Host triacylglycerols shape the lipidome of intracellular trypanosomes and modulate their growth

Intracellular infection and multi-organ colonization by the protozoan parasite, Trypanosoma cruzi, underlie the complex etiology of human Chagas disease. While T. cruzi can establish cytosolic residence in a broad range of mammalian cell types, the molecular mechanisms governing this process remain poorly understood. Despite the anticipated capacity for fatty acid synthesis in this parasite, recent observations suggest that intracellular T. cruzi amastigotes may rely on host fatty acid metabolism to support infection. To investigate this prediction, it was necessary to establish baseline lipidome information for the mammalian-infective stages of T. cruzi and their mammalian host cells. An unbiased, quantitative mass spectrometric analysis of lipid fractions was performed with the identification of 1079 lipids within 30 classes. From these profiles we deduced that T. cruzi amastigotes maintain an overall lipid identity that is distinguishable from mammalian host cells. A deeper analysis of the fatty acid moiety distributions within each lipid subclass facilitated the high confidence assignment of host- and parasite-like lipid signatures. This analysis unexpectedly revealed a strong host lipid signature in the parasite lipidome, most notably within its glycerolipid fraction. The near complete overlap of fatty acid moiety distributions observed for host and parasite triacylglycerols suggested that T. cruzi amastigotes acquired a significant portion of their lipidome from host triacylglycerol pools. Metabolic tracer studies confirmed long-chain fatty acid scavenging by intracellular T. cruzi amastigotes, a capacity that was significantly diminished in host cells deficient for de novo triacylglycerol synthesis via the diacylglycerol acyltransferases (DGAT1/2). Reduced T. cruzi amastigote proliferation in DGAT1/2-deficient fibroblasts further underscored the importance of parasite coupling to host triacylglycerol pools during the intracellular infection cycle. Thus, our comprehensive lipidomic dataset provides a substantially enhanced view of T. cruzi infection biology highlighting the interplay between host and parasite lipid metabolism with potential bearing on future therapeutic intervention strategies

Actividades de crianças do pré-escolar e educadores de infância com o computador em portugal

Muito se tem escrito hoje sobre a integração das tecnologias de informação e comunicação (TIC) na escola e diversas pesquisas têm vindo a demonstrar a importância da familiarização da criança desta idade com a tecnologia, quer porque esta faz parte inquestionável do mundo que a rodeia, quer pela relevância educativa das experiências que lhe pode proporcionar. No entanto existem muito poucos estudos que nos descrevam e analisem processos efectivos de integração da tecnologia na Escola em geral, e ao nível da educação Pré-Escolar em particular. Este foi o ponto de partida da nossa investigação, verificar as práticas de crianças e Educadores de Infância com as TIC, mais especificamente o computador, em Portugal. Elaborámos um questionário que foi enviado, via correio electrónico, para Educadores de Infância de todo o país e como forma de triangulação de dados foram realizadas observações em dois jardins-de-infância, mais concretamente nas aulas de informática das crianças do pré-escolar. Terminamos com conclusões baseadas nos dados recolhidos e algumas considerações finais sobre o tema

Expression And Functional Analysis Of Lipids And Glycolipids From The Mammal-Dwelling Stages Of Trypanosoma Cruzi

Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive. Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF. Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynThesis could eventually be exploited as a potential target for new therapeutic interventions. Glycosylphosphatidylinositol (GPI)-anchoring is a protein post-translational modification ubiquitously found in eukaryotes. There is a growing body of evidence showing that protein-free GPIs (or glycoinositolphospholipids, GIPLs) and GPI-anchored proteins (GPI-APs) are involved in host-protozoan interaction processes, such as host-cell adhesion and invasion, and pathogenesis. Here, we used a highly sensitive and unbiased approach that employs liquid chromatography-tandem mass spectrometry (LC-MSn) for the analysis of the GPIome (GPIomics) of the mammal-dwelling trypomastigote and amastigote stages of Trypanosoma cruzi, the causative agent of Chagas disease. This approach allows for the structural characterization of both the lipid and the glycan moieties of GPI-APs and GIPLs. We have identified over 140 GIPL and GPI-AP species from these two parasite forms, most of which had not been described in the literature. In contrast to epimastigote-derived GIPLs (eGIPLs), trypomastigote-derived and amastigote-derived GIPLs (tGIPLs and aGIPLs, respectively) tend to have longer and structurally more diverse glycan moieties. Similar results were observed for trypomastigote-derived and amastigote-derived GPI-APs (tGPI-APs and aGPI-APs, respectively), although tGPI-APs and aGPI-APs tended to be structurally less diverse than their GIPL counterparts. The lipid moieties of tGIPLs are composed mainly of O-alkyl-2-O-acyl-glycerolipids (AAGs), typically with longer fatty acid chains than those of eGIPLs. Conversely, amastigotes tend to have an approximately equal number of GIPLs containing AAG or ceramide moieties. Interestingly, the majority of the lipid moieties of GPI-APs derived from both mammal-dwelling stages of T. cruzi contain C18:1- or C18:2- fatty acid substituents. The proteomic analysis of fractions enriched in GPI-APs from the three life-stages of this parasite showed much greater protein diversity in the mammal-dwelling stages than in epimastigotes. The observations made in this study will, hopefully, help us further understand possible structure-function correlations of GPIs and their role during chronic infection

