516 research outputs found
Biosynthesis of GDP-fucose and other sugar nucleotides in the blood-stages of Plasmodium falciparum
Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry- based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDPfucose suggests that the metabolite may be used for further fucosylation reactions.Fil: Sanz, Silvia. Hospital Clínico de Barcelona. Centro de Investigación en Salud Internacional de Barcelona; España;Fil: Bandini, Giulia. Wellcome Trust Biocentre. Division of Biological Chemistry and Drug Discovery. University of Dundee. College of Life Sciences; Reino Unido;Fil: Ospina, Diego. Hospital Clínico de Barcelona. Centro de Investigación en Salud Internacional de Barcelona; España;Fil: Bernabeu, Maria. Hospital Clínico de Barcelona. Centro de Investigación en Salud Internacional de Barcelona; España;Fil: Mariño, Karina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Wellcome Trust Biocentre. Division of Biological Chemistry and Drug Discovery. University of Dundee. College of Life Sciences; Reino Unido;Fil: Fernández Becerra, Carmen. Hospital Clínico de Barcelona. Centro de Investigación en Salud Internacional de Barcelona; España;Fil: Izquierdo, Luis. Hospital Clínico de Barcelona. Centro de Investigación en Salud Internacional de Barcelona; España
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Conditional expression of PfAP2-G for controlled massive sexual conversion in Plasmodium falciparum.
Malaria transmission requires that some asexual parasites convert into sexual forms termed gametocytes. The initial stages of sexual development, including sexually committed schizonts and sexual rings, remain poorly characterized, mainly because they are morphologically identical to their asexual counterparts and only a small subset of parasites undergo sexual development. Here, we describe a system for controlled sexual conversion in the human malaria parasite Plasmodium falciparum, based on conditional expression of the PfAP2-G transcription factor. Using this system, ~90 percent of the parasites converted into sexual forms upon induction, enabling the characterization of committed and early sexual stages without further purification. We characterized sexually committed schizonts and sexual rings at the transcriptomic and phenotypic levels, which revealed down-regulation of genes involved in solute transport upon sexual commitment, among other findings. The new inducible lines will facilitate the study of early sexual stages at additional levels, including multiomic characterization and drug susceptibility assays
Conditional expression of PfAP2-G for controlled massive sexual conversion in Plasmodium falciparum
--- - i: - Plasmodium falciparum content: - "Malaria transmission requires that some asexual parasites convert into sexual forms termed gametocytes. The initial stages of sexual development, including sexually committed schizonts and sexual rings, remain poorly haracterized, mainly because they are morphologically identical to their sexual counterparts and only a small subset of parasites undergo sexual development. Here, we describe a system for controlled sexual conversion in the human
malaria parasite " - ", based on conditional expression of the
PfAP2-G transcription factor. Using this system, ~90 percent of
the parasites converted into sexual forms upon induction,
enabling the characterization of committed and early sexual
stages without further purification. We characterized sexually
committed schizonts and sexual rings at the transcriptomic and
phenotypic levels, which revealed down-regulation of genes
involved in solute transport upon sexual commitment, among other
findings. The new inducible lines will facilitate the study of
early sexual stages at additional levels, including multiomic
characterization and drug susceptibility assays.
Increased expression levels of the pvcrt-o and pvmdr1 genes in a patient with severe Plasmodium vivax malaria
<p>Abstract</p> <p>Background</p> <p>There are increasing reports of severe clinical cases exclusively associated with <it>Plasmodium vivax </it>infections. Notably, this severity has been recently suggested to be associated with chloroquine resistance.</p> <p>Patients</p> <p>Two different patients presented at the Hospital Clinic in Barcelona with <it>P. vivax </it>malaria episodes. One patient had severe symptoms and the other mild symptoms. Both patients traveled through the Brazilian Amazon (Manaus) in 2007. For both patients the current diagnosis of malaria was the first. Two other patients with mild symptoms presented to the "Centro de Pesquisa em Medicina Tropical", also in the Brazilian Amazon (Rondônia) in 2000.</p> <p>Methods</p> <p>To exclude the possibility that the patient's severe symptoms were due to <it>Plasmodium falciparum</it>, a nested PCR was performed. A magnetic method was used to purify <it>P. vivax </it>free of human leukocytes. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters likely to be involved in chloroquine resistance in <it>P. vivax</it>, namely the <it>P. vivax </it>chloroquine resistance transporter, <it>pvcrt-o</it>, and the <it>P. vivax </it>multidrug resistance transporter, <it>pvmdr 1</it>.</p> <p>Results</p> <p>Results demonstrated that the severe clinical symptoms were exclusively due to <it>P. vivax</it>. The patient presented acute respiratory conditions requiring admission to the intensive care unit. The magnetic method showed highly purified infected-reticulocytes with mature stages. In addition, it was found that parasites obtained from the severe patient had up to 2.9-fold increase in <it>pvmdr1 </it>levels and up to 21.9-fold increase in <it>pvcrt-o </it>levels compared to expression levels of parasites from the other patients with mild symptoms.</p> <p>Conclusion</p> <p>This is the first clinical case of severe disease exclusively associated with vivax malaria in Spain. Moreover, these findings suggest that clinical severity could be associated with increased expression levels of parasite genes likely involved in chloroquine resistance. It is necessary to further explore the potential of <it>pvmdr1 </it>and particularly <it>pvcrt-o </it>expression levels as molecular markers of severe disease in <it>P. vivax</it>.</p
State-of-the-art in host-derived biomarkers of Chagas disease prognosis and early evaluation of anti-Trypanosoma cruzi treatment response
Chagas disease is caused by infection with the parasite
Trypanosoma cruzi, which might lead to a chronic disease state
and drive to irreversible damage to the heart and/or digestive
tract tissues. Endemic in 21 countries in the Americas, it is
the neglected disease with a highest burden in the region.
