Toxoplasma gondii ist wohl der am besten an den Menschen und Tieren angepasste Parasit der Welt und eine der Hauptursachen für Abtreibungen und opportunistsiche Infektionen. Das Verständnis der Stoffwechselflexibilität dieses Parasiten ist essentiel um seine Anpassungsfähigkeit nachzuvollziehen. Lipide sind Grundbestandteile der biologischen Membranen. Doch in den letzten Jahrzehnten sind neben der Biogenese der Membranen noch weitere Funktionen entdeckt worden. Hier berichten wir zum ersten Mal über Phosphatidylthreonin (PtdThr) als Hauptphospholipid von T. gondii und zeigen seine Beziehung mit seinem archetypischen Analogon, Phosphatidylserin (PtdSer). Wir identifizieren auch ein neues Parasitenenzym (TgPTS), welches von der regulären PtdSer-Synthase-Familie abgewichen ist und dieses sonst selten vorkommende Lipid synthetisiert. Die Gendeletion von TgPTS stoppte nicht nur die Synthese von PtdThr, sondern auch stark beeinträchtigt den lytischen Zyklus von T. gondii, insbesondere beim Aus- und Eintritt in die Wirtszellen, ohne dabei die Replikation des Parasiten zu verhindern. Unsere Arbeit zeigt, dass entweder ein niedriger Ca2+-Pool oder eine verminderte Ca2+-Mobilisierung aus dem endoplasmatischen Retikulum des Parasiten eine reduzierte Motilität verursachen, welche den beobachteten Phänotypen zugrunde liegt. Darüber hinaus fehlt es den mutierten Parasiten an PtdThr (Δtgpts), sie sind avirulent und üben vollständigen Schutz gegen akute und chronische Toxoplasmose in einem Mausmodell aus. Diese Ergebnisse verdeutlichen die Bedeutung der Parasitenlipide als therapeutisches Ziel und von genetisch abgeschwächten Stämmen für die Entwicklung von wirksamen Impfstoffen gegen Kokzidien. / Toxoplasma gondii is arguably the most succesful parasite in Earth and a major cause of abortion and opportunistic infections in humans and livestock. Understanding the metabolic flexibility of this parasite is essential to comprehend its adaptability. Lipids are basic components of biological membranes. However, in the last decades non-customary roles for lipids beyond membrane biogenesis have been recognized. Here we report for the first time phosphatidylthreonine (PtdThr) as a major phospholipid in T. gondii and show its relationship with its archetypical analog, phosphatidylserine (PtdSer). We also identify a novel parasite enzyme (TgPTS), which diverged from the mainstream PtdSer-synthase family to produce this otherwise rare lipid. Genetic ablation of TgPTS not only abolished PtdThr synthesis, but also impaired severely the lytic cycle of T. gondii, particularly the egress but also the invasion into host cells without affecting the parasite replication. Our work indicates that, either a lower Ca2+ pool and/or a diminished Ca2+ mobilization from the parasite endoplasmic reticulum cause a reduced gliding motility, which underlies the observed phenotypes. Moreover, the mutant parasites lacking PtdThr (Δtgpts) are avirulent and exert full protection against both, acute and chronic toxoplasmosis in a murine model. These results highlight the importance of parasite lipids as therapeutic targets and of genetically-attenuated strains in the development of effective vaccines against coccidian parasites.

O mecanismo responsável pela exposição da fosfatidilserina (PS) nas membranas celulares não está bem definido. Uma atividade dependente de ATP está envolvida, provavelmente uma ATPase tipo-P. ATPases tipo P são uma família de proteínas transmembranares envolvidas no transporte de metais, íons e fosfolipídios através da membrana plasmática. As P4 ATPases translocam aminofosfolipidios (APTLs) como a PS durante a apoptose. No entanto, o sentido do transporte de PS pela APLT não está claramente definido. Os macrófagos reconhecem a PS exposta na superfície das células apoptóticas, o que inibe sua capacidade microbicida. Formas promastigotas e amastigotas de Leishmania ssp. sofrem apoptose, porém a exposição de PS na superfície dos promastigotas sempre leva à morte, enquanto que nos amastigotas não está necessariamente associada à morte e permite a internalização desses protozoários e sua sobrevivência no macrófago. Esse trabalho teve como objetivo a caracterização molecular da APLT de L. (L.) amazonensis e a avaliação de seu papel na exposição de PS nesse parasita. / The mechanism responsible for phosphatidylserine (PS) exposure in biological membranes is still an open subject. An ATP-dependent activity is involved, probably a Type P- ATPase. Type P ATPases are a family of transmembrane proteins involved in the transport of metals, ions and phospholipids across plasma membrane. P4 ATPases mediate phospholipid transport (APLT) as PS during the process of cell death by apoptosis. However, the direction (inwards or outwards) of this translocation has not been defined. Macrophages recognize exposed PS on the surface of apoptotic cells, what inhibits their microbicidal capacity. Promastigotes and amastigotes of Leishmania ssp. die by apoptosis, but PS exposure on promastigotes always leads to apoptosis, whereas PS exposure by amastigotes is not necessarily associated to death and allows their internalization and survival in the macrophage. This work aimed to characterize APLT from L. (L.) amazonensis and to evaluate its role in PS exposure in this parasite.