<i>T</i>. <i>cruzi</i> intracellular amastigotes (ICA) scavenge and incorporate exogenous FA, amastigote FA acquisition and proliferation are compromised in DGAT-TG synthesis deficient host cells.

<p>Lipidomic analysis of uninfected HFF (<i>uninfected</i>), <i>T</i>. <i>cruzi</i>-infected HFF <i>(infected</i>) and <i>T</i>. <i>cruzi</i> amastigotes purified from HFF (<i>ICA</i>) show incorporation of exogenous C15:0 FA into <b>(A)</b> TG, <b>(A, inset)</b> total FA, dotted line indicates the average C15:0, C17:0, C17:1 in unlabeled samples, <b>(B)</b> PI, and <b>(C)</b> PE. Representative autoradiographs showing <b>(D)</b> neutral lipid and <b>(E)</b> glycerophospholipid TLC analysis of ¹⁴C-palmitate incorporation into uninfected (lanes 1–3), infected (lanes 4–6), and isolated amastigotes (lanes 7–9) from wild type mouse embryonic fibroblasts, diacylglycerol acyl transferase 1/2 knockout MEF DGAT1/2^-/-, and DGAT1/2^-/- (+DGAT2) cell lines, respectively; <b>(F)</b> Proliferation of <i>T</i>. <i>cruzi</i> amastigotes measured by CFSE intensity at 18 hpi (undivided) and 48 hpi in WT MEF, DGAT1/2^-/- and DGAT1/2^-/- (+DGAT2) cell lines.</p

Overview of experimental design, evaluation of <i>T</i>. <i>cruzi</i> amastigote isolation and LC-MS/MS methods.

<p><b>(A)</b> Schematic overview of the experimental approach that involved parallel lipidomics analysis of <i>T</i>. <i>cruzi</i> parasites isolated from two different mammalian cell types: C2C12 (mouse skeletal myoblast) and HFF (human foreskin fibroblasts). Host cell infection was established with <i>T</i>. <i>cruzi</i> trypomastigotes (TCT) and intracellular <i>T</i>. <i>cruzi</i> amastigotes (ICA) were isolated from infected host cells 48 hours post infection as detailed in the Methods. <i>T</i>. <i>cruzi</i> amastigote purity was evaluated using <b>(B)</b> transmission electron microscopy, scale bar = 2 μm, and <b>(C)</b> western blot analysis using antibodies to <i>T</i>. <i>cruzi</i>, host mitochondria (ATP5B), endoplasmic reticulum (IRE1) and lipid droplets (TIP47) to probe whole cell lysates of control uninfected HFF (<i>HFFu</i>), <i>T</i>. <i>cruzi</i>-infected HFF <i>(HFFi</i>), and <i>T</i>. <i>cruzi</i> amastigotes purified from HFF (<i>hICA</i>). <b>(D)</b> Positive ion mode base peak chromatogram of lipid extracts derived from C2C12, HFF, and cognate <i>T</i>. <i>cruzi</i> amastigote (cICA and hICA, respectively) analyzed by LC-ESI-MS/MS. The major lipid subclasses eluting at different retention times (min) are indicated above the chromatogram. TG–triacylglycerol, DG–diacylglycerol, Cer–ceramide, CerG–hexosylceramide, SM–sphingomyelin, LPC–lysophosphatidylcholine, PC–phosphatidylcholine, LPE–lysophosphatidylethanolamine, PE–phosphatidylethanolamine, LPS–lysophosphatidylserine, PS–phosphatidylserine, PI–phosphatidylinositol, PG–phosphatidylglycerol.</p

FA composition in TG and DG of <i>T</i>. <i>cruzi</i> intracellular amastigotes mirrors host cells.

