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1.
Clin Infect Dis ; 2021 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-33580234

RESUMO

BACKGROUND: In order to expedite the development of new oral treatment regimens for visceral leishmaniasis (VL), there is a need for early markers to evaluate treatment response and predict long-term outcomes. METHODS: Data from three clinical trials were combined in this study, where Eastern African VL patients received various antileishmanial therapies. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative PCR (qPCR) before, during and up to six months after treatment. The predictive performance of pharmacodynamic parameters for clinical relapse was evaluated using receiver-operating characteristic curves. Clinical trial simulations were performed to determine the power associated with the use of blood parasite load as a surrogate endpoint to predict clinical outcome at six months. RESULTS: The absolute parasite density on day 56 after start of treatment was found to be a highly sensitive predictor of relapse within six months of follow-up at a cut-off of 20 parasites/mL (AUC 0.92, specificity 0.91, sensitivity 0.89). Blood parasite loads correlated well with tissue parasite loads (ρ= 0.80) and with microscopy gradings of bone marrow and spleen aspirate smears. Clinical trial simulations indicated a >80% power to detect a difference in cure rate between treatment regimens if this difference was high (>50%) and when minimally 30 patients were included per regimen. CONCLUSION: Blood Leishmania parasite load determined by qPCR is a promising early biomarker to predict relapse in VL patients. Once optimized, it might be useful in dose finding studies of new chemical entities.

2.
J Microbiol Methods ; 173: 105935, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376283

RESUMO

BACKGROUND: Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed. METHODOLOGY: The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection. PRINCIPAL FINDINGS: Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed. CONCLUSIONS/SIGNIFICANCE: The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies.

3.
PLoS Negl Trop Dis ; 14(4): e0008246, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324742

RESUMO

The development of an accurate protein-based antigen detection assay for diagnosis of active visceral leishmaniasis (VL) would represent a major clinical advance. VL is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani. The gold standard confirmatory diagnostic test for VL is the demonstration of parasites or their DNA from aspirates from spleen, lymph node, and bone marrow or from blood buffy coats. Here we describe the production and use of monoclonal antibodies (mAbs) for the development of a sensitive and specific antigen detection capture ELISA for VL diagnosis. This test simultaneously detects six leishmania protein biomarkers that we have previously described (Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1). The initial clinical validation of this new mAb-based multiplexed capture ELISA showed a sensitivity of ≥93%. The test was negative with 35 urine samples from healthy control subjects as well as with 30 patients with confirmed non-VL tropical diseases (cutaneous leishmaniasis, n = 6; Chagas disease, n = 6; schistosomiasis, n = 6; and tuberculosis, n = 12). These results strongly support the possible utility of this mAb-based multiplexed capture ELISA as a promising diagnostic test for active VL as well as for monitoring the treatment efficacy of this disease. The test is ready for upscaling and validation for clinical use.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/urina , Leishmania donovani/química , Leishmania infantum/química , Leishmaniose Visceral/diagnóstico , Urinálise/métodos , Urina/química , Adolescente , Adulto , Idoso , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Biomarcadores/urina , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto Jovem
4.
J Clin Microbiol ; 57(5)2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30787142

RESUMO

Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/urina , Proteínas de Protozoários/urina , Adolescente , Adulto , Idoso , Anticorpos Antiprotozoários , Biomarcadores/urina , Brasil , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Índia , Quênia , Leishmania donovani/isolamento & purificação , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/parasitologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto Jovem
5.
Clin Microbiol Rev ; 31(4)2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30158301

RESUMO

Research in visceral leishmaniasis in the last decade has been focused on how better to use the existing medicines as monotherapy or in combination. Systematic research by geographical regions has shown that a universal treatment is far from today's reality. Substantial progress has been made in the elimination of kala-azar in South Asia, with a clear strategy on first- and second-line therapy options of single-dose liposomal amphotericin B and a combination of paromomycin and miltefosine, respectively, among other interventions. In Eastern Africa, sodium stibogluconate (SSG) and paromomycin in combination offer an advantage compared to the previous SSG monotherapy, although not exempted of limitations, as this therapy requires 17 days of painful double injections and bears the risk of SSG-related cardiotoxicity. In this region, attempts to improve the combination therapy have been unsuccessful. However, pharmacokinetic studies have led to a better understanding of underlying mechanisms, like the underexposure of children to miltefosine treatment, and an improved regimen using an allometric dosage. Given this global scenario of progress and pitfalls, we here review what steps need to be taken with existing medicines and highlight the urgent need for oral drugs. Furthermore, it should be noted that six candidates belonging to five new chemical classes are reaching phase I, ensuring an optimistic near future.


