RESUMEN
Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.
Asunto(s)
Compuestos de Boro/metabolismo , Carboxipeptidasas/metabolismo , Tripanocidas/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma congolense/enzimología , Trypanosoma vivax/enzimología , Tripanosomiasis Africana/veterinaria , Valina/análogos & derivados , Animales , Ácidos Carboxílicos/metabolismo , Resistencia a Medicamentos , Femenino , Ganado , Ratones , Parasitemia/veterinaria , Profármacos/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma congolense/efectos de los fármacos , Trypanosoma vivax/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología , Valina/metabolismoRESUMEN
Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.
Asunto(s)
Variación Antigénica , ADN Polimerasa Dirigida por ADN/metabolismo , Telómero/genética , Trypanosoma brucei brucei/genética , Línea Celular , Núcleo Celular/enzimología , Núcleo Celular/genética , Segregación Cromosómica , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , Regulación de la Expresión Génica , Genoma de Protozoos , Humanos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interferencia de ARN , Telómero/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/patogenicidad , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , ADN Polimerasa thetaRESUMEN
All cells are subject to structural damage that must be addressed for continued growth. A wide range of damage affects the genome, meaning multiple pathways have evolved to repair or bypass the resulting DNA lesions. Though many repair pathways are conserved, their presence or function can reflect the life style of individual organisms. To identify genome maintenance pathways in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens to identify genes important for survival following exposure to the alkylating agent methyl methanesulphonate. Amongst a cohort of broadly conserved and, therefore, early evolved repair pathways, we reveal multiple activities not so far examined functionally in T. brucei, including DNA polymerases, DNA helicases and chromatin factors. In addition, the screens reveal Trypanosoma- or kinetoplastid-specific repair-associated activities. We also provide focused analyses of repair-associated protein kinases and show that loss of at least nine, and potentially as many as 30 protein kinases, including a nuclear aurora kinase, sensitises T. brucei to alkylation damage. Our results demonstrate the potential for synthetic lethal genome-wide screening of gene function in T. brucei and provide an evolutionary perspective on the repair pathways that underpin effective responses to damage, with particular relevance for related kinetoplastid pathogens. By revealing that a large number of diverse T. brucei protein kinases act in the response to damage, we expand the range of eukaryotic signalling factors implicated in genome maintenance activities.
Asunto(s)
Reparación del ADN , Genoma de Protozoos , Proteínas Quinasas/genética , Proteínas Protozoarias/genética , Interferencia de ARN , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Daño del ADN/efectos de los fármacos , Evolución Molecular , Metilmetanosulfonato/análogos & derivados , Metilmetanosulfonato/toxicidad , Mutágenos/toxicidad , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
Kinetoplastid parasites cause lethal diseases in humans and animals. The kinetoplast itself contains the mitochondrial genome, comprising a huge, complex DNA network that is also an important drug target. Isometamidium, for example, is a key veterinary drug that accumulates in the kinetoplast in African trypanosomes. Kinetoplast independence and isometamidium resistance are observed where certain mutations in the F1-γ-subunit of the two-sector F1Fo-ATP synthase allow for Fo-independent generation of a mitochondrial membrane potential. To further explore kinetoplast biology and drug resistance, we screened a genome-scale RNA interference library in African trypanosomes for isometamidium resistance mechanisms. Our screen identified 14 V-ATPase subunits and all 4 adaptin-3 subunits, implicating acidic compartment defects in resistance; V-ATPase acidifies lysosomes and related organelles, whereas adaptin-3 is responsible for trafficking among these organelles. Independent strains with depleted V-ATPase or adaptin-3 subunits were isometamidium resistant, and chemical inhibition of the V-ATPase phenocopied this effect. While drug accumulation in the kinetoplast continued after V-ATPase subunit depletion, acriflavine-induced kinetoplast loss was specifically tolerated in these cells and in cells depleted for adaptin-3 or endoplasmic reticulum membrane complex subunits, also identified in our screen. Consistent with kinetoplast dispensability, V-ATPase defective cells were oligomycin resistant, suggesting ATP synthase uncoupling and bypass of the normal Fo-A6-subunit requirement; this subunit is the only kinetoplast-encoded product ultimately required for viability in bloodstream-form trypanosomes. Thus, we describe 30 genes and 3 protein complexes associated with kinetoplast-dependent growth. Mutations affecting these genes could explain natural cases of dyskinetoplasty and multidrug resistance. Our results also reveal potentially conserved communication between the compartmentalized two-sector rotary ATPases.
