and exported to the host erythrocyte surface upon infection. It is shed in the blood circulation by the activity of an uncharacterized membrane protease(s). The shed Pb TIP could be detected in the host serum during infection. Our results demonstrate that the shed Pb TIP exhibits binding on the surface of macrophages and reduces their inflammatory cytokine response while upregulating the anti-inflammatory cytokines such as TGF-β and IL-10. Such manipulated immune responses are observed in the later stage of malaria infection. Pb TIP induced Th2-type gene transcript changes in macrophages, hinting toward its potential to regulate the host immune responses against the parasite. Therefore, this study highlights the role of a Plasmodium -released protein, Pb TIP, in immune evasion using macrophages, which may represent the critical strategy of the parasite to successfully survive and thrive in its host. This study also indicates the human malaria parasite TIP as a potential diagnostic molecule that could be exploited in lateral flow-based immunochromatographic tests for malaria disease diagnosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kalia, Anand, Quadiri, Bhattacharya, Sahoo and Singh.)
- Published
- 2021
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4. The enteric pathogen Cryptosporidium parvum exports proteins into the cytosol of the infected host cell.
- Author
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Dumaine JE, Sateriale A, Gibson AR, Reddy AG, Gullicksrud JA, Hunter EN, Clark JT, and Striepen B
- Subjects
- Animals, Mice, Cryptosporidiosis parasitology, Cryptosporidium parvum physiology, Cytosol parasitology, Host-Parasite Interactions, Protozoan Proteins physiology
- Abstract
The parasite Cryptosporidium is responsible for diarrheal disease in young children causing death, malnutrition, and growth delay. Cryptosporidium invades enterocytes where it develops in a unique intracellular niche. Infected cells exhibit profound changes in morphology, physiology, and transcriptional activity. How the parasite effects these changes is poorly understood. We explored the localization of highly polymorphic proteins and found members of the Cryptosporidium parvum MEDLE protein family to be translocated into the cytosol of infected cells. All intracellular life stages engage in this export, which occurs after completion of invasion. Mutational studies defined an N-terminal host-targeting motif and demonstrated proteolytic processing at a specific leucine residue. Direct expression of MEDLE2 in mammalian cells triggered an ER stress response, which was also observed during infection. Taken together, our studies reveal the presence of a Cryptosporidium secretion system capable of delivering parasite proteins into the infected enterocyte., Competing Interests: JD, AS, AG, AR, JG, EH, JC, BS No competing interests declared, (© 2021, Dumaine et al.)
- Published
- 2021
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5. Rhoptry kinase protein 39 (ROP39) is a novel factor that recruits host mitochondria to the parasitophorous vacuole of Toxoplasma gondii.
- Author
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Fukumoto J, Sakura T, Matsubara R, Tahara M, Matsuzaki M, and Nagamune K
- Subjects
- Host-Parasite Interactions, Humans, Fibroblasts parasitology, Mitochondria parasitology, Protein Kinases physiology, Protozoan Proteins physiology, Toxoplasma physiology, Vacuoles parasitology
- Abstract
Most intracellular pathogens replicate in a vacuole to avoid the defense system of the host. A few pathogens recruit host mitochondria around those vacuoles, but the molecules responsible for mitochondrial recruitment remain unidentified. It is only in the apicomplexan parasite Toxoplasma gondii, that mitochondrial association factor 1b (MAF1b) has been identified as an association factor for host mitochondria. Here, we show that rhoptry kinase family protein 39 (ROP39) induces host mitochondrial recruitment in T. gondii. We found that the abundance of ROP39 was increased on host mitochondria extracted from human foreskin fibroblasts (HFFs) infected with T. gondii. ROP39 expressed exogenously in HFFs localized on host mitochondria, indicating that it has the potential to bind to host mitochondria without assistance from other parasite factors. Confocal microscopy revealed that ROP39 colocalized with host mitochondria on the membrane of parasitophorous vacuoles, in which the parasites reside. Moreover, we observed about a 10% reduction in the level of mitochondrial association in rop39-knockout parasites compared with a parental strain., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)
- Published
- 2021
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6. Native structure of the RhopH complex, a key determinant of malaria parasite nutrient acquisition.
- Author
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Ho CM, Jih J, Lai M, Li X, Goldberg DE, Beck JR, and Zhou ZH
- Subjects
- Cell Membrane Permeability, Cryoelectron Microscopy, Erythrocyte Membrane parasitology, Humans, Models, Molecular, Nutrients metabolism, Protein Conformation, Proteomics, Protozoan Proteins physiology, Protozoan Proteins ultrastructure, Structure-Activity Relationship, Erythrocyte Membrane metabolism, Plasmodium falciparum metabolism, Protozoan Proteins chemistry
- Abstract
The RhopH complex is implicated in malaria parasites' ability to invade and create new permeability pathways in host erythrocytes, but its mechanisms remain poorly understood. Here, we enrich the endogenous RhopH complex in a native soluble form, comprising RhopH2, CLAG3.1, and RhopH3, directly from parasite cell lysates and determine its atomic structure using cryo-electron microscopy (cryo-EM), mass spectrometry, and the cryoID program. CLAG3.1 is positioned between RhopH2 and RhopH3, which both share substantial binding interfaces with CLAG3.1 but make minimal contacts with each other. The forces stabilizing individual subunits include 13 intramolecular disulfide bonds. Notably, CLAG3.1 residues 1210 to 1223, previously predicted to constitute a transmembrane helix, are embedded within a helical bundle formed by residues 979 to 1289 near the C terminus of CLAG3.1. Buried in the core of the RhopH complex and largely shielded from solvent, insertion of this putative transmembrane helix into the erythrocyte membrane would likely require a large conformational rearrangement. Given the unusually high disulfide content of the complex, it is possible that such a rearrangement could be initiated by the breakage of allosteric disulfide bonds, potentially triggered by interactions at the erythrocyte membrane. This first direct observation of an exported Plasmodium falciparum transmembrane protein-in a soluble, trafficking state and with atomic details of buried putative membrane-insertion helices-offers insights into the assembly and trafficking of RhopH and other parasite-derived complexes to the erythrocyte membrane. Our study demonstrates the potential the endogenous structural proteomics approach holds for elucidating the molecular mechanisms of hard-to-isolate complexes in their native, functional forms., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)
- Published
- 2021
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7. Identification of a protein unique to the genus Plasmodium that contains a WD40 repeat domain and extensive low-complexity sequence.
- Author
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Cortés GT, Beltran MMG, Gómez-Alegría CJ, and Wiser MF
- Subjects
- Amino Acid Sequence, Animals, Birds, Cloning, Molecular, Epitopes chemistry, Gene Expression Regulation, Models, Chemical, Parasites metabolism, Peptide Hydrolases chemistry, Plasmodium classification, Proteins, Protozoan Proteins chemistry, Protozoan Proteins genetics, Protozoan Proteins physiology, Two-Hybrid System Techniques, Plasmodium genetics, Protozoan Proteins isolation & purification, WD40 Repeats
- Abstract
Proteins containing WD40 domains play important roles in the formation of multiprotein complexes. Little is known about WD40 proteins in the malaria parasite. This report contains the initial description of a WD40 protein that is unique to the genus Plasmodium and possibly closely related genera. The N-terminal portion of this protein consists of seven WD40 repeats that are highly conserved in all Plasmodium species. Following the N-terminal region is a central region that is conserved within the major Plasmodium clades, such as parasites of great apes, monkeys, rodents, and birds, but partially conserved across all Plasmodium species. This central region contains extensive low-complexity sequence and is predicted to have a disordered structure. Proteins with disordered structure generally function in molecular interactions. The C-terminal region is semi-conserved across all Plasmodium species and has no notable features. This WD40 repeat protein likely functions in some aspect of parasite biology that is unique to Plasmodium and this uniqueness makes the protein a possible target for therapeutic intervention.
- Published
- 2021
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8. Plasmodium falciparum phenotypic and genotypic resistance profile during the emergence of Piperaquine resistance in Northeastern Thailand.
- Author
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Boonyalai N, Thamnurak C, Sai-Ngam P, Ta-Aksorn W, Arsanok M, Uthaimongkol N, Sundrakes S, Chattrakarn S, Chaisatit C, Praditpol C, Fagnark W, Kirativanich K, Chaorattanakawee S, Vanachayangkul P, Lertsethtakarn P, Gosi P, Utainnam D, Rodkvamtook W, Kuntawunginn W, Vesely BA, Spring MD, Fukuda MM, Lanteri C, Walsh D, Saunders DL, Smith PL, Wojnarski M, Sirisopana N, Waters NC, Jongsakul K, and Gaywee J
- Subjects
- Adolescent, Adult, Aged, Antimalarials administration & dosage, Antimalarials therapeutic use, Artemisinins administration & dosage, Artemisinins therapeutic use, DNA Copy Number Variations, DNA, Protozoan genetics, Drug Therapy, Combination, Endemic Diseases, Female, Genetic Association Studies, Genotype, Haplotypes genetics, Humans, Malaria, Falciparum epidemiology, Male, Middle Aged, Parasitemia drug therapy, Parasitemia epidemiology, Plasmodium falciparum genetics, Plasmodium falciparum growth & development, Plasmodium falciparum isolation & purification, Protozoan Proteins genetics, Protozoan Proteins physiology, Quinolines administration & dosage, Quinolines therapeutic use, Thailand epidemiology, Young Adult, Antimalarials pharmacology, Drug Resistance genetics, Malaria, Falciparum drug therapy, Plasmodium falciparum drug effects, Quinolines pharmacology
- Abstract
Malaria remains a public health problem in Thailand, especially along its borders where highly mobile populations can contribute to persistent transmission. This study aimed to determine resistant genotypes and phenotypes of 112 Plasmodium falciparum isolates from patients along the Thai-Cambodia border during 2013-2015. The majority of parasites harbored a pfmdr1-Y184F mutation. A single pfmdr1 copy number had CVIET haplotype of amino acids 72-76 of pfcrt and no pfcytb mutations. All isolates had a single pfk13 point mutation (R539T, R539I, or C580Y), and increased % survival in the ring-stage survival assay (except for R539I). Multiple copies of pfpm2 and pfcrt-F145I were detected in 2014 (12.8%) and increased to 30.4% in 2015. Parasites containing either multiple pfpm2 copies with and without pfcrt-F145I or a single pfpm2 copy with pfcrt-F145I exhibited elevated IC
90 values of piperaquine. Collectively, the emergence of these resistance patterns in Thailand near Cambodia border mirrored the reports of dihydroartemisinin-piperaquine treatment failures in the adjacent province of Cambodia, Oddar Meanchey, suggesting a migration of parasites across the border. As malaria elimination efforts ramp up in Southeast Asia, host nations militaries and other groups in border regions need to coordinate the proposed interventions.- Published
- 2021
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9. Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila.