Assuntos
Antiprotozoários/uso terapêutico , Descoberta de Drogas/tendências , Leishmaniose Visceral/tratamento farmacológico , Pesquisa Biomédica/tendências , Humanos
6.
PLoS Negl Trop Dis ; 12(2): e0006264, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29444079

RESUMO

BACKGROUND: Confirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp Leishmania Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results. METHODOLOGY/PRINCIPAL FINDINGS: The Loopamp Leishmania Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite Leishmania rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%. CONCLUSIONS/SIGNIFICANCE: Due to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration.


Assuntos
Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Adolescente , Adulto , Antígenos de Protozoários/genética , Criança , Pré-Escolar , Feminino , Humanos , Leishmania donovani/genética , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Masculino , Pessoa de Meia-Idade , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Sudão , Temperatura , Adulto Jovem
7.
J Mol Diagn ; 20(2): 253-263, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29355825

RESUMO

Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA real-time quantitative PCR (qPCR) assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood, and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0.005 and 0.002 parasites per mg liver and spleen, respectively) and was not prone to false-positive reactions. Evaluation of the SL-RNA assay on clinical samples demonstrated lower threshold cycle values than the kDNA qPCR, an excellent interrun stability of 97%, a 93% agreement with the kDNA assay, and an estimated sensitivity, specificity, and accuracy of 93.2%, 94.3%, and 93.8%, respectively. The SL-RNA qPCR assay was equally efficient for detecting Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guayensis, Leishmania panamensis, Leishmania braziliensis, L. infantum, and Leishmania donovani and revealed similar SL-RNA levels in the different species and the occurrence of polycistronic SL-containing transcripts in Viannia species. Collectively, this single SL-RNA qPCR assay enables universal Leishmania detection and represents a particularly useful addition to the widely used kDNA assay in clinical studies in which the detection of viable parasites is pivotal to assess parasitological cure.


Assuntos
DNA de Cinetoplasto/análise , Leishmania infantum/genética , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Mesocricetus/parasitologia , RNA Líder para Processamento/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Criança , Pré-Escolar , Cricetinae , Confiabilidade dos Dados , Feminino , Humanos , Fígado/parasitologia , Sensibilidade e Especificidade , Baço/parasitologia
8.
PLoS Negl Trop Dis ; 11(7): e0005779, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28727723

RESUMO

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/µL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/µL of CL Brener DNA and 1 fg/µl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.


Assuntos
Doença de Chagas/diagnóstico , DNA de Protozoário/sangue , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Trypanosoma cruzi/isolamento & purificação , Doença de Chagas/parasitologia , Feminino , Humanos , Recém-Nascido , Masculino , Sensibilidade e Especificidade , Trypanosoma cruzi/genética
9.
Clin Infect Dis ; 65(1): 150-153, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28520851

RESUMO

We compared xenodiagnosis with quantitative polymerase chain reaction in skin biopsies from 3 patients with maculopapular or nodular post-kala-azar dermal leishmaniasis (PKDL). All patients infected sand flies. Parasite loads in skin varied from 1428 to 63 058 parasites per microgram. PKDL detection and treatment are important missing components of the kala-azar elimination program.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Psychodidae/parasitologia , Adulto , Animais , DNA de Protozoário/análise , DNA de Protozoário/genética , Feminino , Humanos , Índia , Leishmania donovani/genética , Leishmania donovani/patogenicidade , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/transmissão , Masculino , Reação em Cadeia da Polimerase , Pele/parasitologia , Xenodiagnóstico , Adulto Jovem
10.
PLoS Negl Trop Dis ; 9(1): e3404, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25568942

RESUMO

Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.