Asunto(s)
ADN de Cinetoplasto/metabolismo , Resistencia a Medicamentos , Mitocondrias/enzimología , ATPasas de Translocación de Protón/metabolismo , Trypanosoma brucei brucei/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Ácidos/metabolismo , Animales , Compartimento Celular/efectos de los fármacos , Resistencia a Medicamentos/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Mitocondrias/efectos de los fármacos , Fenantridinas/química , Fenantridinas/farmacología , Reacción en Cadena de la Polimerasa , Subunidades de Proteína/metabolismo , Interferencia de ARN/efectos de los fármacos , Reproducibilidad de los Resultados , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genéticaRESUMEN
The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His(99) and Cys(136)), and an Asp (Asp(134)) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.
Asunto(s)
Alelos , Proteasas de Cisteína , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas Protozoarias , Trypanosoma brucei brucei , Animales , Dominio Catalítico , Proteasas de Cisteína/biosíntesis , Proteasas de Cisteína/química , Proteasas de Cisteína/genética , Ratones , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genéticaRESUMEN
The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.
Asunto(s)
Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Línea Celular , Fibroblastos/parasitología , Humanos , Orgánulos/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/genética , Vías SecretorasRESUMEN
Thorough bioinformatic and phylogenetic analyses of Plasmodium falciparum tyrosine kinase-like kinase (TKL) sequences revealed a clear evolutionary relationship of PF3D7_1121300 (thereafter called PfTKL2) to the IL-1 receptor-associated kinase (IRAK)/receptor-like kinase (RLK)/Pelle protein family. We identified a novel conserved motif that is unique to this family, as well as an insertion whose length allows distribution of its members into two distinct subfamilies, in a way that matches exactly the dichotomy between 'Tube/Tube-like kinases' (TTLKs) and 'Pelle-like kinases' (PLKs) distinguished previously on the basis of features in accessory domains. The PfTKL2 protein is expressed ubiquitously in asexual blood stages and in gametocytes, and the recombinant enzyme displays kinase activity in vitro. The protein is exported to the host erythrocyte; furthermore, in accordance with data from a previous study of the extracellular proteome of Plasmodium-infected erythrocytes, we show that PfTKL2 is secreted into the culture medium. Considering the functions of other members of the RLK/Pelle family in immunity, and its secretion to the extracellular medium, we speculate that PfTKL2 functions may include an immunomodulatory role promoting parasite survival in the human host.
Asunto(s)
Plasmodium falciparum/enzimología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Biología Computacional , Eritrocitos/parasitología , Humanos , Plasmodium falciparum/genéticaRESUMEN
Background: Soft tissue sarcomas are rare and often go undetected until a later stage, particularly when they present as intra-articular or tenosynovial lesions mimicking benign synovial pathologies. The failure to distinguish between malignant and benign synovial disease can have a significant impact on patient outcomes and limit alternatives for local control surgery and limb salvage. Case Description: In this case series, we present two cases of soft tissue sarcomas, one being an intraarticular synovial chondrosarcoma, and the other a pleomorphic spindle cell sarcoma centred along tendon sheaths. Radiologically, the initial clinical presentation of these cases resembled benign synovial pathologies, leading to a delay in diagnosis and treatment. Conclusion: Our study underscores the importance of maintaining a low threshold of suspicion for surveillance, a multidisciplinary approach, and early histological diagnosis to ensure appropriate timely treatment and a favourable prognosis for patients with soft tissue sarcomas.