- Author
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Nabeel-Shah S, Garg J, Saettone A, Ashraf K, Lee H, Wahab S, Ahmed N, Fine J, Derynck J, Pu S, Ponce M, Marcon E, Zhang Z, Greenblatt JF, Pearlman RE, Lambert JP, and Fillingham J
- Subjects
- Amino Acid Sequence, Bacterial Proteins metabolism, Biological Evolution, Conserved Sequence, DNA metabolism, DNA-Binding Proteins metabolism, Epigenesis, Genetic, Gene Expression, HEK293 Cells, Histone Chaperones chemistry, Histone Chaperones physiology, Histones metabolism, Humans, Neoplasms metabolism, Neoplasms mortality, Oncogenes, Protozoan Proteins chemistry, Protozoan Proteins physiology, Retinoblastoma-Binding Protein 4 metabolism, Retinoblastoma-Binding Protein 7 metabolism, Tetrahymena thermophila genetics, Tetrahymena thermophila growth & development, Histone Chaperones metabolism, Protozoan Proteins metabolism, Tetrahymena thermophila metabolism
- Abstract
Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2021
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10. Trypanosome RNAEditing Substrate Binding Complex integrity and function depends on the upstream action of RESC10.
- Author
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Dubey AP, Tylec BL, McAdams NM, Sortino K, and Read LK
- Subjects
- RNA, Guide, Kinetoplastida metabolism, RNA, Messenger chemistry, RNA, Mitochondrial chemistry, RNA-Binding Proteins metabolism, Trypanosoma brucei brucei growth & development, Uridine metabolism, Protozoan Proteins physiology, RNA Editing, RNA, Messenger metabolism, RNA, Mitochondrial metabolism, RNA-Binding Proteins physiology, Trypanosoma brucei brucei genetics
- Abstract
Uridine insertion/deletion editing of mitochondrial mRNAs is a characteristic feature of kinetoplastids, including Trypanosoma brucei. Editing is directed by trans-acting gRNAs and catalyzed by related RNA Editing Core Complexes (RECCs). The non-catalytic RNA Editing Substrate Binding Complex (RESC) coordinates interactions between RECC, gRNA and mRNA. RESC is a dynamic complex comprising GRBC (Guide RNA Binding Complex) and heterogeneous REMCs (RNA Editing Mediator Complexes). Here, we show that RESC10 is an essential, low abundance, RNA binding protein that exhibits RNase-sensitive and RNase-insensitive interactions with RESC proteins, albeit its minimal in vivo interaction with RESC13. RESC10 RNAi causes extensive RESC disorganization, including disruption of intra-GRBC protein-protein interactions, as well as mRNA depletion from GRBC and accumulation on REMCs. Analysis of mitochondrial RNAs at single nucleotide resolution reveals transcript-specific effects: RESC10 dramatically impacts editing progression in pan-edited RPS12 mRNA, but is critical for editing initiation in mRNAs with internally initiating gRNAs, pointing to distinct initiation mechanisms for these RNA classes. Correlations between sites at which editing pauses in RESC10 depleted cells and those in knockdowns of previously studied RESC proteins suggest that RESC10 acts upstream of these factors and that RESC is particularly important in promoting transitions between uridine insertion and deletion RECCs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2021
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11. Metalloaminopeptidases of the Protozoan Parasite Plasmodium falciparum as Targets for the Discovery of Novel Antimalarial Drugs.
- Author
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Mills B, Isaac RE, and Foster R
- Subjects
- Amino Acid Sequence, Aminopeptidases chemistry, Aminopeptidases physiology, Animals, Antimalarials chemistry, Catalytic Domain, Cell Line, Drug Discovery, Humans, Parasitic Sensitivity Tests, Plasmodium falciparum enzymology, Protozoan Proteins chemistry, Protozoan Proteins physiology, Aminopeptidases antagonists & inhibitors, Antimalarials pharmacology, Plasmodium falciparum drug effects, Protozoan Proteins antagonists & inhibitors
- Abstract
Malaria poses a significant threat to approximately half of the world's population with an annual death toll close to half a million. The emergence of resistance to front-line antimalarials in the most lethal human parasite species, Plasmodium falciparum ( Pf ), threatens progress made in malaria control. The prospect of losing the efficacy of antimalarial drugs is driving the search for small molecules with new modes of action. Asexual reproduction of the parasite is critically dependent on the recycling of amino acids through catabolism of hemoglobin (Hb), which makes metalloaminopeptidases (MAPs) attractive targets for the development of new drugs. The Pf genome encodes eight MAPs, some of which have been found to be essential for parasite survival. In this article, we discuss the biological structure and function of each MAP within the Pf genome, along with the drug discovery efforts that have been undertaken to identify novel antimalarial candidates of therapeutic value.
- Published
- 2021
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12. The MRN complex promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes.
- Author
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Mehnert AK, Prorocic M, Dujeancourt-Henry A, Hutchinson S, McCulloch R, and Glover L
- Subjects
- DNA Breaks, Double-Stranded, Trypanosoma brucei brucei immunology, Antigenic Variation, DNA-Binding Proteins physiology, MRE11 Homologue Protein physiology, Protozoan Proteins physiology, Recombinational DNA Repair, Trypanosoma brucei brucei genetics
- Abstract
Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 and MRE11 are required for RAD51-dependent homologous recombination and phosphorylation of histone H2A following a DNA double strand break (DSB), but neither MRE11 nor RAD50 substantially influence DSB resection at a chromosome-internal locus. In addition, we reveal intrinsic separation-of-function between T. brucei RAD50 and MRE11, with only RAD50 suppressing DSB repair using donors with short stretches of homology at a subtelomeric locus, and only MRE11 directing DSB resection at the same locus. Finally, we show that loss of either MRE11 or RAD50 causes a greater diversity of expressed VSG variants following DSB repair. We conclude that MRN promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2021
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13. Plasmodium falciparum replication factor C subunit 1 is involved in genotoxic stress response.
- Author
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Sheriff O, Yaw A, Lai SK, Loo HL, Sze SK, and Preiser PR
- Subjects
- Artesunate pharmacology, Cell Death, Chloroquine pharmacology, DNA Repair, DNA Replication, DNA, Protozoan, Erythrocytes parasitology, Gene Expression Regulation, Host-Parasite Interactions, Humans, Malaria, Falciparum drug therapy, Malaria, Falciparum parasitology, Protozoan Proteins physiology, Reactive Oxygen Species metabolism, Antimalarials pharmacology, DNA Damage, Plasmodium falciparum drug effects, Plasmodium falciparum physiology, Replication Protein C physiology
- Abstract
About half the world's population is at risk of malaria, with Plasmodium falciparum malaria being responsible for the most malaria related deaths globally. Antimalarial drugs such as chloroquine and artemisinin are directed towards the proliferating intra-erythrocytic stages of the parasite, which is responsible for all the clinical symptoms of the disease. These antimalarial drugs have been reported to function via multiple pathways, one of which induces DNA damage via the generation of free radicals and reactive oxygen species. An urgent need to understand the mechanistic details of drug response and resistance is highlighted by the decreasing clinical efficacy of the front line drug, Artemisinin. The replication factor C subunit 1 is an important component of the DNA replication machinery and DNA damage response mechanism. Here we show the translocation of PfRFC1 from an intranuclear localisation to the nuclear periphery, indicating an orchestrated progression of distinct patterns of replication in the developing parasites. PfRFC1 responds to genotoxic stress via elevated protein levels in soluble and chromatin bound fractions. Reduction of PfRFC1 protein levels upon treatment with antimalarials suggests an interplay of replication, apoptosis and DNA repair pathways leading to cell death. Additionally, mislocalisation of the endogenously tagged protein confirmed its essential role in parasites' replication and DNA repair. This study provides key insights into DNA replication, DNA damage response and cell death in P. falciparum., (© 2020 John Wiley & Sons Ltd.)
- Published
- 2021
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14. Host-pathogen interaction in the tissue environment during Plasmodium blood-stage infection.
- Author
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Yui K and Inoue SI
- Subjects
- Animals, Anopheles parasitology, Erythrocytes parasitology, Humans, Membrane Proteins physiology, Mice, Protozoan Proteins physiology, Cytokines immunology, Host-Parasite Interactions, Malaria immunology, Plasmodium falciparum physiology, Spleen immunology
- Abstract
Human malarial infection occurs after an infectious Anopheles mosquito bites. Following the initial liver-stage infection, parasites transform into merozoites, infecting red blood cells (RBCs). Repeated RBC infection then occurs during the blood-stage infection, while patients experience various malarial symptoms. Protective immune responses are elicited by this systemic infection, but excessive responses are sometimes harmful for hosts. As parasites infect only RBCs and their immediate precursors during this stage, direct parasite-host interactions occur primarily in the environment surrounded by endothelial lining of blood vessels. The spleen is the major organ where the immune system encounters infected RBCs, causing immunological responses. Its tissue structure is markedly altered during malarial infection in mice and humans. Plasmodium falciparum parasites inside RBCs express proteins, such as PfEMP-1 and RIFIN, transported to the RBC surfaces in order to evade immunological attack by sequestering themselves in the peripheral vasculature avoiding spleen or by direct immune cell inhibition through inhibitory receptors. Host cell production of regulatory cytokines IL-10 and IL-27 limits excessive immune responses, avoiding tissue damage. The regulation of the protective and inhibitory immune responses through host-parasite interactions allows chronic Plasmodium infection. In this review, we discuss underlying interaction mechanisms relevant for developing effective strategies against malaria., (© 2020 John Wiley & Sons Ltd.)
- Published
- 2021
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15. Novel IM-associated protein Tim54 plays a role in the mitochondrial import of internal signal-containing proteins in Trypanosoma brucei.
