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1.
Int J Mol Sci ; 24(8)2023 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-37108334

RESUMO

Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally catalyzed by three types of prenyl transferases, which include farnesyl transferase (FT), geranylgeranyl transferase (GGT-1) and Rab geranylgeranyl transferase (GGT-2). Studies in malaria parasites showed that these parasites contain prenylated proteins, which are proposed to play multiple functions in parasites. However, the prenyl transferases have not been functionally characterized in parasites of subphylum Apicomplexa. Here, we functionally dissected functions of three of the prenyl transferases in the Apicomplexa model organism Toxoplasma gondii (T. gondii) using a plant auxin-inducible degron system. The homologous genes of the beta subunit of FT, GGT-1 and GGT-2 were endogenously tagged with AID at the C-terminus in the TIR1 parental line using a CRISPR-Cas9 approach. Upon depletion of these prenyl transferases, GGT-1 and GGT-2 had a strong defect on parasite replication. Fluorescent assay using diverse protein markers showed that the protein markers ROP5 and GRA7 were diffused in the parasites depleted with GGT-1 and GGT-2, while the mitochondrion was strongly affected in parasites depleted with GGT-1. Importantly, depletion of GGT-2 caused the stronger defect to the sorting of rhoptry protein and the parasite morphology. Furthermore, parasite motility was observed to be affected in parasites depleted with GGT-2. Taken together, this study functionally characterized the prenyl transferases, which contributed to an overall understanding of protein prenylation in T. gondii and potentially in other related parasites.


Assuntos
Parasitos , Toxoplasma , Animais , Transferases/metabolismo , Parasitos/metabolismo , Toxoplasma/metabolismo , Farnesiltranstransferase/metabolismo , Prenilação de Proteína , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
2.
PLoS Pathog ; 13(5): e1006379, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28475612

RESUMO

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion.


Assuntos
Calmodulina/metabolismo , Modelos Moleculares , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Calmodulina/genética , Movimento Celular , Citoesqueleto/metabolismo , Técnicas de Inativação de Genes , Interações Hospedeiro-Parasita , Espectrometria de Massas , Miosinas/genética , Miosinas/metabolismo , Organismos Geneticamente Modificados , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/citologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade
3.
Infect Immun ; 84(5): 1262-1273, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26755159

RESUMO

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division in T. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes in T. gondii.


Assuntos
Cálcio/metabolismo , Técnicas de Inativação de Genes/métodos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Deleção de Sequência , Toxoplasma/enzimologia , Toxoplasma/genética , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Feminino , Genes Essenciais , Genes de Protozoários , Camundongos Endogâmicos C57BL , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/parasitologia , Toxoplasmose/patologia
4.
Antimicrob Agents Chemother ; 60(1): 570-9, 2016 01.
Artigo em Inglês | MEDLINE | ID: mdl-26552986

RESUMO

Cryptosporidiosis is a serious diarrheal disease in immunocompromised patients and malnourished children, and treatment is complicated by a lack of adequate drugs. Recent studies suggest that the natural occurrence of a small gatekeeper residue in serine threonine calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium parvum might be exploited to target this enzyme and block parasite growth. Here were explored the potency with which a series of pyrazolopyrimidine analogs, which are selective for small gatekeeper kinases, inhibit C. parvum CDPK1 and block C. parvum growth in tissue culture in vitro. Although these compounds potently inhibited kinase activity in vitro, most had no effect on parasite growth. Moreover, among those that were active against parasite growth, there was a very poor correlation with their 50% inhibitory concentrations against the enzyme. Active compounds also had no effect on cell invasion, unlike the situation in Toxoplasma gondii, where these compounds block CDPK1, prevent microneme secretion, and disrupt cell invasion. These findings suggest that CPDK1 is not essential for C. parvum host cell invasion or growth and therefore that it is not the optimal target for therapeutic intervention. Nonetheless, several inhibitors with low micromolar 50% effective concentrations were identified, and these may affect other essential targets in C. parvum that are worthy of further exploration.