RESUMEN
Introduction: Chondrosarcoma of the synovium is a rare and malignant form of cartilaginous tumor that originates in synovial tissue. There have only been a limited number of reported cases of malignant transformation of synovial chondromatosis (SC) into secondary chondrosarcoma (SCH), primarily in the hip and knee, in patients with resistant illness. The occurrence of chondrosarcoma in SC of the wrist is highly uncommon, as evidenced by only a single previous case study that has been documented in the literature. Case Report: This study presents a case series of two patients with primary SC who developed SCH at the wrist joint. Conclusion: Clinicians treating localized swellings of the hand and wrist should be alert to the likelihood of a sarcoma diagnosis to minimize delays to definitive therapy.
RESUMEN
Aurora kinases compose a family of conserved Ser/Thr protein kinases playing essential roles in eukaryotic cell division. To date, Aurora homologues remain uncharacterized in the protozoan phylum Apicomplexa. In malaria parasites, the characterization of Aurora kinases may help understand the cell cycle control during erythrocytic schizogony where asynchronous nuclear divisions occur. In this study, we revisited the kinome of Plasmodium falciparum and identified three Aurora-related kinases, Pfark-1, -2, -3. Among these, Pfark-1 is highly conserved in malaria parasites and also appears to be conserved across Apicomplexa. By tagging the endogenous Pfark-1 gene with the green fluorescent protein (GFP) in live parasites, we show that the Pfark-1-GFP protein forms paired dots associated with only a subset of nuclei within individual schizonts. Immunofluorescence analysis using an anti-α-tubulin antibody strongly suggests a recruitment of Pfark-1 at duplicated spindle pole bodies at the entry of the M phase of the cell cycle. Unsuccessful attempts at disrupting the Pfark-1 gene with a knockout construct further indicate that Pfark-1 is required for parasite growth in red blood cells. Our study provides new insights into the cell cycle control of malaria parasites and reports the importance of Aurora kinases as potential targets for new antimalarials.
Asunto(s)
Eritrocitos/parasitología , Plasmodium falciparum/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Huso Acromático/metabolismo , Aurora Quinasas , Secuencia Conservada , Eliminación de Gen , Genes Esenciales , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Filogenia , Plasmodium falciparum/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue.
Asunto(s)
Variación Antigénica , Antígenos de Protozoos/metabolismo , Reparación del ADN , Mapeo de Interacción de Proteínas , Proteínas Protozoarias/metabolismo , Recombinasa Rad51/metabolismo , Trypanosoma brucei brucei/fisiología , Antígenos de Protozoos/genética , Secuencia Conservada , Eliminación de Gen , Proteínas Protozoarias/genética , Recombinasa Rad51/genética , Recombinación Genética , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/inmunologíaRESUMEN
Thyroid mucoepidermoid carcinoma (MEC) is a rare thyroid malignancy first documented in 1977. The majority of thyroid MECs are indolent, low-grade tumours with excellent prognosis. A woman in her 60s presented with an ongoing sensation of a lump in the left neck. There were no swallowing, voice or airway concerns. Ultrasound of the neck showed an enlarged thyroid with U5 and U3 features on the right and left lobes, respectively. Right fine needle aspiration cytology (FNAC) demonstrated certain features of Hurthle cell or anaplastic carcinoma (Thy5). Left FNAC showed Hurthle cell changes with atypical cells and prominent nucleoli (Thy3a). Following total thyroidectomy, histopathology revealed synchronous right low-grade MEC and left papillary thyroid microcarcinomas (pT2(m) N0 M0) on a background of Hashimoto's thyroiditis. This case adds to the literature and details the key histopathological features for a rare but important differential in patients with thyroid carcinoma due to synchronous histological types.