- Author
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Singha UK, Tripathi A, Smith JT Jr, Quinones L, Saha A, Singha T, and Chaudhuri M
- Subjects
- Mitochondria metabolism, Mitochondrial Membranes metabolism, Protein Transport, Membrane Transport Proteins physiology, Mitochondrial Proteins physiology, Protozoan Proteins physiology, Trypanosoma brucei brucei metabolism
- Abstract
Background: The translocase of the mitochondrial inner membrane (TIM) imports most of the nucleus-encoded proteins that are destined for the matrix, inner membrane (IM) and the intermembrane space (IMS). Trypanosoma brucei, the infectious agent for African trypanosomiasis, possesses a unique TIM complex consisting of several novel proteins in association with a relatively conserved protein TbTim17. Tandem affinity purification of the TbTim17 protein complex revealed TbTim54 as a potential component of this complex., Results: TbTim54, a trypanosome-specific IMS protein, is peripherally associated with the IM and is present in a protein complex slightly larger than the TbTim17 complex. TbTim54 knockdown (KD) reduced the import of TbTim17 and compromised the integrity of the TbTim17 complex. TbTim54 KD inhibited the in vitro mitochondrial import and assembly of the internal signal-containing mitochondrial carrier proteins MCP3, MCP5 and MCP11 to a greater extent than TbTim17 KD. Furthermore, TbTim54 KD, but not TbTim17 KD, significantly hampered the mitochondrial targeting of ectopically expressed MCP3 and MCP11. These observations along with our previous finding that the mitochondrial import of N-terminal signal-containing proteins like cytochrome oxidase subunit 4 and MRP2 was affected to a greater extent by TbTim17 KD than TbTim54 KD indicating a substrate-specificity of TbTim54 for internal-signal containing mitochondrial proteins. In other organisms, small Tim chaperones in the IMS are known to participate in the translocation of MCPs. We found that TbTim54 can directly interact with at least two of the six known small TbTim proteins, TbTim11 and TbTim13, as well as with the N-terminal domain of TbTim17., Conclusion: TbTim54 interacts with TbTim17. It also plays a crucial role in the mitochondrial import and complex assembly of internal signal-containing IM proteins in T. brucei., Significance: We are the first to characterise TbTim54, a novel TbTim that is involved primarily in the mitochondrial import of MCPs and TbTim17 in T. brucei., (© 2020 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)
- Published
- 2021
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16. Role of inflammasomes in innate host defense against Entamoeba histolytica.
- Author
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Begum S, Gorman H, Chadha A, and Chadee K
- Subjects
- Caspases physiology, Cysteine Proteases physiology, Entamoeba histolytica pathogenicity, Gastrointestinal Microbiome, Humans, Lectins physiology, Macrophages physiology, NLR Family, Pyrin Domain-Containing 3 Protein physiology, Protozoan Proteins physiology, Virulence, Dysentery, Amebic immunology, Entamoeba histolytica immunology, Entamoebiasis immunology, Host-Parasite Interactions immunology, Immunity, Innate, Inflammasomes immunology
- Abstract
Intestinal amebiasis is the disease caused by the extracellular protozoan parasite Entamoeba histolytica (Eh) that induces a dynamic and heterogeneous interaction profile with the host immune system during disease pathogenesis. In 90% of asymptomatic infection, Eh resides with indigenous microbiota in the outer mucus layer of the colon without prompting an immune response. However, for reasons that remain unclear, in a minority of the Eh-infected individuals, this fine tolerated relationship is switched to a pathogenic phenotype and advanced to an increasingly complex host-parasite interaction. Eh disease susceptibility depends on parasite virulence factors and their interactions with indigenous bacteria, disruption of the mucus bilayers, and adherence to the epithelium provoking host immune cells to evoke a robust pro-inflammatory response mediated by inflammatory caspases and inflammasome activation. To understand Eh pathogenicity and innate host immune responses, this review highlights recent advances in our understanding of how Eh induces outside-in signaling via Mϕs to activate inflammatory caspases and inflammasome to regulate pro-inflammatory responses., (©2020 Society for Leukocyte Biology.)
- Published
- 2020
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17. Thrombospondin-1 Plays an Essential Role in Yes-Associated Protein Nuclear Translocation during the Early Phase of Trypanosoma cruzi Infection in Heart Endothelial Cells.
- Author
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Arun A, Rayford KJ, Cooley A, Rachakonda G, Villalta F, Pratap S, Lima MF, Sheibani N, and Nde PN
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Animals, Cell Cycle Proteins metabolism, DNA-Binding Proteins metabolism, Endothelial Cells metabolism, Gene Knockout Techniques, Mice, Myoblasts metabolism, Protein Serine-Threonine Kinases metabolism, Rats, Signal Transduction physiology, Thrombospondin 1 deficiency, Trans-Activators physiology, Active Transport, Cell Nucleus physiology, Endothelial Cells parasitology, Myoblasts parasitology, Myocardium cytology, Protozoan Proteins physiology, Thrombospondin 1 physiology, Trypanosoma cruzi physiology
- Abstract
The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This neglected tropical disease causes severe morbidity and mortality in endemic regions. About 30% of T. cruzi infected individuals will present with cardiac complications. Invasive trypomastigotes released from infected cells can be carried in the vascular endothelial system to infect neighboring and distant cells. During the process of cellular infection, the parasite induces host cells, to increase the levels of host thrombospondin-1 (TSP-1), to facilitate the process of infection. TSP-1 plays important roles in the functioning of vascular cells, including vascular endothelial cells with important implications in cardiovascular health. Many signal transduction pathways, including the yes-associated protein 1 (YAP)/transcriptional coactivator, with PDZ-binding motif (TAZ) signaling, which are upstream of TSP-1, have been linked to the pathophysiology of heart damage. The molecular mechanisms by which T. cruzi signals, and eventually infects, heart endothelial cells remain unknown. To evaluate the importance of TSP-1 expression in heart endothelial cells during the process of T. cruzi infection, we exposed heart endothelial cells prepared from Wild Type and TSP-1 Knockout mouse to invasive T . cruzi trypomastigotes at multiple time points, and evaluated changes in the hippo signaling cascade using immunoblotting and immunofluorescence assays. We found that the parasite turned off the hippo signaling pathway in TSP-1KO heart endothelial cells. The levels of SAV1 and MOB1A increased to a maximum of 2.70 ± 0.23 and 5.74 ± 1.45-fold at 3 and 6 h, respectively, in TSP-1KO mouse heart endothelial cells (MHEC), compared to WT MHEC, following a parasite challenge. This was accompanied by a significant continuous increase in the nuclear translocation of downstream effector molecule YAP, to a maximum mean nuclear fluorescence intensity of 10.14 ± 0.40 at 6 h, compared to wild type cells. Furthermore, we found that increased nuclear translocated YAP significantly colocalized with the transcription co-activator molecule pan-TEAD, with a maximum Pearson's correlation coefficient of 0.51 ± 0.06 at 6 h, compared to YAP-Pan-TEAD colocalization in the WT MHEC, which decreased significantly, with a minimum Pearson's correlation coefficient of 0.30 ± 0.01 at 6 h. Our data indicate that, during the early phase of infection, upregulated TSP-1 is essential for the regulation of the hippo signaling pathway. These studies advance our understanding of the molecular interactions occurring between heart endothelial cells and T. cruzi , in the presence and absence of TSP-1, providing insights into processes linked to parasite dissemination and pathogenesis.
- Published
- 2020
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18. The proofreading activity of Pfprex from Plasmodium falciparum can prevent mutagenesis of the apicoplast genome by oxidized nucleotides.
- Author
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Sharma M, Narayanan N, and Nair DT
- Subjects
- Apicoplasts genetics, Genome, Protozoan genetics, Multienzyme Complexes metabolism, Oxidation-Reduction, Plasmodium falciparum genetics, Protozoan Proteins metabolism, Apicoplasts metabolism, DNA Repair, Deoxyguanine Nucleotides metabolism, Multienzyme Complexes physiology, Mutagenesis genetics, Nucleotides metabolism, Plasmodium falciparum metabolism, Protozoan Proteins physiology
- Abstract
The DNA polymerase module of the Pfprex enzyme (PfpPol) is responsible for duplication of the genome of the apicoplast organelle in the malaria parasite. We show that PfpPol can misincorporate oxidized nucleotides such as 8oxodGTP opposite dA. This event gives rise to transversion mutations that are known to lead to adverse physiological outcomes. The apicoplast genome is particularly vulnerable to the harmful effects of 8oxodGTP due to very high AT content (~ 87%). We show that the proofreading activity of PfpPol has the unique ability to remove the oxidized nucleotide from the primer terminus. Due to this property, the proofreading domain of PfpPol is able to prevent mutagenesis of the AT-rich apicoplast genome and neutralize the deleterious genotoxic effects of ROS generated in the apicoplast due to normal metabolic processes. The proofreading activity of the Pfprex enzyme may, therefore, represent an attractive target for therapeutic intervention. Also, a survey of DNA repair pathways shows that the observed property of Pfprex constitutes a novel form of dynamic error correction wherein the repair of promutagenic damaged nucleotides is concomitant with DNA replication.
- Published
- 2020
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19. Unilateral Cleavage Furrows in Multinucleate Cells.
- Author
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Bindl J, Molnar ES, Ecke M, Prassler J, Müller-Taubenberger A, and Gerisch G
- Subjects
- Cell Division genetics, Cell Division physiology, Cell Fusion methods, Cell Membrane physiology, Cytokinesis genetics, Dictyostelium genetics, Gene Knockout Techniques, Genes, Protozoan, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Microfilament Proteins genetics, Microfilament Proteins physiology, Myosin Type II deficiency, Myosin Type II genetics, Myosin Type II physiology, Protozoan Proteins genetics, Protozoan Proteins physiology, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Cytokinesis physiology, Dictyostelium cytology, Dictyostelium physiology
- Abstract
Multinucleate cells can be produced in Dictyostelium by electric pulse-induced fusion. In these cells, unilateral cleavage furrows are formed at spaces between areas that are controlled by aster microtubules. A peculiarity of unilateral cleavage furrows is their propensity to join laterally with other furrows into rings to form constrictions. This means cytokinesis is biphasic in multinucleate cells, the final abscission of daughter cells being independent of the initial direction of furrow progression. Myosin-II and the actin filament cross-linking protein cortexillin accumulate in unilateral furrows, as they do in the normal cleavage furrows of mononucleate cells. In a myosin-II-null background, multinucleate or mononucleate cells were produced by cultivation either in suspension or on an adhesive substrate. Myosin-II is not essential for cytokinesis either in mononucleate or in multinucleate cells but stabilizes and confines the position of the cleavage furrows. In fused wild-type cells, unilateral furrows ingress with an average velocity of 1.7 µm × min
-1 , with no appreciable decrease of velocity in the course of ingression. In multinucleate myosin-II-null cells, some of the furrows stop growing, thus leaving space for the extensive broadening of the few remaining furrows.- Published
- 2020
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20. Leishmania dual-specificity tyrosine-regulated kinase 1 (DYRK1) is required for sustaining Leishmania stationary phase phenotype.