Assuntos
Antiprotozoários/farmacologia , Cryptosporidium parvum/efeitos dos fármacos , Proteínas Quinases/química , Proteínas de Protozoários/química , Pirazóis/farmacologia , Pirimidinas/farmacologia , Esporozoítos/efeitos dos fármacos , Animais , Antiprotozoários/síntese química , Bovinos , Linhagem Celular , Cryptosporidium parvum/enzimologia , Cryptosporidium parvum/genética , Cryptosporidium parvum/crescimento & desenvolvimento , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/parasitologia , Fezes/parasitologia , Expressão Gênica , Humanos , Concentração Inibidora 50 , Masculino , Testes de Sensibilidade Parasitária , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Pirazóis/síntese química , Pirimidinas/síntese química , Esporozoítos/enzimologia , Esporozoítos/crescimento & desenvolvimento , Relação Estrutura-Atividade
5.
Proc Natl Acad Sci U S A ; 109(26): 10426-31, 2012 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-22699510

RESUMO

Iron/sulfur cluster (ISC)-containing proteins are essential components of cells. In most eukaryotes, Fe/S clusters are synthesized by the mitochondrial ISC machinery, the cytosolic iron/sulfur assembly system, and, in photosynthetic species, a plastid sulfur-mobilization (SUF) system. Here we show that the anaerobic human protozoan parasite Blastocystis, in addition to possessing ISC and iron/sulfur assembly systems, expresses a fused version of the SufC and SufB proteins of prokaryotes that it has acquired by lateral transfer from an archaeon related to the Methanomicrobiales, an important lineage represented in the human gastrointestinal tract microbiome. Although components of the Blastocystis ISC system function within its anaerobic mitochondrion-related organelles and can functionally replace homologues in Trypanosoma brucei, its SufCB protein has similar biochemical properties to its prokaryotic homologues, functions within the parasite's cytosol, and is up-regulated under oxygen stress. Blastocystis is unique among eukaryotic pathogens in having adapted to its parasitic lifestyle by acquiring a SUF system from nonpathogenic Archaea to synthesize Fe/S clusters under oxygen stress.


Assuntos
Evolução Biológica , Blastocystis/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Anaerobiose , Animais , Dados de Sequência Molecular , Filogenia
6.
Mol Microbiol ; 89(1): 135-51, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23675735

RESUMO

Ferredoxins are highly conserved proteins that function universally as electron transporters. They not only require Fe-S clusters for their own activity, but are also involved in Fe-S formation itself. We identified two homologues of ferredoxin in the genome of the parasitic protist Trypanosoma brucei and named them TbFdxA and TbFdxB. TbFdxA protein, which is homologous to other eukaryotic mitochondrial ferredoxins, is essential in both the procyclic (= insect-transmitted) and bloodstream (mammalian) stage, but is more abundant in the active mitochondrion of the former stage. Depletion of TbFdxA caused disruption of Fe-S cluster biogenesis and lowered the level of intracellular haem. However, TbFdxB, which is present exclusively within kinetoplastid flagellates, was non-essential for the procyclic stage, and double knock-down with TbFdxA showed this was not due to functional redundancy between the two homologues. Heterologous expressions of human orthologues HsFdx1 and HsFdx2 fully rescued the growth and Fe-S-dependent enzymatic activities of TbFdxA knock-down. In both cases, the genuine human import signals allowed efficient import into the T. brucei mitochondrion. Given the huge evolutionary distance between trypanosomes and humans, ferredoxins clearly have ancestral and highly conserved function in eukaryotes and both human orthologues have retained the capacity to participate in Fe-S cluster assembly.