Asunto(s)
Carcinoma Mucoepidermoide , Neoplasias de la Tiroides , Femenino , Humanos , Carcinoma Mucoepidermoide/diagnóstico por imagen , Carcinoma Mucoepidermoide/cirugía , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/cirugía , Neoplasias de la Tiroides/patología , Biopsia con Aguja FinaRESUMEN
Synthetic biology and metabolic engineering offer potentially green and attractive routes to the production of high value compounds. The provision of high-quality parts and pathways is crucial in enabling the biosynthesis of chemicals using synthetic biology. While a number of regulatory parts that provide control at the transcriptional and translational level have been developed, relatively few exist at the protein level. Single domain antibodies (sdAb) such as camelid heavy chain variable fragments (VHH) possess binding characteristics which could be exploited for their development and use as novel parts for regulating metabolic pathways at the protein level in microbial cell factories. Here, a platform for the use of VHH as tools in Escherichia coli is developed and subsequently used to modulate linalool production in E. coli. The coproduction of a Design of Experiments (DoE) optimized pBbE8k His6-VHHCyDisCo system alongside a heterologous linalool production pathway facilitated the identification of anti-bLinS VHH that functioned as modulators of bLinS. This resulted in altered product profiles and significant variation in the titers of linalool, geraniol, nerolidol, and indole obtained. The ability to alter the production levels of high value terpenoids, such as linalool, in a tunable manner at the protein level could represent a significant step forward for the development of improved microbial cell factories. This study serves as a proof of principle indicating that VHH can be used to modulate enzyme activity in engineered pathways within E. coli. Given their almost limitless binding potential, we posit that single domain antibodies could emerge as powerful regulatory parts in synthetic biology applications.
Asunto(s)
Monoterpenos Acíclicos/metabolismo , Proteínas Bacterianas/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroliasas/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Ingeniería Metabólica/métodos , Anticuerpos de Dominio Único/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Reactores Biológicos , Camélidos del Nuevo Mundo/inmunología , Codón , Activación Enzimática/inmunología , Hidroliasas/genética , Hidroliasas/metabolismo , Streptomyces/enzimología , Streptomyces/genética , Biología Sintética/métodosRESUMEN
BACKGROUND: Post-transcriptional control of gene expression is suspected to play an important role in malaria parasites. In yeast and metazoans, part of the stress response is mediated through phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), which results in the selective translation of mRNAs encoding stress-response proteins. METHODS: The impact of starvation on the phosphorylation state of PfeIF2alpha was examined. Bioinformatic methods were used to identify plasmodial eIF2alpha kinases. The activity of one of these, PfeIK1, was investigated using recombinant protein with non-physiological substrates and recombinant PfeIF2alpha. Reverse genetic techniques were used to disrupt the pfeik1 gene. RESULTS: The data demonstrate that the Plasmodium falciparum eIF2alpha orthologue is phosphorylated in response to starvation, and provide bioinformatic evidence for the presence of three eIF2alpha kinases in P. falciparum, only one of which (PfPK4) had been described previously. Evidence is provided that one of the novel eIF2alpha kinases, PfeIK1, is able to phosphorylate the P. falciparum eIF2alpha orthologue in vitro. PfeIK1 is not required for asexual or sexual development of the parasite, as shown by the ability of pfeik1- parasites to develop into sporozoites. However, eIF2alpha phosphorylation in response to starvation is abolished in pfeik1- asexual parasites CONCLUSION: This study strongly suggests that a mechanism for versatile regulation of translation by several kinases with a similar catalytic domain but distinct regulatory domains, is conserved in P. falciparum.