- Author
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Rocha VPC, Dacher M, Young SA, Kolokousi F, Efstathiou A, Späth GF, Soares MBP, and Smirlis D
- Subjects
- Animals, DNA, Protozoan genetics, Female, Gene Deletion, Gene Knockout Techniques, Genetic Fitness, Lipid Droplets metabolism, Macrophages parasitology, Male, Mice, Mice, Inbred BALB C, Morphogenesis, Dyrk Kinases, Cell Cycle, Leishmania infantum genetics, Leishmania infantum growth & development, Protein Serine-Threonine Kinases physiology, Protein-Tyrosine Kinases physiology, Protozoan Proteins physiology
- Abstract
Although the multiplicative and growth-arrested states play key roles in Leishmania development, the regulators of these transitions are largely unknown. In an attempt to gain a better understanding of these processes, we characterised one member of a family of protein kinases with dual specificity, LinDYRK1, which acts as a stasis regulator in other organisms. LinDYRK1 overexpressing parasites displayed a decrease in proliferation and in cell cycle re-entry of arrested cells. Parasites lacking LinDYRK1 displayed distinct fitness phenotypes in logarithmic and stationary growth phases. In logarithmic growth phase, LinDYRK1
-/- parasites proliferated better than control lines, supporting a role of this kinase in stasis, while in stationary growth phase, LinDYRK1-/- parasites had important defects as they rounded up, accumulated vacuoles and lipid bodies and displayed subtle but consistent differences in lipid composition. Moreover, they expressed less metacyclic-enriched transcripts, displayed increased sensitivity to complement lysis and a significant reduction in survival within peritoneal macrophages. The distinct LinDYRK1-/- growth phase phenotypes were mirrored by the distinct LinDYRK1 localisations in logarithmic (mainly in flagellar pocket area and endosomes) and late stationary phase (mitochondrion). Overall, this work provides first evidence for the role of a DYRK family member in sustaining promastigote stationary phase phenotype and infectivity., (© 2020 John Wiley & Sons Ltd.)- Published
- 2020
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21. Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure in Toxoplasma gondii .
- Author
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O'Shaughnessy WJ, Hu X, Beraki T, McDougal M, and Reese ML
- Subjects
- Cilia physiology, Cilia ultrastructure, Extracellular Signal-Regulated MAP Kinases physiology, Protozoan Proteins metabolism, Protozoan Proteins physiology, Toxoplasma metabolism, Toxoplasma physiology, Cilia metabolism, Extracellular Signal-Regulated MAP Kinases metabolism, Toxoplasma enzymology
- Abstract
Primary cilia are important organizing centers that control diverse cellular processes. Apicomplexan parasites like Toxoplasma gondii have a specialized cilium-like structure called the conoid that organizes the secretory and invasion machinery critical for the parasites' lifestyle. The proteins that initiate the biogenesis of this structure are largely unknown. We identified the Toxoplasma orthologue of the conserved kinase ERK7 as essential to conoid assembly. Parasites in which ERK7 has been depleted lose their conoids late during maturation and are immotile and thus unable to invade new host cells. This is the most severe phenotype to conoid biogenesis yet reported, and is made more striking by the fact that ERK7 is not a conoid protein, as it localizes just basal to the structure. ERK7 has been recently implicated in ciliogenesis in metazoan cells, and our data suggest that this kinase has an ancient and central role in regulating ciliogenesis throughout Eukaryota.
- Published
- 2020
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22. Autophagy-related protein PfATG18 participates in food vacuole dynamics and autophagy-like pathway in Plasmodium falciparum.
- Author
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Agrawal P, Manjithaya R, and Surolia N
- Subjects
- Autophagy, Autophagy-Related Proteins physiology, Membrane Proteins physiology, Plasmodium falciparum metabolism, Protozoan Proteins physiology, Vacuoles metabolism
- Abstract
Plasmodium falciparum has a limited repertoire of autophagy-related genes (ATGs), and the functions of various proteins of the autophagy-like pathway are not fully established in this protozoan parasite. Studies suggest that some of the autophagy proteins are crucial for parasite growth. PfATG18, for example, is essential for parasite replication and has a noncanonical role in apicoplast biogenesis. In this study, we demonstrate the conserved functions of PfATG18 in food vacuole (FV) dynamics and autophagy. Intriguingly, the P. falciparum FV is found to undergo fission and fusion and PfATG18 gets enriched at the interfaces of the newly generated multilobed FV during the process. In addition, expression of PfATG18 is induced upon starvation, both at the mRNA and protein level indicating its participation in the autophagy-like pathway, which is independent of its role in apicoplast biogenesis. The study also shows that PfATG18 is transported to the FV via the haemoglobin trafficking pathway. Overall, this study establishes the conserved functions of Atg18 in this important apicomplexan., (© 2019 John Wiley & Sons Ltd.)
- Published
- 2020
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23. A patatin-like phospholipase functions during gametocyte induction in the malaria parasite Plasmodium falciparum.
- Author
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Flammersfeld A, Panyot A, Yamaryo-Botté Y, Aurass P, Przyborski JM, Flieger A, Botté C, and Pradel G
- Subjects
- Animals, Cytoplasm genetics, Cytoplasm metabolism, Female, Gene Expression Regulation, Gene Knockdown Techniques, Genome, Protozoan, Life Cycle Stages, Lipid Metabolism, Mice, Phospholipases genetics, Plasmodium falciparum enzymology, Protozoan Proteins genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Phospholipases physiology, Plasmodium falciparum physiology, Protozoan Proteins physiology
- Abstract
Patatin-like phospholipases (PNPLAs) are highly conserved enzymes of prokaryotic and eukaryotic organisms with major roles in lipid homeostasis. The genome of the malaria parasite Plasmodium falciparum encodes four putative PNPLAs with predicted functions during phospholipid degradation. We here investigated the role of one of the plasmodial PNPLAs, a putative PLA
2 termed PNPLA1, during blood stage replication and gametocyte development. PNPLA1 is present in the asexual and sexual blood stages and here localizes to the cytoplasm. PNPLA1-deficiency due to gene disruption or conditional gene-knockdown had no effect on intraerythrocytic growth, gametocyte development and gametogenesis. However, parasites lacking PNPLA1 were impaired in gametocyte induction, while PNPLA1 overexpression promotes gametocyte formation. The loss of PNPLA1 further leads to transcriptional down-regulation of genes related to gametocytogenesis, including the gene encoding the sexual commitment regulator AP2-G. Additionally, lipidomics of PNPLA1-deficient asexual blood stage parasites revealed overall increased levels of major phospholipids, including phosphatidylcholine (PC), which is a substrate of PLA2 . PC synthesis is known to be pivotal for erythrocytic replication, while the reduced availability of PC precursors drives the parasite into gametocytogenesis; we thus hypothesize that the higher PC levels due to PNPLA1-deficiency prevent the blood stage parasites from entering the sexual pathway., (© 2019 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)- Published
- 2020
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24. Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission.
- Author
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Depoix D, Marques SR, Ferguson DJ, Chaouch S, Duguet T, Sinden RE, Grellier P, and Kohl L
- Subjects
- Animals, Culicidae parasitology, Female, Gene Knockout Techniques, Genes, Protozoan, Life Cycle Stages, Malaria parasitology, Mice, Mitosis, Models, Animal, Mosquito Vectors parasitology, Organisms, Genetically Modified, Protozoan Proteins genetics, Axoneme physiology, Kinesins genetics, Kinesins physiology, Plasmodium berghei physiology, Protozoan Proteins physiology
- Abstract
Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin-8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal '9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation., (© 2019 John Wiley & Sons Ltd.)
- Published
- 2020
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25. Identification of Fis1 Interactors in Toxoplasma gondii Reveals a Novel Protein Required for Peripheral Distribution of the Mitochondrion.
- Author
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Jacobs K, Charvat R, and Arrizabalaga G
- Subjects
- Life Cycle Stages, Monensin pharmacology, Protozoan Proteins genetics, Toxoplasma drug effects, Toxoplasma genetics, Mitochondria physiology, Protozoan Proteins physiology, Toxoplasma cytology
- Abstract
Toxoplasma gondii 's single mitochondrion is very dynamic and undergoes morphological changes throughout the parasite's life cycle. During parasite division, the mitochondrion elongates, enters the daughter cells just prior to cytokinesis, and undergoes fission. Extensive morphological changes also occur as the parasite transitions from the intracellular environment to the extracellular environment. We show that treatment with the ionophore monensin causes reversible constriction of the mitochondrial outer membrane and that this effect depends on the function of the fission-related protein Fis1. We also observed that mislocalization of the endogenous Fis1 causes a dominant-negative effect that affects the morphology of the mitochondrion. As this suggests that Fis1 interacts with proteins critical for maintenance of mitochondrial structure, we performed various protein interaction trap screens. In this manner, we identified a novel outer mitochondrial membrane protein, LMF1, which is essential for positioning of the mitochondrion in intracellular parasites. Normally, while inside a host cell, the parasite mitochondrion is maintained in a lasso shape that stretches around the parasite periphery where it has regions of coupling with the parasite pellicle, suggesting the presence of membrane contact sites. In intracellular parasites lacking LMF1, the mitochondrion is retracted away from the pellicle and instead is collapsed, as normally seen only in extracellular parasites. We show that this phenotype is associated with defects in parasite fitness and mitochondrial segregation. Thus, LMF1 is necessary for mitochondrial association with the parasite pellicle during intracellular growth, and proper mitochondrial morphology is a prerequisite for mitochondrial division. IMPORTANCE Toxoplasma gondii is an opportunistic pathogen that can cause devastating tissue damage in the immunocompromised and congenitally infected. Current therapies are not effective against all life stages of the parasite, and many cause toxic effects. The single mitochondrion of this parasite is a validated drug target, and it changes its shape throughout its life cycle. When the parasite is inside a cell, the mitochondrion adopts a lasso shape that lies in close proximity to the pellicle. The functional significance of this morphology is not understood and the proteins involved are currently not known. We have identified a protein that is required for proper mitochondrial positioning at the periphery and that likely plays a role in tethering this organelle. Loss of this protein results in dramatic changes to the mitochondrial morphology and significant parasite division and propagation defects. Our results give important insight into the molecular mechanisms regulating mitochondrial morphology., (Copyright © 2020 Jacobs et al.)
- Published
- 2020
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26. The PHIST protein GEXP02 targets the host cytoskeleton during sexual development of Plasmodium falciparum.