Assuntos
Genoma de Protozoário , Filogenia , Proteínas de Protozoários/metabolismo , Trypanosoma/classificação , Trypanosoma/genética , Regulação da Expressão Gênica , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Análise de Componente Principal , Proteínas de Protozoários/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
11.
Eur J Med Chem ; 74: 541-51, 2014 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-24530464

RESUMO

The natural product, convolutamine I (1), has anti-trypanosomal activity however it has a high molecular weight of 473 due to a presence of 3 bromine atoms. The synthesis of the natural product convolutamine I (1) together with its analogues are presented. A SAR study against Trypanosoma brucei brucei led to compounds with improved physico-chemical properties: lower molecular weight and lower log P while maintaining potency (with a slight 2-fold improvement).


Assuntos
Diaminas/química , Fenetilaminas/química , Tripanossomicidas/uso terapêutico , Tripanossomíase Africana/tratamento farmacológico , Diaminas/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Fenetilaminas/farmacologia , Espectrometria de Massas por Ionização por Electrospray
12.
Eukaryot Cell ; 13(4): 504-16, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24562907

RESUMO

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and ß) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Ciclo Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/enzimologia , Descoberta de Drogas , Álcoois Graxos/farmacologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética
13.
Mol Cell Biol ; 33(24): 4872-88, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24126051

RESUMO

The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA synthetase (MARS) complex contained at least six aaRS enzymes and three additional non-aaRS proteins. Steady-state kinetic studies showed that association in the MARS complex enhances tRNA-aminoacylation efficiency, which is in part dependent on a MARS complex-associated protein (MCP), named MCP2, that binds tRNAs and increases their aminoacylation by the complex. Conditional repression of MCP2 in T. brucei bloodstream forms resulted in reduced parasite growth and infectivity in mice. Thus, association in a MARS complex enhances tRNA-aminoacylation and contributes to parasite fitness. The MARS complex may be part of a cellular regulatory system and a target for drug development.


Assuntos
Aminoacil-tRNA Sintetases/fisiologia , Proteínas de Protozoários/fisiologia , RNA de Protozoário/metabolismo , RNA de Transferência/metabolismo , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Animais , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Complexos Multienzimáticos/fisiologia , Transporte Proteico , Aminoacilação de RNA de Transferência , Trypanosoma brucei brucei/fisiologia
14.
PLoS One ; 8(6): e67327, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23805309

RESUMO

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.


Assuntos
Ciclinas , Complexos Multiproteicos , Proteínas Proto-Oncogênicas c-crk , Proteínas de Protozoários , Trypanosoma brucei brucei , Animais , Proliferação de Células/genética , Ciclinas/genética , Ciclinas/metabolismo , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/genética , Tripanossomíase Africana/metabolismo
15.
BMC Res Notes ; 2: 46, 2009 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-19309510

RESUMO

BACKGROUND: The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis. Its cell cycle is complex and not fully understood at the molecular level. The T. brucei genome contains over 6000 protein coding genes with >50% having no predicted function. A small scale RNA interference (RNAi) screen was carried out in Trypanosoma brucei to evaluate the prospects for identifying novel cycle regulators. RESULTS: Procyclic form T. brucei were transfected with a genomic RNAi library and 204 clones isolated. However, only 76 RNAi clones were found to target a protein coding gene of potential interest. These clones were screened for defects in proliferation and cell cycle progression following RNAi induction. Sixteen clones exhibited proliferation defects upon RNAi induction, with eight clones displaying potential cell cycle defects. To confirm the phenotypes, new RNAi cell lines were generated and characterised for five genes targeted in these clones. While we confirmed that the targeted genes are essential for proliferation, we were unable to unambiguously classify them as cell cycle regulators. CONCLUSION: Our study identified genes essential for proliferation, but did not, as hoped, identify novel cell cycle regulators. Screening of the RNAi library for essential genes was extremely labour-intensive, which was compounded by the suboptimal quality of the library. For such a screening method to be viable for a large scale or genome wide screen, a new, significantly improved RNAi library will be required, and automated phenotyping approaches will need to be incorporated.