Asunto(s)
Secuencia de Aminoácidos/genética , Aminoácidos/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Plasmodium falciparum/genética , eIF-2 Quinasa/metabolismo , Aminoácidos/genética , Animales , Southern Blotting , Clonación Molecular/métodos , Biología Computacional , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/fisiología , Humanos , Datos de Secuencia Molecular , Fosforilación , Plasmodium falciparum/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inanición , eIF-2 Quinasa/genética , eIF-2 Quinasa/fisiologíaRESUMEN
In addition to its role in erythrocyte invasion, Plasmodium falciparum actin is implicated in endocytosis, cytokinesis and inheritance of the chloroplast-like organelle called the apicoplast. Previously, the inability to visualise filamentous actin (F-actin) dynamics had restricted the characterisation of both F-actin and actin regulatory proteins, a limitation we recently overcame for Toxoplasma (Periz et al, 2017). Here, we have expressed and validated actin-binding chromobodies as F-actin-sensors in Plasmodium falciparum and characterised in-vivo actin dynamics. F-actin could be chemically modulated, and genetically disrupted upon conditionally deleting actin-1. In a comparative approach, we demonstrate that Formin-2, a predicted nucleator of F-actin, is responsible for apicoplast inheritance in both Plasmodium and Toxoplasma, and additionally mediates efficient cytokinesis in Plasmodium. Finally, time-averaged local intensity measurements of F-actin in Toxoplasma conditional mutants revealed molecular determinants of spatiotemporally regulated F-actin flow. Together, our data indicate that Formin-2 is the primary F-actin nucleator during apicomplexan intracellular growth, mediating multiple essential functions.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Citocinesis/genética , Forminas/química , Malaria Falciparum/genética , Citoesqueleto de Actina/química , Actinas/genética , Actinas/metabolismo , Apicoplastos/química , Apicoplastos/metabolismo , Endocitosis/genética , Eritrocitos/química , Eritrocitos/parasitología , Forminas/genética , Regulación de la Expresión Génica/genética , Humanos , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Unión Proteica , Toxoplasma/metabolismo , Toxoplasma/patogenicidadRESUMEN
BACKGROUND: Malaria, caused by the parasitic protist Plasmodium falciparum, represents a major public health problem in the developing world. The P. falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets. We report an exhaustive analysis of the P. falciparum genomic database (PlasmoDB) aimed at identifying and classifying all protein phosphatases (PP) in this organism. RESULTS: Using a variety of bioinformatics tools, we identified 27 malarial putative PP sequences within the four major established PP families, plus 7 sequences that we predict to dephosphorylate "non-protein" substrates. We constructed phylogenetic trees to position these sequences relative to PPs from other organisms representing all major eukaryotic phyla except Cercozoans (for which no full genome sequence is available). Predominant observations were: (i) P. falciparum possessed the smallest phosphatome of any of the organisms investigated in this study; (ii) no malarial PP clustered with the tyrosine-specific subfamily of the PTP group (iii) a cluster of 7 closely related members of the PPM/PP2C family is present, and (iv) some P. falciparum protein phosphatases are present in clades lacking any human homologue. CONCLUSION: The considerable phylogenetic distance between Apicomplexa and other Eukaryotes is reflected by profound divergences between the phosphatome of malaria parasites and those of representative organisms from all major eukaryotic phyla, which might be exploited in the context of efforts for the discovery of novel targets for antimalarial chemotherapy.
Asunto(s)
Fosfoproteínas Fosfatasas/aislamiento & purificación , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Animales , Genómica , Humanos , Cadenas de Markov , FilogeniaRESUMEN
BACKGROUND: Trypanosomes are coated with a variant surface glycoprotein (VSG) that is so densely packed that it physically protects underlying proteins from effectors of the host immune system. Periodically cells expressing a distinct VSG arise in a population and thereby evade immunity. The main structural feature of VSGs are two long alpha-helices that form a coiled coil, and sets of relatively unstructured loops that are distal to the plasma membrane and contain most or all of the protective epitopes. The primary structure of different VSGs is highly variable, typically displaying only ~20% identity with each other. The genome has nearly 2000 VSG genes, which are located in subtelomeres. Only one VSG gene is expressed at a time, and switching between VSGs primarily involves gene conversion events. The archive of silent VSGs undergoes diversifying evolution rapidly, also involving gene conversion. The VSG family is a paradigm for alpha helical coiled coil structures, epitope variation and GPI-anchor signals. At the DNA level, the genes are a paradigm for diversifying evolutionary processes and for the role of subtelomeres and recombination mechanisms in generation of diversity in multigene families. To enable ready availability of VSG sequences for addressing these general questions, and trypanosome-specific questions, we have created VSGdb, a database of all known sequences. DESCRIPTION: VSGdb contains fully annotated VSG sequences from the genome sequencing project, with which it shares all identifiers and annotation, and other available sequences. The database can be queried in various ways. Sequence retrieval, in FASTA format, can deliver protein or nucleotide sequence filtered by chromosomes or contigs, gene type (functional, pseudogene, etc.), domain and domain sequence family. Retrieved sequences can be stored as a temporary database for BLAST querying, reports from which include hyperlinks to the genome project database (GeneDB) CDS Info and to individual VSGdb pages for each VSG, containing annotation and sequence data. Queries (text search) with specific annotation terms yield a list of relevant VSGs, displayed as identifiers leading again to individual VSG web pages. CONCLUSION: VSGdb http://www.vsgdb.org/ is a freely available, web-based platform enabling easy retrieval, via various filters, of sets of VSGs that will enable detailed analysis of a number of general and trypanosome-specific questions, regarding protein structure potential, epitope variability, sequence evolution and recombination events.