- Author
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Warncke JD, Passecker A, Kipfer E, Brand F, Pérez-Martínez L, Proellochs NI, Kooij TWA, Butter F, Voss TS, and Beck HP
- Subjects
- Host-Parasite Interactions, Humans, Erythrocyte Membrane metabolism, Germ Cells cytology, Malaria, Falciparum parasitology, Plasmodium falciparum physiology, Protozoan Proteins physiology
- Abstract
A hallmark of the biology of Plasmodium falciparum blood stage parasites is their extensive host cell remodelling, facilitated by parasite proteins that are exported into the erythrocyte. Although this area has received extensive attention, only a few exported parasite proteins have been analysed in detail, and much of this remodelling process remains unknown, particularly for gametocyte development. Recent advances to induce high rates of sexual commitment enable the production of large numbers of gametocytes. We used this approach to study the Plasmodium helical interspersed subtelomeric (PHIST) protein GEXP02, which is expressed during sexual development. We show by immunofluorescence that GEXP02 is exported to the gametocyte-infected host cell periphery. Co-immunoprecipitation revealed potential interactions between GEXP02 and components of the erythrocyte cytoskeleton as well as other exported parasite proteins. This indicates that GEXP02 targets the erythrocyte cytoskeleton and is likely involved in its remodelling. GEXP02 knock-out parasites show no obvious phenotype during gametocyte maturation, transmission through mosquitoes, and hepatocyte infection, suggesting auxiliary or redundant functions for this protein. In summary, we performed a detailed cellular and biochemical analysis of a sexual stage-specific exported parasite protein using a novel experimental approach that is broadly applicable to study the biology of P. falciparum gametocytes., (© 2019 John Wiley & Sons Ltd.)
- Published
- 2020
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27. The Sec1/Munc18-like proteins TgSec1 and TgVps45 play pivotal roles in assembly of the pellicle and sub-pellicle network in Toxoplasma gondii.
- Author
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Cao S, Chen H, Liang X, Fu J, Wang S, Zheng J, Zhang Z, Pang Y, Wang J, Shen B, and Jia H
- Subjects
- Fibroblasts, HEK293 Cells, Humans, Munc18 Proteins physiology, Organelles metabolism, Protozoan Proteins physiology, Toxoplasma metabolism
- Abstract
Post-Golgi vesicle trafficking is indispensable for precise movement of proteins to the pellicle, the sub-pellicle network and apical secretory organelles in Apicomplexa. However, only a small number of molecular complexes involved in trafficking, tethering and fusion of vesicles have been identified in Toxoplasma gondii. Consequently, it is unclear how complicated vesicle trafficking is accomplished in this parasite. Sec1/Munc18-like (SM) proteins are essential components of protein complexes involved in vesicle fusion. Here, we found that depletion of the SM protein TgSec1 using an auxin-inducible degron-based conditional knockout strategy led to mislocalization of plasma membrane proteins. By contrast, conditional depletion of the SM protein TgVps45 led to morphological changes, asymmetrical loss of the inner membrane complex and defects in nucleation of sub-pellicular microtubules, polarization and symmetrical assembly of daughter parasites during repeated endodyogeny. TgVps45 interacts with the SNARE protein TgStx16 and TgVAMP4-1. Conditional ablation of TgStx16 causes the similar growth defect like TgVps45 deficiency suggested they work together for the vesicle fusion at TGN. These findings indicate that these two SM proteins are crucial for assembly of pellicle and sub-pellicle network in T. gondii respectively., (© 2019 John Wiley & Sons Ltd.)
- Published
- 2020
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28. Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion.
- Author
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Del Rosario M, Periz J, Pavlou G, Lyth O, Latorre-Barragan F, Das S, Pall GS, Stortz JF, Lemgruber L, Whitelaw JA, Baum J, Tardieux I, and Meissner M
- Subjects
- Actins genetics, Active Transport, Cell Nucleus physiology, Animals, Cell Nucleus parasitology, Cell Nucleus physiology, Cells, Cultured, Gene Knockout Techniques, Humans, Merozoites genetics, Merozoites pathogenicity, Merozoites physiology, Models, Biological, Mutation, Plasmodium falciparum genetics, Protozoan Proteins genetics, Signal Transduction, Toxoplasma genetics, Virulence physiology, Actins physiology, Host-Parasite Interactions physiology, Plasmodium falciparum pathogenicity, Plasmodium falciparum physiology, Protozoan Proteins physiology, Toxoplasma parasitology, Toxoplasma pathogenicity
- Abstract
The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. Super-resolution microscopy and real-time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites., (© 2019 The Author. Published under the terms of the CC BY 4.0 license.)
- Published
- 2019
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29. Male-Specific Protein Disulphide Isomerase Function is Essential for Plasmodium Transmission and a Vulnerable Target for Intervention.
- Author
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Angrisano F, Sala KA, Tapanelli S, Christophides GK, and Blagborough AM
- Subjects
- Animals, Female, Male, Mice, Plasmodium berghei drug effects, Plasmodium berghei pathogenicity, Protozoan Proteins physiology, Antimalarials pharmacology, Antimalarials therapeutic use, Bacitracin pharmacology, Bacitracin therapeutic use, Malaria prevention & control, Malaria transmission, Malaria Vaccines pharmacology, Malaria Vaccines therapeutic use, Plasmodium berghei enzymology, Protein Disulfide-Isomerases physiology
- Abstract
Inhibiting transmission of Plasmodium is an essential strategy in malaria eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. Lack of knowledge of the mechanisms underlying fertilization have been a hindrance in the development of transmission-blocking interventions. Here we describe a protein disulphide isomerase essential for malarial transmission (PDI-Trans/PBANKA_0820300) to the mosquito. We show that PDI-Trans activity is male-specific, surface-expressed, essential for fertilization/transmission, and exhibits disulphide isomerase activity which is up-regulated post-gamete activation. We demonstrate that PDI-Trans is a viable anti-malarial drug and vaccine target blocking malarial transmission with the use of PDI inhibitor bacitracin (98.21%/92.48% reduction in intensity/prevalence), and anti-PDI-Trans antibodies (66.22%/33.16% reduction in intensity/prevalence). To our knowledge, these results provide the first evidence that PDI function is essential for malarial transmission, and emphasize the potential of anti-PDI agents to act as anti-malarials, facilitating the future development of novel transmission-blocking interventions.
- Published
- 2019
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30. Functional analyses of an axonemal inner-arm dynein complex in the bloodstream form of Trypanosoma brucei uncover its essential role in cytokinesis initiation.
- Author
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Zhang X, Hu H, Lun ZR, and Li Z
- Subjects
- Axonemal Dyneins physiology, Axoneme metabolism, Cell Division genetics, Cell Division physiology, Cell Line, Cell Movement, Contractile Proteins genetics, Contractile Proteins physiology, Dyneins metabolism, Dyneins physiology, Flagella metabolism, Flagella physiology, Life Cycle Stages, Protozoan Proteins genetics, Protozoan Proteins physiology, RNA Interference, Trypanosoma brucei brucei metabolism, Axonemal Dyneins metabolism, Contractile Proteins metabolism, Cytokinesis physiology, Protozoan Proteins metabolism
- Abstract
The flagellated eukaryote Trypanosoma brucei alternates between the insect vector and the mammalian host and proliferates through an unusual mode of cell division. Cell division requires flagellum motility-generated forces, but flagellum motility exerts distinct effects between different life cycle forms. Motility is required for the final cell abscission of the procyclic form in the insect vector, but is necessary for the initiation of cell division of the bloodstream form in the mammalian host. The underlying mechanisms remain elusive. Here we carried out functional analyses of a flagellar axonemal inner-arm dynein complex in the bloodstream form and investigated its mechanistic role in cytokinesis initiation. We showed that the axonemal inner-arm dynein heavy chain TbIAD5-1 and TbCentrin3 form a complex, localize to the flagellum, and are required for viability in the bloodstream form. We further demonstrated the interdependence between TbIAD5-1 and TbCentrin3 for maintenance of protein stability. Finally, we showed that depletion of TbIAD5-1 and TbCentrin3 arrested cytokinesis initiation and disrupted the localization of multiple cytokinesis initiation regulators. These findings identified the essential role of an axonemal inner-arm dynein complex in cell division, and provided molecular insights into the flagellum motility-mediated cytokinesis initiation in the bloodstream form of T. brucei., (© 2019 John Wiley & Sons Ltd.)
- Published
- 2019
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31. A Plasmodium falciparum C -mannosyltransferase is dispensable for parasite asexual blood stage development.
- Author
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López-Gutiérrez B, Cova M, and Izquierdo L
- Subjects
- Blood parasitology, CRISPR-Cas Systems, Glycosylation, Loss of Function Mutation, Mannosyltransferases genetics, Plasmodium falciparum genetics, Protozoan Proteins genetics, Reproduction, Asexual, Salivary Glands parasitology, Thrombospondins genetics, Thrombospondins physiology, Life Cycle Stages, Mannosyltransferases physiology, Plasmodium falciparum enzymology, Plasmodium falciparum physiology, Protozoan Proteins physiology
- Abstract
C-mannosylation was recently identified in the thrombospondin-related anonymous protein (TRAP) from Plasmodium falciparum salivary gland sporozoites. A candidate P. falciparum C-mannosyltransferase (PfDPY-19) was demonstrated to modify thrombospondin type 1 repeat (TSR) domains in vitro, exhibiting a different acceptor specificity than their mammalian counterparts. According to the described minimal acceptor of PfDPY19, several TSR domain-containing proteins of P. falciparum could be C-mannosylated in vivo. However, the relevance of this protein modification for the parasite viability remains unknown. In the present study, we used CRISPR/Cas9 technology to generate a PfDPY19 null mutant, demonstrating that this glycosyltransferase is not essential for the asexual blood development of the parasite. PfDPY19 gene disruption was not associated with a growth phenotype, not even under endoplasmic reticulum-stressing conditions that could impair protein folding. The data presented in this work strongly suggest that PfDPY19 is unlikely to play a critical role in the asexual blood stages of the parasite, at least under in vitro conditions.
- Published
- 2019
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32. Evaluation of clan CD C11 peptidase PNT1 and other Leishmania mexicana cysteine peptidases as potential drug targets.