<p>FA area % is plotted for <b>(A)</b> TG and <b>(B)</b> DG classes for HFF-derived samples, (C2C12 plotted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006800#ppat.1006800.s006" target="_blank">S5 Fig</a>): uninfected HFF (<i>HFFu</i>), <i>T</i>. <i>cruzi</i>-infected HFF <i>(HFFi</i>) and <i>T</i>. <i>cruzi</i> amastigotes purified from HFF (<i>hICA</i>). Data are represented as mean ± standard deviation.</p

Role of the Apt1 protein in polysaccharide secretion by Cryptococcus neoformans

Submitted by Fabricia Pimenta ([email protected]) on 2019-02-01T15:47:25Z No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013f.pdf: 1895669 bytes, checksum: 6dea90228d254715230bf0e6f7e4def6 (MD5)Approved for entry into archive by Fabricia Pimenta ([email protected]) on 2019-03-07T20:40:52Z (GMT) No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013f.pdf: 1895669 bytes, checksum: 6dea90228d254715230bf0e6f7e4def6 (MD5)Made available in DSpace on 2019-03-07T20:40:52Z (GMT). No. of bitstreams: 1 ve_Marcio_Rodrigues_etal_CDTS_2013f.pdf: 1895669 bytes, checksum: 6dea90228d254715230bf0e6f7e4def6 (MD5) Previous issue date: 2014-06Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.The University of British Columbia. Faculty of Land and Food Systems. Department of Microbiology and Immunology. Michael Smith Laboratories. Vancouver, Canada.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.The University of British Columbia. Faculty of Land and Food Systems. Department of Microbiology and Immunology. Michael Smith Laboratories. Vancouver, Canada.The University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.The University of Texas at El Paso. Department of Biological Sciences. Border Biomedical Research Center. El Paso, Texas, USA.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer. Rio de Janeiro, RJ, BrazilThe University of British Columbia. Faculty of Land and Food Systems. Department of Microbiology and Immunology. Michael Smith Laboratories. Vancouver, Canada.Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Professor Paulo de Góes. Rio de Janeiro, RJ, Brasil.Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1Δ mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1Δ mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitro growth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1 caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformans via processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection

Glucosylceramide Transferase Activity is Critical for Encystation and Viable Cyst Production by an Intestinal Protozoan, Giardia lamblia

The production of viable cysts by Giardia is essential for its survival in the environment and spreading the infection via contaminated food and water. The hallmark of cyst production (also known as encystation) is the biogenesis of encystation-specific vesicles (ESVs) that transport cyst-wall proteins to trophozoite’s plasma membrane before laying down the protective cyst wall. However, the molecules that regulate ESV biogenesis and maintain cyst viability have never before been identified. Here, we report that giardial glucosylceramide transferase-1 (gGlcT1), an enzyme of sphingolipid biosynthesis, plays a key role in ESV biogenesis and maintaining cyst viability. We find that overexpression of this enzyme induced the formation of aggregated/enlarged ESVs and generated clustered cysts with reduced viability. The silencing of gGlcT1 synthesis by anti-sense morpholino oligonucleotide abolished ESV production and generated mostly non-viable cysts. Interestingly, when gGlcT1-overexpressed Giardia was transfected with anti-gGlcT1 morpholino, the enzyme activity, vesicle biogenesis, and cyst viability returned to normal, suggesting that the regulated expression of gGlcT1 is important for encystation and viable cyst production. Furthermore, the overexpression of gGlcT1 increased the influx of membrane lipids and fatty acids without altering the fluidity of plasma membranes, indicating that the expression of gGlcT1 activity is linked to lipid internalization and maintaining the overall lipid balance in this parasite. Taken together, our results suggest that gGlcT1 is a key player of ESV biogenesis and cyst viability and therefore could be targeted for developing new anti-giardial therapie

ESI-MS analysis of cell wall neutral glycolipids from Pb3 and Pb18 cultivated in the presence (pl) or absence of plasma.

<p>A, Full-scan spectra in the positive-ion mode show the identified glycolipids Hex-C16∶0-OH/d19∶2-Cer (<i>m/z</i> 848.7), Hex-C18∶0-OH/d18∶2-Cer (<i>m/z</i> 862.8), Hex-C18∶0-OH/d18∶1-Cer (<i>m/z</i> 864.9), Hex-C18∶1-OH/d19∶2-Cer (<i>m/z</i> 874.9), and Hex-C18∶0-OH/d19∶2-Cer (<i>m/z</i> 876.9). B, Tandem-MS spectrum of the major glycolipid species identified (<i>m/z</i> 876.9). <i>m/z</i>, mass to charge ratio.</p