Asunto(s)
Bases de Datos Factuales , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Secuencia de Bases/genética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína/genética , Análisis de Secuencia de ADN/métodosRESUMEN
Protein kinases (PKs) are a class of druggable targets in Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (sleeping sickness), yet little is known about which PKs are essential for survival in mammals. A recent kinome-wide RNAi screen with 176 individual bloodstream form Trypanosoma brucei lines identified PKs required for proliferation in culture. In order to assess which PKs are also potential virulence factors essential in vivo, lines were pooled, inoculated into mice, and screened for loss of fitness after 48 h RNAi. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to growth in culture. We identified 49 PKs with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. Nine PKs had a more pronounced growth defect in vivo, than in vitro. Amongst these PKs were several with putative functions related to stress responses mediated through the PI3K/TOR or MAPK signaling cascades, which act to protect the parasite from complement-mediated and osmotic lysis. Identification of these virulence-associated PKs provides new insights into T. brucei-host interaction and reveals novel potential protein kinase drug targets.
Asunto(s)
Proteínas Quinasas/genética , Análisis de Secuencia de ARN/métodos , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/parasitología , Animales , Genes Esenciales , Ratones , Proteínas Protozoarias/genética , Interferencia de ARN , Transducción de Señal , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/genética , Tripanosomiasis Africana/veterinaria , Factores de Virulencia/genéticaRESUMEN
Trypanosomes cause a variety of diseases in man and domestic animals in Africa, Latin America, and Asia. In the Trypanozoon subgenus, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause human African trypanosomiasis, whereas Trypanosoma brucei brucei, Trypanosoma evansi, and Trypanosoma equiperdum are responsible for nagana, surra, and dourine in domestic animals, respectively. The genetic relationships between T. evansi and T. equiperdum and other Trypanozoon species remain unclear because the majority of phylogenetic analyses has been based on only a few genes. In this study, we have conducted a phylogenetic analysis based on genome-wide SNP analysis comprising 56 genomes from the Trypanozoon subgenus. Our data reveal that T. equiperdum has emerged at least once in Eastern Africa and T. evansi at two independent occasions in Western Africa. The genomes within the T. equiperdum and T. evansi monophyletic clusters show extremely little variation, probably due to the clonal spread linked to the independence from tsetse flies for their transmission.
Asunto(s)
Filogenia , Polimorfismo de Nucleótido Simple , Trypanosoma/genética , África Oriental , África Occidental , Genes Protozoarios , Genoma de Protozoos , Estudio de Asociación del Genoma CompletoRESUMEN
Synthetic biology is opening up new opportunities for the sustainable and efficient production of valuable chemicals in engineered microbial factories. Here we review the application of synthetic biology approaches to the engineering of monoterpene/monoterpenoid production, highlighting the discovery of novel catalytic building blocks, their accelerated assembly into functional pathways, general strategies for product diversification, and new methods for the optimization of productivity to economically viable levels. Together, these emerging tools allow the rapid creation of microbial production systems for a wide range of monoterpenes and their derivatives for a diversity of industrial applications.