- Author
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Grewal JS, Catta-Preta CMC, Brown E, Anand J, and Mottram JC
- Subjects
- Gene Deletion, Genes, Essential, Humans, Leishmania mexicana genetics, Leishmania mexicana pathogenicity, Virulence genetics, Cysteine Endopeptidases genetics, Cysteine Endopeptidases physiology, Leishmania mexicana enzymology, Leishmaniasis, Cutaneous parasitology, Protozoan Proteins genetics, Protozoan Proteins physiology
- Abstract
Leishmania mexicana is one of the causative agents of cutaneous leishmaniasis in humans. There is an urgent need to identify new drug targets to combat the disease. Cysteine peptidases play crucial role in pathogenicity and virulence in Leishmania spp. and are promising targets for developing new anti-leishmanial drugs. Genetic drug target validation has been performed on a number of cysteine peptidases, but others have yet to be characterized. We targeted 16 L. mexicana cysteine peptidases for gene deletion and tagging using CRISPR-Cas9 in order to identify essential genes and ascertain their cellular localization. Our analysis indicates that two clan CA, family C2 calpains (LmCAL27.1, LmCAL31.6) and clan CD, family C11 PNT1 are essential for survival in the promastigote stage. The other peptidases analysed, namely calpains LmCAL4.1, LmCAL25.1, and members of clan CA C51, C78, C85 and clan CP C97 were found to be non-essential. We generated a gene deletion mutant (Δpnt1) which was severely compromised in its cell growth and a conditional gene deletion mutant of PNT1 (Δpnt1: PNT1
flox /Δ pnt1:HYG [SSU DiCRE]). PNT1 localizes to distinct foci on the flagellum and on the surface of the parasite. The conditional gene deletion of PNT1 induced blebs and pits on the cell surface and eventual cell death. Over-expression of PNT1, but not an active site mutant PNT1C134A , was lethal, suggesting that active PNT1 peptidase is required for parasite survival. Overall, our data suggests that PNT1 is an essential gene and one of a number of cysteine peptidases that are potential drug targets in Leishmania., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)- Published
- 2019
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33. Eimeria tenella: specific EtAMA1-binding peptides inhibit sporozoite entry into host cells.
- Author
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Ma D, Huang Y, Ma C, Zhang L, Wang J, Wang D, Li J, and Dalloul RA
- Subjects
- Animals, Coccidiosis prevention & control, Molecular Docking Simulation, Protozoan Proteins physiology, Random Allocation, Sporozoites physiology, Chickens, Coccidiosis veterinary, Eimeria tenella physiology, Poultry Diseases prevention & control
- Abstract
Avian coccidiosis caused by Eimeria inflicts high economic losses to the poultry industry. Application of drugs and live vaccines presents particular challenges in pathogen resistance and cost, hence alternative anti-coccidial strategies are needed. In this study, peptides that specifically bind E. tenella AMA1 (EtAMA1) were screened from a phage display peptide library. The positive clones of target phages were characterized by ELISA after four rounds of biopanning. The binding capabilities with EtAMA1 and sporozoite proteins for the two selected peptides were detected by ELISA. The role of the two target peptides in inhibiting sporozoite invasion of MDBK cells was evaluated in vitro and the anti-coccidial effects of the two phages were assessed by an animal experiment. The three-dimensional (3D) structural model of EtAMA1 extracellular domain (EctoAMA1) protein was constructed based on the crystal template of TgAMA1 (PDB ID: 2 × 2Z), and the molecular docking between target peptides and EctoAMA1 model was analyzed. The results showed that two selected phages strongly interacted with EctoAMA1 and sporozoites protein. Two corresponding specific EtAMA1-binding peptide (named L and C) showed significant effects on inhibiting sporozoite invasion of MDBK cells. Chickens orally fed the two target phages showed partial protection against homologous challenge. Homology modeling analysis showed an apical hydrophobic groove was shaped on the top of the EctoAMA1 model. Molecular docking indicated the interaction between the EctoAMA1 protein and the two peptides, which was mainly reflected by the hydrophobic interaction and formation of intermolecular hydrogen bond. The above results suggest that the peptides L and C, especially L peptide, competed with E. tenella rhotry neck protein 2 (EtRON2) for binding to EtAMA1 located on the surface of sporozoites, and therefore inhibited the parasite invasion into cells., (© 2019 Poultry Science Association Inc.)
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- 2019
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34. Neutrophil elastase promotes Leishmania donovani infection via interferon-β.
- Author
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Dias BT, Dias-Teixeira KL, Godinho JP, Faria MS, Calegari-Silva T, Mukhtar MM, Lopes U, Mottram JC, and Lima APCA
- Subjects
- Animals, Animals, Genetically Modified, Leishmania donovani genetics, Leishmania donovani physiology, Leishmaniasis, Visceral metabolism, Leishmaniasis, Visceral parasitology, Leukocyte Elastase deficiency, Leukocyte Elastase genetics, Macrophages metabolism, Macrophages parasitology, Mice, Mice, Inbred C57BL, Mice, Knockout, Protozoan Proteins genetics, Protozoan Proteins physiology, Toll-Like Receptor 2 deficiency, Toll-Like Receptor 2 genetics, Toll-Like Receptor 2 metabolism, Toll-Like Receptor 4 deficiency, Toll-Like Receptor 4 genetics, Toll-Like Receptor 4 metabolism, Interferon-beta metabolism, Leishmania donovani pathogenicity, Leishmaniasis, Visceral etiology, Leukocyte Elastase metabolism
- Abstract
Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major , named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF-α and IFN-β production, and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knockout mice for NE ( ela
-/- ), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela-/- mice were reduced and accompanied by increased NO and decreased TGF-β production. Expression of ISP2 was not detected in L. donovani , and a transgenic line constitutively expressing ISP 2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela-/- macrophages displayed significantly lower IFN-β mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN-β. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN-β necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway.-Dias, B. T., Dias-Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari-Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes Leishmania donovani infection via interferon-β.- Published
- 2019
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35. Malaria transmission through the mosquito requires the function of the OMD protein.
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Currà C, Kehrer J, Lemgruber L, Silva PAGC, Bertuccini L, Superti F, Pace T, Ponzi M, Frischknecht F, Siden-Kiamos I, and Mair GR
- Subjects
- Animals, Female, Fluorescent Antibody Technique, Indirect, Gene Knockdown Techniques, Malaria parasitology, Mice, Microscopy, Electron, Scanning, Protozoan Proteins genetics, Reverse Transcriptase Polymerase Chain Reaction, Anopheles parasitology, Malaria transmission, Plasmodium berghei physiology, Protozoan Proteins physiology
- Abstract
Ookinetes, one of the motile and invasive forms of the malaria parasite, rely on gliding motility in order to establish an infection in the mosquito host. Here we characterize the protein PBANKA_0407300 which is conserved in the Plasmodium genus but lacks significant similarity to proteins of other eukaryotes. It is expressed in gametocytes and throughout the invasive mosquito stages of P. berghei, but is absent from asexual blood stages. Mutants lacking the protein developed morphologically normal ookinetes that were devoid of productive motility although some stretching movement could be detected. We therefore named the protein Ookinete Motility Deficient (OMD). Several key factors known to be involved in motility however were normally expressed and localized in the mutant. Importantly, the mutant failed to establish an infection in the mosquito which resulted in a total malaria transmission blockade., Competing Interests: The authors have declared that no competing interests exist.
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- 2019
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36. Rhoptry neck protein 11 has crucial roles during malaria parasite sporozoite invasion of salivary glands and hepatocytes.
- Author
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Bantuchai S, Nozaki M, Thongkukiatkul A, Lorsuwannarat N, Tachibana M, Baba M, Matsuoka K, Tsuboi T, Torii M, and Ishino T
- Subjects
- Animals, Blotting, Southern, DNA, Protozoan chemistry, DNA, Protozoan isolation & purification, Erythrocytes parasitology, Female, Mice, Mice, Inbred C57BL, Mice, Inbred ICR, Microscopy, Electron, Transmission, Microscopy, Immunoelectron, Protozoan Proteins immunology, Rabbits, Rats, Rats, Wistar, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Salivary Glands parasitology, Sporozoites physiology, Anopheles parasitology, Hepatocytes parasitology, Plasmodium berghei physiology, Protozoan Proteins physiology
- Abstract
The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells - mosquito salivary glands and mammalian hepatocytes - via involvement in sporozoite motility., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2019
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37. The host cell secretory pathway mediates the export of Leishmania virulence factors out of the parasitophorous vacuole.
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Arango Duque G, Jardim A, Gagnon É, Fukuda M, and Descoteaux A
- Subjects
- Animals, Endoplasmic Reticulum parasitology, Female, Glycosphingolipids physiology, Humans, Leishmania growth & development, Leishmaniasis parasitology, Metalloendopeptidases physiology, Mice, Mice, Inbred C57BL, Phagocytes parasitology, Phagocytosis, Phagosomes parasitology, Qa-SNARE Proteins physiology, R-SNARE Proteins physiology, Secretory Pathway, Vacuoles parasitology, Virulence, Host-Parasite Interactions physiology, Leishmania pathogenicity, Leishmania physiology, Protozoan Proteins physiology, Virulence Factors physiology
- Abstract
To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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38. Toxoplasma F-box protein 1 is required for daughter cell scaffold function during parasite replication.
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Baptista CG, Lis A, Deng B, Gas-Pascual E, Dittmar A, Sigurdson W, West CM, and Blader IJ
- Subjects
- Animals, F-Box Proteins antagonists & inhibitors, F-Box Proteins genetics, Gene Knockdown Techniques, Genes, Protozoan, Humans, Protein Interaction Domains and Motifs, Protozoan Proteins antagonists & inhibitors, Protozoan Proteins genetics, S-Phase Kinase-Associated Proteins physiology, Toxoplasma genetics, F-Box Proteins physiology, Protozoan Proteins physiology, Toxoplasma growth & development, Toxoplasma pathogenicity
- Abstract
By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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39. Essential role of GEXP15, a specific Protein Phosphatase type 1 partner, in Plasmodium berghei in asexual erythrocytic proliferation and transmission.
- Author
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Hollin T, De Witte C, Fréville A, Guerrera IC, Chhuon C, Saliou JM, Herbert F, Pierrot C, and Khalife J
- Subjects
- Animals, Anopheles parasitology, Erythrocytes parasitology, Female, Genes, Protozoan, Host-Parasite Interactions genetics, Host-Parasite Interactions physiology, Humans, Malaria parasitology, Malaria transmission, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Mosquito Vectors parasitology, Plasmodium berghei genetics, Protein Binding, Protein Interaction Domains and Motifs, Protein Phosphatase 1 chemistry, Protein Phosphatase 1 genetics, Proteomics, Protozoan Proteins chemistry, Protozoan Proteins genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Plasmodium berghei growth & development, Plasmodium berghei physiology, Protein Phosphatase 1 physiology, Protozoan Proteins physiology
- Abstract
The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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40. A post-transcriptional respiratome regulon in trypanosomes.