Assuntos
Ferredoxinas/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/metabolismo , Análise por Conglomerados , Transporte de Elétrons , Ferredoxinas/genética , Técnicas de Silenciamento de Genes , Teste de Complementação Genética , Humanos , Filogenia , Transporte Proteico , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/genética
7.
Commun Biol ; 7(1): 596, 2024 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-38762629

RESUMO

Apicomplexan parasites harbor a complex endomembrane system as well as unique secretory organelles. These complex cellular structures require an elaborate vesicle trafficking system, which includes Rab GTPases and their regulators, to assure the biogenesis and secretory of the organelles. Here we exploit the model apicomplexan organism Toxoplasma gondii that encodes a family of Rab GTPase Activating Proteins, TBC (Tre-2/Bub2/Cdc16) domain-containing proteins. Functional profiling of these proteins in tachyzoites reveals that TBC9 is the only essential regulator, which is localized to the endoplasmic reticulum (ER) in T. gondii strains. Detailed analyses demonstrate that TBC9 is required for normal distribution of proteins targeting to the ER, and the Golgi apparatus in the parasite, as well as for the normal formation of daughter inner membrane complexes (IMCs). Pull-down assays show a strong protein interaction between TBC9 and specific Rab GTPases (Rab11A, Rab11B, and Rab2), supporting the role of TBC9 in daughter IMC formation and early vesicular transport. Thus, this study identifies the only essential TBC domain-containing protein TBC9 that regulates early vesicular transport and IMC formation in T. gondii and potentially in closely related protists.


Assuntos
Retículo Endoplasmático , Proteínas Ativadoras de GTPase , Proteínas de Protozoários , Toxoplasma , Proteínas rab de Ligação ao GTP , Toxoplasma/metabolismo , Toxoplasma/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Retículo Endoplasmático/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Complexo de Golgi/metabolismo , Transporte Proteico , Animais , Vesículas Transportadoras/metabolismo
8.
Trends Parasitol ; 40(5): 416-426, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38637184

RESUMO

The micropore, a mysterious structure found in apicomplexan species, was recently shown to be essential for nutrient acquisition in Plasmodium falciparum and Toxoplasma gondii. However, the differences between the micropores of these two parasites questions the nature of a general apicomplexan micropore structure and whether the formation process model from Plasmodium can be applied to other apicomplexans. We analyzed the literature on different apicomplexan micropores and found that T. gondii probably harbors a more representative micropore type than the more widely studied ones in Plasmodium. Using recent knowledge of the Kelch 13 (K13) protein interactome and gene depletion phenotypes in the T. gondii micropore, we propose a model of micropore formation, thus enriching our wider understanding of micropore protein function.


Assuntos
Apicomplexa , Plasmodium falciparum , Toxoplasma , Apicomplexa/fisiologia , Apicomplexa/genética , Toxoplasma/genética , Toxoplasma/fisiologia , Plasmodium falciparum/fisiologia , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética
9.
Microbiol Spectr ; : e0066124, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39162521

RESUMO

Toxoplasma gondii, the causative parasite of toxoplasmosis, is an apicomplexan parasite that infects warm-blooded mammals. The ability of the calcium-binding proteins (CBPs) to transport large amounts of Ca2+ appears to be critical for the biological activity of T. gondii. However, the functions of some members of the CBP family have not yet been deciphered. Here, we characterized a putative CBP of T. gondii, TgpCaBP (TGME49_229480), which is composed of four EF-hand motifs with Ca2+-binding capability. TgpCaBP was localized in the cytosol and ER of T. gondii, and parasites lacking the TgpCaBP gene exhibited diminished abilities in cell invasion, intracellular growth, egress, and motility. These phenomena were due to the abnormalities in intracellular Ca2+ efflux and ER Ca2+ storage, and the reduction in motility was associated with a decrease in the discharge of secretory proteins. Therefore, we propose that TgpCaBP is a Ca2+ transporter and signaling molecule involved in Ca2+ regulation and parasitization in the hosts.IMPORTANCECa2+ signaling is essential in the development of T. gondii. In this study, we identified a calcium-binding protein in T. gondii, named TgpCaBP, which actively regulates intracellular Ca2+ levels in the parasite. Deletion of the gene coding for TgpCaBP caused serious deficits in the parasite's ability to maintain a stable intracellular calcium environment, which also impaired the secretory protein discharged from the parasite, and its capacity of gliding motility, cell invasion, intracellular growth, and egress from host cells. In summary, we have identified a novel calcium-binding protein, TgpCaBP, in the zoonotic parasite T. gondii, which is a potential therapeutic target for toxoplasmosis.