16.
Curr Opin Microbiol ; 10(6): 520-7, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18023244

RESUMO

The process of cytokinesis, where the cytoplasm of one cell is divided to produce two daughter cells, is intricate in trypanosomatids because of the requirement to replicate and segregate a number of single copy organelles, including the nucleus, kinetoplast, Golgi apparatus, and flagellum. Identifying regulators of the three stages of cytokinesis, initiation, furrow ingression, and abscission is complicated by the fact that cell division in trypanosomatids is easily perturbed and aberrant cells are readily produced during functional characterization of gene products. In this review, we discuss direct and indirect effects on cytokinesis, using Trypanosoma brucei as a model.


Assuntos
Citocinese/fisiologia , Trypanosomatina/fisiologia , Animais , Regulação da Expressão Gênica , Estágios do Ciclo de Vida , Microscopia Eletrônica de Varredura , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/fisiologia , Trypanosoma brucei brucei/ultraestrutura , Trypanosomatina/ultraestrutura
17.
Mol Biochem Parasitol ; 134(2): 233-43, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15003843

RESUMO

Little is known about the relation between the genome organization and gene expression in Leishmania. Bioinformatic analysis can be used to predict genes and find homologies with known proteins. A model was proposed, in which genes are organized into large clusters and transcribed from only one strand, in the form of large polycistronic primary transcripts. To verify the validity of this model, we studied gene expression at the transcriptional, post-transcriptional and translational levels in a unique locus of 34kb located on chr27 and represented by cosmid L979. Sequence analysis revealed 115 ORFs on either DNA strand. Using computer programs developed for Leishmania genes, only nine of these ORFs, localized on the same strand, were predicted to code for proteins, some of which show homologies with known proteins. Additionally, one pseudogene, was identified. We verified the biological relevance of these predictions. mRNAs from nine predicted genes and proteins from seven were detected. Nuclear run-on analyses confirmed that the top strand is transcribed by RNA polymerase II and suggested that there is no polymerase entry site. Low levels of transcription were detected in regions of the bottom strand and stable transcripts were identified for four ORFs on this strand not predicted to be protein-coding. In conclusion, the transcriptional organization of the Leishmania genome is complex, raising the possibility that computer predictions may not be comprehensive.


Assuntos
Regulação da Expressão Gênica , Genoma de Protozoário , Leishmania major/genética , Animais , Northern Blotting , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Ordem dos Genes , Genes de Protozoários , Dados de Sequência Molecular , Família Multigênica , Fases de Leitura Aberta , Biossíntese de Proteínas , Proteínas de Protozoários/análise , Pseudogenes , RNA Polimerase II/metabolismo , RNA Mensageiro/análise , RNA de Protozoário/análise , Análise de Sequência de DNA , Homologia de Sequência , Transcrição Genética
18.
Int J Parasitol ; 33(9): 965-75, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12906880

RESUMO

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by two genes, HIS-1.1 and HIS-1.2. These genes are separated by approximately 20 kb of sequence and are located on the same DNA strand of chromosome 27. When Northern blots of parasite RNA were probed with HIS-1 strand-specific riboprobes, we detected sense and antisense transcripts that were polyadenylated and developmentally regulated. When the HIS-1.2 coding region was replaced with the coding region of the neomycin phosphotransferase gene, antisense transcription of this gene was unaffected, indicating that the regulatory elements controlling antisense transcription were located outside of the HIS-1.2 gene, and that transcription in Leishmania can occur from both DNA strands even in the presence of transcription of a selectable marker in the complementary strand. A search for other antisense transcripts within the HIS-1 locus identified an additional transcript (SC-1) within the intervening HIS-1 sequence, downstream of adenine and thymine-rich sequences. These results show that gene expression in Leishmania is not only regulated polycistronically from the sense strand of genomic DNA, but that the complementary strand of DNA also contains sequences that could drive expression of open reading frames from the antisense strand of DNA. These findings suggest that the parasite has evolved in such a way as to maximise the transcription of its genome, a mechanism that might be important for it to maintain virulence.


Assuntos
DNA Antissenso/análise , DNA Complementar/análise , Histonas/genética , Leishmania major/genética , Leishmaniose Cutânea/transmissão , Animais , Northern Blotting/métodos , Humanos , Reação em Cadeia da Polimerase/métodos , Transcrição Genética , Virulência
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