- Author
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Trenaman A, Glover L, Hutchinson S, and Horn D
- Subjects
- Adaptation, Physiological, Amino Acid Sequence, Electron Transport physiology, Gene Silencing, Humans, Mitochondria metabolism, Parasitemia parasitology, Protein Interaction Mapping, Proteomics methods, Proton-Translocating ATPases physiology, RNA Interference, Sequence Alignment, Sequence Homology, Amino Acid, Transcriptome, Trypanosoma brucei brucei isolation & purification, Trypanosomiasis, African parasitology, Variant Surface Glycoproteins, Trypanosoma biosynthesis, Variant Surface Glycoproteins, Trypanosoma genetics, Cell Respiration physiology, Gene Expression Regulation genetics, Protozoan Proteins physiology, RNA-Binding Proteins physiology, Regulon physiology, Trypanosoma brucei brucei physiology
- Abstract
Post-transcriptional regulons coordinate the expression of groups of genes in eukaryotic cells, yet relatively few have been characterized. Parasitic trypanosomatids are particularly good models for studies on such mechanisms because they exhibit almost exclusive polycistronic, and unregulated, transcription. Here, we identify the Trypanosoma brucei ZC3H39/40 RNA-binding proteins as regulators of the respiratome; the mitochondrial electron transport chain (complexes I-IV) and the FoF1-ATP synthase (complex V). A high-throughput RNAi screen initially implicated both ZC3H proteins in variant surface glycoprotein (VSG) gene silencing. This link was confirmed and both proteins were shown to form a cytoplasmic ZC3H39/40 complex. Transcriptome and mRNA-interactome analyses indicated that the impact on VSG silencing was indirect, while the ZC3H39/40 complex specifically bound and stabilized transcripts encoding respiratome-complexes. Quantitative proteomic analyses revealed specific positive control of >20 components from complexes I, II and V. Our findings establish a link between the mitochondrial respiratome and VSG gene silencing in bloodstream form T. brucei. They also reveal a major respiratome regulon controlled by the conserved trypanosomatid ZC3H39/40 RNA-binding proteins., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2019
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41. Plasmodium myosin A drives parasite invasion by an atypical force generating mechanism.
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Robert-Paganin J, Robblee JP, Auguin D, Blake TCA, Bookwalter CS, Krementsova EB, Moussaoui D, Previs MJ, Jousset G, Baum J, Trybus KM, and Houdusse A
- Subjects
- Cell Movement, Crystallography, X-Ray, Erythrocytes parasitology, Nonmuscle Myosin Type IIA chemistry, Nonmuscle Myosin Type IIA metabolism, Phosphorylation, Protozoan Proteins chemistry, Protozoan Proteins metabolism, Nonmuscle Myosin Type IIA physiology, Plasmodium falciparum pathogenicity, Protozoan Proteins physiology
- Abstract
Plasmodium parasites are obligate intracellular protozoa and causative agents of malaria, responsible for half a million deaths each year. The lifecycle progression of the parasite is reliant on cell motility, a process driven by myosin A, an unconventional single-headed class XIV molecular motor. Here we demonstrate that myosin A from Plasmodium falciparum (PfMyoA) is critical for red blood cell invasion. Further, using a combination of X-ray crystallography, kinetics, and in vitro motility assays, we elucidate the non-canonical interactions that drive this motor's function. We show that PfMyoA motor properties are tuned by heavy chain phosphorylation (Ser19), with unphosphorylated PfMyoA exhibiting enhanced ensemble force generation at the expense of speed. Regulated phosphorylation may therefore optimize PfMyoA for enhanced force generation during parasite invasion or for fast motility during dissemination. The three PfMyoA crystallographic structures presented here provide a blueprint for discovery of specific inhibitors designed to prevent parasite infection.
- Published
- 2019
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42. The Nuclear Export Receptors TbMex67 and TbMtr2 Are Required for Ribosome Biogenesis in Trypanosoma brucei.
- Author
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Rink C, Ciganda M, and Williams N
- Subjects
- Protozoan Proteins genetics, RNA, Messenger, Ribosomal Proteins genetics, Trypanosoma brucei brucei physiology, Active Transport, Cell Nucleus, Organelle Biogenesis, Protozoan Proteins physiology, Ribosomes physiology, Trypanosoma brucei brucei genetics
- Abstract
Ribosomal maturation is a complex and highly conserved biological process involving migration of a continuously changing RNP across multiple cellular compartments. A critical point in this process is the translocation of individual ribosomal subunits (60S and 40S) from the nucleus to the cytoplasm, and a number of export factors participate in this process. In this study, we characterize the functional role of the auxiliary export receptors TbMex67 and TbMtr2 in ribosome biogenesis in the parasite Trypanosoma brucei We demonstrate that depletion of each of these proteins dramatically impacts the steady-state levels of other proteins involved in ribosome biogenesis, including the trypanosome-specific factors P34 and P37. In addition, we observe that the loss of TbMex67 or TbMtr2 leads to aberrant ribosome formation, rRNA processing, and polysome formation. Although the TbMex67-TbMtr2 heterodimer is structurally distinct from Mex67-Mtr2 complexes previously studied, our data show that they retain a conserved function in ribosome biogenesis. IMPORTANCE The nuclear export of ribosomal subunits (60S and 40S) depends in part on the activity of the essential auxiliary export receptors TbMtr2 and TbMex67. When these proteins are individually depleted from the medically and agriculturally significant parasite Trypanosoma brucei , distinct alterations in the processing of the rRNAs of the large subunit (60S) are observed as well as aberrations in the assembly of functional ribosomes (polysomes). We also established that TbMex67 and TbMtr2 interact directly or indirectly with the protein components of the 5S RNP, including the trypanosome-specific P34/P37. The critical role that TbMex67 and TbMtr2 play in this essential biological process together with their parasite-specific interactions may provide new therapeutic targets against this important parasite., (Copyright © 2019 Rink et al.)
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- 2019
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43. Poly(ADP-ribose) polymerase-1 (PARP-1) regulates developmental morphogenesis and chemotaxis in Dictyostelium discoideum.
- Author
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Jubin T, Kadam A, and Begum R
- Subjects
- Cyclic AMP metabolism, Dictyostelium genetics, Dictyostelium physiology, Gene Knockout Techniques, Isoenzymes genetics, Isoenzymes physiology, Morphogenesis, Poly (ADP-Ribose) Polymerase-1 genetics, Protozoan Proteins genetics, Signal Transduction, Transcriptome, Chemotaxis, Dictyostelium growth & development, Poly (ADP-Ribose) Polymerase-1 physiology, Protozoan Proteins physiology
- Abstract
Background Information: Poly(ADP-ribose) polymerase-1 (PARP-1) has been attributed to varied roles in DNA repair, cell cycle, cell death, etc. Our previous reports demonstrate the role of PARP-1 during Dictyostelium discoideum development by its constitutive downregulation as well as by PARP-1 ortholog, ADP ribosyl transferase 1 A (ADPRT1A) overexpression. The current study analyses and strengthens the function of ADPRT1A in multicellular morphogenesis of D. discoideum. ADPRT1A was knocked out, and its effect was studied on cAMP signalling, chemotaxis and development of D. discoideum., Results: We report that ADPRT1A is essential in multicellular development of D. discoideum, particularly at the aggregation stage. Genetic alterations of ADPRT1A and chemical inhibition of its activity affects the intracellular and extracellular cAMP levels during aggregation along with chemotaxis. Exogenous cAMP pulses could rescue this defect in the ADPRT1A knockout (ADPRT1A KO). Expression analysis of genes involved in cAMP signalling reveals altered transcript levels of four essential genes (PDSA, REGA, ACAA and CARA). Moreover, ADPRT1A KO affects prespore- and prestalk-specific gene expression and prestalk tendency is favoured in the ADPRT1A KO., Conclusion: ADPRT1A plays a definite role in regulating developmental morphogenesis via cAMP signalling., Significance: This study helps in understanding the role of PARP-1 in multicellular development and differentiation in higher complex organisms., (© 2019 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)
- Published
- 2019
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44. Participation of Trypanosoma cruzi gp63 molecules on the interaction with Rhodnius prolixus.
- Author
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Rebello KM, Uehara LA, Ennes-Vidal V, Garcia-Gomes AS, Britto C, Azambuja P, Menna-Barreto RFS, Santos ALS, Branquinha MH, and d'Avila-Levy CM
- Subjects
- Animals, Antibodies, Protozoan metabolism, Chagas Disease parasitology, Host-Parasite Interactions, Insect Vectors parasitology, Metalloendopeptidases physiology, Protozoan Proteins physiology, Rhodnius parasitology, Trypanosoma cruzi physiology
- Abstract
Trypanosoma cruzi is the causative agent of Chagas disease, a vector-borne disease. The parasite molecules involved in vector interaction have been little investigated. Metallopeptidases and gp63 molecules have been implicated in parasite adhesion of several trypanosomatids to the insect midgut. Although gp63 homologues are highly expanded in the T. cruzi genome, and are implicated in parasite-mammalian host interaction, its role in the insect vector has never been explored. Here, we showed that divalent metal chelators or anti-Tcgp63-I antibodies impaired T. cruzi adhesion to Rhodnius prolixus midgut. Parasites isolated after insect colonization presented a drastic enhancement in the expression of Tcgp63-I. These data highlight, for the first time, that Tcgp63-I and Zn-dependent enzymes contribute to the interaction of T. cruzi with the insect vector.
- Published
- 2019
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45. Nuclear Factor Y (NF-Y) Modulates Encystation in Entamoeba via Stage-Specific Expression of the NF-YB and NF-YC Subunits.