10.
Elife ; 122024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38502570

RESUMO

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here, we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.


Assuntos
Apicoplastos , Transportadores de Ácidos Monocarboxílicos , Parasitos , Toxoplasma , Animais , Camundongos , Apicoplastos/metabolismo , Ácidos Graxos/metabolismo , Compostos Orgânicos/metabolismo , Parasitos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo
11.
Parasit Vectors ; 17(1): 142, 2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38500196

RESUMO

BACKGROUND: The protozoan parasite Toxoplasma gondii encodes dozens of phosphatases, among which a plant-like phosphatase absent from mammalian genomes named PPKL, which is involved in regulating brassinosteroid signaling in Arabidopsis, was identified in the genome. Among the Apicomplexa parasites, T. gondii is an important and representative pathogen in humans and animals. PPKL was previously identified to modulate the apical integrity and morphology of the ookinetes and parasite motility and transmission in another important parasite, Plasmodium falciparum. However, the exact function of PPKL in the asexual stages of T. gondii remains unknown. METHODS: The plant auxin-inducible degron (AID) system was applied to dissect the phenotypes of PPKL in T. gondii. We first analyzed the phenotypes of the AID parasites at an induction time of 24 h, by staining of different organelles using their corresponding markers. These analyses were further conducted for the parasites grown in auxin for 6 and 12 h using a quantitative approach and for the type II strain ME49 of AID parasites. To further understand the phenotypes, the potential protein interactions were analyzed using a proximity biotin labeling approach. The essential role of PPKL in parasite replication was revealed. RESULTS: PPKL is localized in the apical region and nucleus and partially distributed in the cytoplasm of the parasite. The phenotyping of PPKL showed its essentiality for parasite replication and morphology. Further dissections demonstrate that PPKL is required for the maturation of daughter parasites in the mother cells, resulting in multiple nuclei in a single parasite. The phenotype of the daughter parasites and parasite morphology were observed in another type of T. gondii strain ME49. The substantial defect in parasite replication and morphology could be rescued by genetic complementation, thus supporting its essential function for PPKL in the formation of parasites. The protein interaction analysis showed the potential interaction of PPKL with diverse proteins, thus explaining the importance of PPKL in the parasite. CONCLUSIONS: PPKL plays an important role in the formation of daughter parasites, revealing its subtle involvement in the proper maturation of the daughter parasites during division. Our detailed analysis also demonstrated that depletion of PPKL resulted in elongated tubulin fibers in the parasites. The important roles in the parasites are potentially attributed to the protein interaction mediated by kelch domains on the protein. Taken together, these findings contribute to our understanding of a key phosphatase involved in parasite replication, suggesting the potential of this phosphatase as a pharmaceutic target.


Assuntos
Parasitos , Toxoplasma , Humanos , Animais , Toxoplasma/fisiologia , Proteínas de Plantas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Ácidos Indolacéticos/metabolismo , Mamíferos
12.
Parasit Vectors ; 16(1): 409, 2023 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-37941035