Current estimates point at ~6 million people infected, of which
~30% will progress onto the symptomatic tissue disruptive stage.
There is no vaccine but there are two anti-parasitic drugs
available: benznidazole and nifurtimox. However, their efficacy
is variable at the chronic symptomatic stage and both have
frequent adverse effects. Since there are no prognosis markers,
drugs should be administered to all T. cruzi-infected
individuals in the indeterminate and early symptomatic stages.
Nowadays, there are no tests-of-cure either, which greatly
undermines patients' follow-up and the search of safer and more
efficacious drugs. Therefore, the identification and validation
of biomarkers of disease progression and/or treatment response
on which to develop tests of prognosis and/or cure is a major
research priority. Both parasite- and host-derived markers have
been investigated. In the present manuscript we present an
updated outlook of the latter
Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries
BACKGROUND: The chemical treatment of Plasmodium falciparum for
human infections is losing efficacy each year due to the rise of
resistance. One possible strategy to find novel anti-malarial
drugs is to access the largest reservoir of genomic biodiversity
source on earth present in metagenomes of environmental
microbial communities. METHODS: A bioluminescent P. falciparum
parasite was used to quickly detect shifts in viability of
microcultures grown in 96-well plates. A synthetic gene encoding
the Dermaseptin 4 peptide was designed and cloned under tight
transcriptional control in a large metagenomic insert context
(30 kb) to serve as proof-of-principle for the screening
platform. RESULTS: Decrease in parasite viability consistently
correlated with bioluminescence emitted from parasite
microcultures, after their exposure to bacterial extracts
containing a plasmid or fosmid engineered to encode the
Dermaseptin 4 anti-malarial peptide. CONCLUSIONS: Here, a new
technical platform to access the anti-malarial potential in
microbial environmental metagenomes has been developed
Extracellular vesicles in parasitic diseases
Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite–parasite inter-communication as well as in parasite–host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens
Sudden spleen rupture in a Plasmodium vivax-infected patient undergoing malaria treatment
BACKGROUND: Splenomegaly is one of the most common features of
malaria. However, spontaneous splenic rupture, although unusual,
represents a severe complication often leading to death. It is
mostly seen in acute infection and primary attack, and it is
most commonly associated with Plasmodium vivax. Here, a case of
spontaneous splenic rupture diagnosed with a portable ultrasound
apparatus shortly after starting treatment and with recurrent
parasitaemia after splenectomy, is reported. CASE DESCRIPTION:
In November 2015, a 45-year-old Brazilian man presented to the
hospital in Manaus with fever, headache and myalgia. He was
diagnosed with P. vivax malaria and, after a normal G6PD test,
he started treatment with chloroquine and primaquine and was
discharged. Two days later, he went back to the hospital with
abdominal pain, dyspnea, dry cough, pallor, oliguria and fever.
Using a portable ultrasound, he was diagnosed of rupture of the
spleen, which was removed by emergency surgery. After this
episode, he suffered two more malaria episodes with high
parasitaemia at approximately 2-month intervals. DNA from
different portions of the spleen was extracted and a qualitative
PCR was performed to detect P. vivax. CONCLUSIONS: The splenic
rupture suffered by this patient occurred 2 days after starting
the treatment. Having a portable ultrasound apparatus may have
saved the patient's life, as it revealed a haemorrhage needing
an urgent surgery. Parasites were detected by PCR in the
extracted spleen. This patient suffered two more vivax malaria
diagnosed episodes in spite of receiving and completing
treatment with chloroquine and primaquine for each clinical
attack. Splenic rupture during acute malaria is uncommon, but it
is likely underdiagnosed and underreported, because the lack of
means and equipment hinders diagnostic confirmation, especially
in endemic areas
Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries
Ajuts: Departamento Administrativo de Ciencias, Tecnología e Innovación (Colciencias), República de Colombia; Convocatoria 489 - 2009, Código 657048925406, Contrato de financiación RC. 427 - 2009 Colciencias - CorpoGen; Programa de Asistencias Graduadas de Universidad de los Andes, Bogotá, Colombia; i Programa Jóvenes Investigadores de ColcienciasBACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been develope
Proteomics study of human cord blood reticulocyte-derived exosomes
Reticulocyte-derived exosomes (Rex), extracellular vesicles of
endocytic origin, were initially discovered as a cargo-disposal
mechanism of obsolete proteins in the maturation of
reticulocytes into erythrocytes. In this work, we present the
first mass spectrometry-based proteomics of human Rex (HuRex).
HuRex were isolated from cultures of human reticulocyte-enriched
cord blood using different culture conditions and exosome
isolation methods. The newly described proteome consists of 367
proteins, most of them related to exosomes as revealed by gene
ontology over-representation analysis and include multiple
transporters as well as proteins involved in exosome biogenesis
and erythrocytic disorders. Immunoelectron microscopy validated
the presence of the transferrin receptor. Moreover, functional
assays demonstrated active capture of HuRex by mature dendritic
cells. As only seven proteins have been previously associated
with HuRex, this resource will facilitate studies on the role of
human reticulocyte-derived exosomes in normal and pathological
conditions affecting erythropoiesis
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