- Author
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Manna D and Singh U
- Subjects
- Amino Acid Sequence, CCAAT-Binding Factor genetics, Entamoeba genetics, Gene Expression Regulation, Humans, Promoter Regions, Genetic, Protein Binding, Protozoan Proteins genetics, CCAAT-Binding Factor physiology, Entamoeba physiology, Protozoan Proteins physiology
- Abstract
Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of three subunits, namely, NF-YA, NF-YB, and NF-YC, which are conserved throughout evolution. In higher eukaryotes, NF-Y plays important roles in several cellular processes (development, cell cycle regulation, apoptosis, and response to growth, stress, and DNA damage) by controlling gene expression through binding to a CCAAT promoter motif. We demonstrated that NF-Y subunits in the protist Entamoeba , while significantly divergent from those of higher eukaryotes, have well-conserved domains important for subunit interactions and DNA binding and that NF-YB and NF-YC are developmentally expressed during encystation. Electrophoretic mobility shift assays confirmed that the NF-Y protein(s) from Entamoeba cysts binds to a CCAAT motif. Consistent with a role as a transcription factor, the NF-Y proteins show nuclear localization during development. Additionally, we demonstrated that NF-YC localizes to the chromatoid body (an RNA processing center) during development, indicating that it may have a role in RNA processing. Finally, silencing of the NF-YC subunit resulted in reduced stability of the NF-Y complex and decreased encystation efficiency. We demonstrated that the NF-Y complex functions at a time point subsequent to the NAD
+ flux and expression of the transcription factor e ncystation r egulatory m otif-binding protein, both of which are early regulators of Entamoeba development. Taken together, our results demonstrate that the NF-Y complex plays an important role in regulating encystation in Entamoeba and add to our understanding of the transcriptional networks and signals that control this essential developmental pathway in an important human pathogen. IMPORTANCE The human parasite Entamoeba histolytica is an important pathogen with significant global impact and is a leading cause of parasitic death in humans. Since only the cyst form can be transmitted, blocking encystation would prevent new infections, making the encystation pathway an attractive target for the development of new drugs. Identification of the genetic signals and transcriptional regulatory networks that control encystation would be an important advance in understanding the developmental cascade. We show that the Entamoeba NF-Y complex plays a crucial role in regulating the encystation process in Entamoeba ., (Copyright © 2019 Manna and Singh.)- Published
- 2019
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46. Analysis of DrkA kinase's role in STATa activation.
- Author
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Saga Y, Iwade Y, Araki T, Ishikawa M, and Kawata T
- Subjects
- Animals, Dictyostelium cytology, Dictyostelium genetics, Morphogenesis, Mutation, Phosphorylation, Protein Kinases metabolism, Protein-Tyrosine Kinases metabolism, Protozoan Proteins genetics, Protozoan Proteins physiology, STAT Transcription Factors genetics, STAT Transcription Factors physiology, Tyrosine metabolism, Dictyostelium metabolism, Protozoan Proteins metabolism, STAT Transcription Factors metabolism
- Abstract
Dictyostelium STATa is a homologue of metazoan signal transducers and activators of transcription (STATs) and is important for morphogenesis. STATa is activated by phosphorylation on Tyr702 when cells are exposed to extracellular cAMP. Although two tyrosine kinase-like (TKL) proteins, Pyk2 and Pyk3, have been definitively identified as STATc kinases, no kinase is known for STATa activation. Based on homology to the previously identified tyrosine-selective TKLs, we identified DrkA, a member of the TKL family and the Dictyostelium receptor-like kinase (DRK) subfamily, as a candidate STATa kinase. The drkA gene is almost exclusively expressed in prestalk A (pstA) cells, where STATa is activated. Transient over-expression of DrkA increased STATa phosphorylation, although over-expression of the protein causes a severe growth defect and cell death. Furthermore, recombinant DrkA protein is auto-phosphorylated on tyrosine and threonine residues, and an in vitro kinase assay shows that DrkA can phosphorylate STATa on Tyr702 in a STATa-SH2 (phosphotyrosine binding) domain-dependent manner. These observations strongly suggest that DrkA is one of the key regulators of STATa tyrosine phosphorylation and is consistent with it being the kinase that directly activates STATa., (© 2019 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)
- Published
- 2019
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47. An essential contractile ring protein controls cell division in Plasmodium falciparum.
- Author
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Rudlaff RM, Kraemer S, Streva VA, and Dvorin JD
- Subjects
- Animals, Erythrocytes parasitology, Intravital Microscopy methods, Luminescent Proteins chemistry, Luminescent Proteins genetics, Microscopy, Electron, Transmission, Plasmodium falciparum ultrastructure, Schizonts physiology, Time-Lapse Imaging, Red Fluorescent Protein, Cell Division physiology, Contractile Proteins physiology, Plasmodium falciparum physiology, Protozoan Proteins physiology
- Abstract
During the blood stage of human malaria, Plasmodium falciparum parasites divide by schizogony-a process wherein components for several daughter cells are produced within a common cytoplasm and then segmentation, a synchronized cytokinesis, produces individual invasive daughters. The basal complex is hypothesized to be required for segmentation, acting as a contractile ring to establish daughter cell boundaries. Here we identify an essential component of the basal complex which we name PfCINCH. Using three-dimensional reconstructions of parasites at electron microscopy resolution, we show that while parasite organelles form and divide normally, PfCINCH-deficient parasites develop inviable conjoined daughters that contain components for multiple cells. Through biochemical evaluation of the PfCINCH-containing complex, we discover multiple previously undescribed basal complex proteins. Therefore, this work provides genetic evidence that the basal complex is required for precise segmentation and lays the groundwork for a mechanistic understanding of how the parasite contractile ring drives cell division.
- Published
- 2019
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48. Plasmodium falciparum sexual differentiation in malaria patients is associated with host factors and GDV1-dependent genes.
- Author
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Usui M, Prajapati SK, Ayanful-Torgby R, Acquah FK, Cudjoe E, Kakaney C, Amponsah JA, Obboh EK, Reddy DK, Barbeau MC, Simons LM, Czesny B, Raiciulescu S, Olsen C, Abuaku BK, Amoah LE, and Williamson KC
- Subjects
- Age Factors, Child, Child, Preschool, Female, Gametogenesis physiology, Genes, Protozoan physiology, Ghana, Humans, Lysophosphatidylcholines blood, Malaria, Falciparum blood, Male, Parasitemia parasitology, Plasmodium falciparum isolation & purification, Host-Parasite Interactions physiology, Malaria, Falciparum parasitology, Plasmodium falciparum physiology, Protozoan Proteins physiology, Sex Differentiation physiology
- Abstract
Plasmodium sexual differentiation is required for malaria transmission, yet much remains unknown about its regulation. Here, we quantify early gametocyte-committed ring (gc-ring) stage, P. falciparum parasites in 260 uncomplicated malaria patient blood samples 10 days before maturation to transmissible stage V gametocytes using a gametocyte conversion assay (GCA). Seventy six percent of the samples have gc-rings, but the ratio of gametocyte to asexual-committed rings (GCR) varies widely (0-78%). GCR correlates positively with parasitemia and is negatively influenced by fever, not hematocrit, age or leukocyte counts. Higher expression levels of GDV1-dependent genes, ap2-g, msrp1 and gexp5, as well as a gdv1 allele encoding H
217 are associated with high GCR, while high plasma lysophosphatidylcholine levels are associated with low GCR in the second study year. The results provide a view of sexual differentiation in the field and suggest key regulatory roles for clinical factors and gdv1 in gametocytogenesis in vivo.- Published
- 2019
- Full Text
- View/download PDF
49. Oscillatory cAMP cell-cell signalling persists during multicellular Dictyostelium development.
- Author
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Singer G, Araki T, and Weijer CJ
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases deficiency, 3',5'-Cyclic-AMP Phosphodiesterases genetics, 3',5'-Cyclic-AMP Phosphodiesterases physiology, Biological Clocks, Chemotaxis, Dictyostelium cytology, Dictyostelium growth & development, Fluorescence Resonance Energy Transfer, Genes, Reporter, Guanine Nucleotide Exchange Factors genetics, Guanine Nucleotide Exchange Factors physiology, Microscopy, Confocal, Microscopy, Fluorescence, Morphogenesis, Protozoan Proteins genetics, Protozoan Proteins physiology, Subcellular Fractions chemistry, Cyclic AMP physiology, Dictyostelium physiology, Second Messenger Systems physiology
- Abstract
Propagating waves of cAMP, periodically initiated in the aggregation centre, are known to guide the chemotactic aggregation of hundreds of thousands of starving individual Dictyostelium discoideum cells into multicellular aggregates. Propagating optical density waves, reflecting cell periodic movement, have previously been shown to exist in streaming aggregates, mounds and migrating slugs. Using a highly sensitive cAMP-FRET reporter, we have now been able to measure periodically propagating cAMP waves directly in these multicellular structures. In slugs cAMP waves are periodically initiated in the tip and propagate backward through the prespore zone. Altered cAMP signalling dynamics in mutants with developmental defects strongly support a key functional role for cAMP waves in multicellular Dictyostelium morphogenesis. These findings thus show that propagating cAMP not only control the initial aggregation process but continue to be the long range cell-cell communication mechanism guiding cell movement during multicellular Dictyostelium morphogenesis at the mound and slugs stages., Competing Interests: The authors declare no competing interests.
- Published
- 2019
- Full Text
- View/download PDF
50. Actin-binding domains mediate the distinct distribution of two Dictyostelium Talins through different affinities to specific subsets of actin filaments during directed cell migration.
- Author
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Tsujioka M, Uyeda TQP, Iwadate Y, Patel H, Shibata K, Yumoto T, and Yonemura S
- Subjects
- Actin Cytoskeleton metabolism, Binding Sites, Lipid Metabolism, Lipids chemistry, Protein Domains, Protozoan Proteins chemistry, Protozoan Proteins physiology, Talin chemistry, Talin physiology, Cell Movement, Dictyostelium metabolism, Protozoan Proteins analysis, Talin analysis
- Abstract
Although the distinct distribution of certain molecules along the anterior or posterior edge is essential for directed cell migration, the mechanisms to maintain asymmetric protein localization have not yet been fully elucidated. Here, we studied a mechanism for the distinct localizations of two Dictyostelium talin homologues, talin A and talin B, both of which play important roles in cell migration and adhesion. Using GFP fusion, we found that talin B, as well as its C-terminal actin-binding region, which consists of an I/LWEQ domain and a villin headpiece domain, was restricted to the leading edge of migrating cells. This is in sharp contrast to talin A and its C-terminal actin-binding domain, which co-localized with myosin II along the cell posterior cortex, as reported previously. Intriguingly, even in myosin II-null cells, talin A and its actin-binding domain displayed a specific distribution, co-localizing with stretched actin filaments. In contrast, talin B was excluded from regions rich in stretched actin filaments, although a certain amount of its actin-binding region alone was present in those areas. When cells were sucked by a micro-pipette, talin B was not detected in the retracting aspirated lobe where acto-myosin, talin A, and the actin-binding regions of talin A and talin B accumulated. Based on these results, we suggest that talin A predominantly interacts with actin filaments stretched by myosin II through its C-terminal actin-binding region, while the actin-binding region of talin B does not make such distinctions. Furthermore, talin B appears to have an additional, unidentified mechanism that excludes it from the region rich in stretched actin filaments. We propose that these actin-binding properties play important roles in the anterior and posterior enrichment of talin B and talin A, respectively, during directed cell migration., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
- Full Text
- View/download PDF
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