RESUMO

BACKGROUND: The protozoan parasite Toxoplasma gondii encodes a dozen Rab proteins, which are parts of the small GTPase superfamily and regulate intracellular membrane trafficking. Our previous study showed that depletion of Rab1B caused severe defects regarding parasite growth and morphological structure, yet early defects of endocytic trafficking and vesicle sorting to the rhoptry in T. gondii are not expected to have a strong effect. To understand this discrepancy, we performed an integrated analysis at the level of transcriptomics and metabolomics. METHODS: In the study, tetracycline-inducible TATi/Ty-Rab1B parasite line treated with ATc at three different time points (0, 18 and 24 h) was used. We first observed the morphological changes caused by Rab1B depletion via transmission electron technology. Then, high-throughput transcriptome along with non-targeted metabolomics were performed to analyze the RNA expression and metabolite changes in the Rab1B-depleted parasite. The essential nature of Rab1B in the parasite was revealed by the integrated omics approach. RESULTS: Transmission electron micrographs showed a strong disorganization of endo-membranes in the Rab1B-depleted parasites. Our deep analysis of transcriptome and metabolome identified 2181 and 2374 differentially expressed genes (DEGs) and 30 and 83 differentially expressed metabolites (DEMs) at 18 and 24 h of induction in the tetracycline-inducible parasite line, respectively. These DEGs included key genes associated with crucial organelles that contain the rhoptry, microneme, endoplasmic reticulum and Golgi apparatus. The analysis of qRT-PCR verified some of the key DEGs identified by RNA-Seq, supporting that the key vesicular regulator Rab1B was involved in biogenesis of multiple parasite organelles. Functional enrichment analyses revealed pathways related to central carbon metabolisms and lipid metabolisms, such as the TCA cycle, glycerophospholipid metabolism and fatty acid biosynthesis and elongation. Further correlation analysis of the major DEMs and DEGs supported the role of Rab1B in biogenesis of fatty acids (e.g. myrisoleic acid and oleic acid) (R > 0.95 and P < 0.05), which was consistent with the scavenging role in biotin via the endocytic process. CONCLUSIONS: Rab1B played an important role in parasite growth and morphology, which was supported by the replication assay and transmission electron microscopy observation. Our multi-omics analyses provided detailed insights into the overall impact on the parasite upon depletion of the protein. These analyses reinforced the role of Rab1B in the endocytic process, which has an impact on fatty acid biogenesis and the TCA cycle. Taken together, these findings contribute to our understanding of a key vesicular regulator, Rab1B, on parasite metabolism and morphological formation in T. gondii.


Assuntos
Parasitos , Toxoplasma , Animais , Toxoplasma/genética , Toxoplasma/metabolismo , Transcriptoma , Perfilação da Expressão Gênica , Parasitos/genética , Ácidos Graxos/metabolismo , Tetraciclinas/metabolismo , Proteínas de Protozoários/genética
13.
Nat Commun ; 14(1): 977, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36813769

RESUMO

Apicomplexan parasite growth and replication relies on nutrient acquisition from host cells, in which intracellular multiplication occurs, yet the mechanisms that underlie the nutrient salvage remain elusive. Numerous ultrastructural studies have documented a plasma membrane invagination with a dense neck, termed the micropore, on the surface of intracellular parasites. However, the function of this structure remains unknown. Here we validate the micropore as an essential organelle for endocytosis of nutrients from the host cell cytosol and Golgi in the model apicomplexan Toxoplasma gondii. Detailed analyses demonstrated that Kelch13 is localized at the dense neck of the organelle and functions as a protein hub at the micropore for endocytic uptake. Intriguingly, maximal activity of the micropore requires the ceramide de novo synthesis pathway in the parasite. Thus, this study provides insights into the machinery underlying acquisition of host cell-derived nutrients by apicomplexan parasites that are otherwise sequestered from host cell compartments.


Assuntos
Toxoplasma , Toxoplasma/metabolismo , Endocitose , Complexo de Golgi/metabolismo , Transporte Biológico , Proteínas de Protozoários/metabolismo
14.
mBio ; 14(4): e0130923, 2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37548452

RESUMO

In the apicomplexans, endocytosed cargos (e.g., hemoglobin) are trafficked to a specialized organelle for digestion. This follows a unique endocytotic process at the micropore/cytostome in these parasites. However, the mechanism underlying endocytic trafficking remains elusive, due to the repurposing of classical endocytic proteins for the biogenesis of apical organelles. To resolve this issue, we have exploited the genetic tractability of the model apicomplexan Toxoplasma gondii, which ingests host cytosolic materials (e.g., green fluorescent protein[GFP]). We determined an association between protein prenylation and endocytic trafficking, and using an alkyne-labeled click chemistry approach, the prenylated proteome was characterized. Genome editing, using clustered regularly interspaced short palindromic repaet/CRISPR-associated nuclease 9 (CRISPR/Cas9), was efficiently utilized to generate genetically modified lines for the functional screening of 23 prenylated candidates. This identified four of these proteins that regulate the trafficking of endocytosed GFP vesicles. Among these proteins, Rab1B and YKT6.1 are highly conserved but are non-classical endocytic proteins in eukaryotes. Confocal imaging analysis showed that Rab1B and Ras are substantially localized to both the trans-Golgi network and the endosome-like compartments in the parasite. Conditional knockdown of Rab1B caused a rapid defect in secretory trafficking to the rhoptry bulb, suggesting a trafficking intersection role for the key regulator Rab1B. Further experiments confirmed a critical role for protein prenylation in regulating the stability/activity of these proteins (i.e., Rab1B and YKT6.1) in the parasite. Our findings define the molecular basis of endocytic trafficking and reveal a potential intersection function of Rab1B on membrane trafficking in T. gondii. This might extend to other related protists, including the malarial parasites. IMPORTANCE The protozoan Toxoplasma gondii establishes a permissive niche, in host cells, that allows parasites to acquire large molecules such as proteins. Numerous studies have demonstrated that the parasite repurposes the classical endocytic components for secretory sorting to the apical organelles, leaving the question of endocytic transport to the lysosome-like compartment unclear. Recent studies indicated that endocytic trafficking is likely to associate with protein prenylation in malarial parasites. This information promoted us to examine this association in the model apicomplexan T. gondii and to identify the key components of the prenylated proteome that are involved. By exploiting the genetic tractability of T. gondii and a host GFP acquisition assay, we reveal four non-classical endocytic proteins that regulate the transport of endocytosed cargos (e.g., GFP) in T. gondii. Thus, we extend the principle that protein prenylation regulates endocytic trafficking and elucidate the process of non-classical endocytosis in T. gondii and potentially in other related protists.


Assuntos
Toxoplasma , Toxoplasma/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/genética , Transporte Proteico , Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo
15.
Mol Microbiol ; 81(6): 1403-18, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21790804

RESUMO

IscA/Isa proteins function as alternative scaffolds for the assembly of Fe-S clusters and/or provide iron for their assembly in prokaryotes and eukaryotes. Isa are usually non-essential and in most organisms are confined to the mitochondrion. We have studied the function of TbIsa1 and TbIsa2 in Trypanosoma brucei, where the requirement for both of them to sustain cell growth depends on the life cycle stage. The TbIsa proteins are abundant in the procyclic form, which contains an active organelle. Both proteins are indispensable for growth, as they are required for the assembly of Fe-S clusters in mitochondrial aconitase, fumarase and succinate dehydrogenase. Reactive oxygen species but not iron accumulate in the procyclic mitochondrion upon ablation of the TbIsa proteins, but their depletion does not influence the assembly of Fe-S clusters in cytosolic proteins. In the bloodstream form, which has a downregulated mitochondrion, the TbIsa proteins are non-essential. The Isa2 orthologue of the anaerobic protist Blastocystis partially rescued the growth and enzymatic activities of TbIsa1/2 knock-down. Rescues of single knock-downs as well as heterologous rescues with human Isa orthologues partially recovered the activities of aconitase and fumarase. These results show that the Isa1 and Isa2 proteins of diverse eukaryotes have overlapping functions.


Assuntos
Deleção de Genes , Teste de Complementação Genética , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Blastocystis/genética , Sobrevivência Celular , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo
16.
Proc Natl Acad Sci U S A ; 105(36): 13468-73, 2008 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-18768799

RESUMO

Trypanosoma brucei, the agent of human sleeping sickness and ruminant nagana, is the most genetically tractable representative of the domain Excavata. It is evolutionarily very distant from humans, with a last common ancestor over 1 billion years ago. Frataxin, a highly conserved small protein involved in iron-sulfur cluster synthesis, is present in both organisms, and its deficiency is responsible for Friedreich's ataxia in humans. We have found that T. brucei growth-inhibition phenotype caused by down-regulated frataxin is rescued by means of human frataxin. The rescue is fully dependent on the human frataxin being imported into the trypanosome mitochondrion. Processing of the imported protein by mitochondrial processing peptidase can be blocked by mutations in the signal peptide, as in human cells. Although in human cells frataxin must be processed to execute its function, the same protein in the T. brucei mitochondrion is functional even in the absence of processing. Our results illuminate remarkable conservation of the mechanisms of mitochondrial protein import and processing.


Assuntos
Proteínas de Ligação ao Ferro/metabolismo , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Linhagem Celular , Citosol/metabolismo , Humanos , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/genética , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Frataxina
17.
Mol Microbiol ; 69(1): 94-109, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18433447

RESUMO

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe-S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin-depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe-S cluster biogenesis and protection from reactive oxygen species.


Assuntos
Evolução Molecular , Expressão Gênica , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Mitocondriais/metabolismo , Trichomonas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Eucarióticas/classificação , Células Eucarióticas/fisiologia , Humanos , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/genética , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Fenótipo , Filogenia , Células Procarióticas/classificação , Células Procarióticas/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , Alinhamento de Sequência , Trichomonas/química , Trichomonas/classificação , Trichomonas/genética , Frataxina
18.
Colloids Surf B Biointerfaces ; 180: 473-480, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31102851

RESUMO

Evolutionary pressure has pushed many extant plants and animals to develop micro/nanostructures on their surfaces to keep them clean. These structures have become ideal models for bio-inspired design. Although microstructures on biological surfaces have been widely studied, little attention has been paid to the combined role of microstructures and animal's active cleaning behaviors in keeping their surfaces clean. In this study, we explored the relationship between these micro/nanostructures and wettability as well as the role of the housefly cleaning behaviors in keeping their wings clean. Hierarchical structures consisting of microscale macrotrichias with nanoscale grooves on the wings were observed under scanning electron microscope. The wings were hydrophobic (CA = 133.3°) but with high adhesion to water (CAH = 87.5°), indicating that they were non-self-cleaning surfaces. Macroscale droplets standing on the wings could be best described as being in a transitional wetting state between Wenzel and Cassie-Baxter states due to the presence of the nanoscale grooves, which increased the resistance to water penetration. The hydrophobicity decreased (CA = 109.9°) when the nanostructures were removed by coating the wings with a thick layer of polydimethylsiloxane (PDMS). The houseflies could highly efficiently remove the microscale droplets atop the macrotrichias, and reduce bacterial contamination on their wings through grooming and flutter activities. These active cleaning behaviors could offset the absence of self-cleaning properties and play a key role in keeping the wings clean. The results indicate that housefly wings could be used as a template for the design of special functional surfaces. The present findings not only improve our understanding of the wettability and cleaning properties of natural surfaces, but also provide important insights into the design of bio-inspired materials.


Assuntos
Comportamento Animal/fisiologia , Moscas Domésticas/anatomia & histologia , Produtos Domésticos , Asas de Animais/anatomia & histologia , Animais , Dimetilpolisiloxanos/química , Moscas Domésticas/ultraestrutura , Modelos Teóricos , Molhabilidade , Asas de Animais/ultraestrutura
19.
Mol Biochem Parasitol ; 162(1): 100-4, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18765259

RESUMO

Frataxin is a conserved mitochondrial protein, almost universally present in prokaryotes and eukaryotes, where it is implicated in Fe-S cluster assembly and several other processes. Here we show that frataxins from the diatom Thalassiosira pseudonana and the plant Arabidopsis thaliana are efficiently targeted and processed in the mitochondrion of the evolutionary distant excavate kinetoplastid flagellate Trypanosoma brucei. Moreover, both heterologous frataxins are able to rescue a lethal deficiency for T. brucei frataxin.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Diatomáceas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Diatomáceas/genética , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/genética , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestrutura , Frataxina
20.
Bio Protoc ; 8(4)2018 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-29644255

RESUMO

Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii's 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to functionally investigate hundreds of putative essential protein coding genes. Accordingly, we recently implemented the auxin-inducible degron (AID) system for studying essential proteins in T. gondii. Here we provide a step-by-step protocol for examining protein function in T. gondii using the AID system in a tissue culture setting.

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