A galectina-1 (Gal-1) é uma lectina homodimérica multifuncional capaz de reconhecer e se ligar a beta-galactosídeos por meio de um domínio denominado carbohydrate recognition domain (CRD). A Gal-1 humana (Gal-1h) e a Gal-1 de camundongo (Gal-1c) mantêm 88,15% de homologia e, apesar de não existirem mutações em aminoácidos-chave do CRD, há substituições próximas a esses resíduos. Considerando as implicações dessas diferenças em estrutura e função, e que é comum a utilização de modelos murinos para estudar a função Gal-1, o presente trabalho objetiva analisar comparativamente a Gal-1c e a Gal-1h por meio de ensaios de cristalização e determinação estrutural da Gal-1c, além da avaliação comparativa da atividade lectínica da Gal-1h e da Gal-1c por glycan array e hemaglutinação. Também foi avaliada a capacidade de ambas as Gal-1 em induzir a exposição de fosfatidilserina (FS) em neutrófilos ativados provenientes de medula de camundongos normais ou deficientes de ?-2 integrina (Mac-1), de modo a investigar se a interação Gal-1/Mac-1 estaria envolvida nesse processo. Preparações homogêneas e ativas de Gal-1c e Gal-1h foram utilizadas nos ensaios. Os cristais de Gal-1c foram obtidos em 20% de polietilenoglicol 3350 e 0,2 M de fluoreto de amônio. Os dados de difração de raios X foram coletados e processados, obtendo-se uma estrutura com resolução de 2,4 Å. Observou-se que substituições de aminoácidos entre a Gal-1c e a Gal-1h estão localizadas em regiões expostas ao solvente, próximas do CRD e distantes da interface de dimerização. A análise comparativa entre Gal-1c e Gal-1h mostrou que estas substituições conferem a Gal-1c um caráter mais polar, com consequente aumento da distribuição de volume molecular. Nos ensaios de hemaglutinação, pode-se observar que é necessária uma concentração 2 vezes maior de Gal-1c para aglutinar eritrócitos humanos, de carneiro e de coelho na mesma proporção que a Gal-1h. Por meio do glycan array, pode-se determinar o perfil de ligação a glicanas de ambas as Gal-1. As duas Gal-1 apresentam afinidade por glicanas ramificadas contendo galactose terminal, e a Gal-1h apresentou maior intensidade de ligação às glicanas quando comparada à Gal-1c. Preparações de Gal-1c e Gal-1h induzem níveis semelhantes de exposição de FS na superfície de neutrófilos deficientes ou não de Mac-1, sugerindo que a interação Gal-1/Mac-1 não esteja envolvida no processo de exposição de FS na superfície de neutrófilos ativados. Assim, a diferença sequencial entre a Gal-1c e a Gal-1h é capaz de gerar diferenças estruturais consideráveis que implicam no reconhecimento diferencial de glicanas, o que, entretanto, não se reflete na capacidade de indução de FS na superfície de neutrófilos ativados. Além disso, a interação Gal-1/Mac-1 parece não participar desse processo, o que pode indicar que o papel da Gal-1 no turnover de neutrófilos, via reconhecimento fagocítico, seja um processo complexo e independente dessa interação. / Galectin-1 (Gal-1) is a homodimeric and multifunctional lectin that recognizes and binds to beta-galactoside by a carbohydrate recognition domain (CRD). Human Gal-1 (hGal-1) and mouse Gal-1 (mGal-1) are 88.15% identical, and although there are no mutations in key amino acids within the CRD, there are differences in the amino acids sequence near the CRD. Given the potential of these differences to alter overall structure and function, and the common utilization of murine models to study Gal-1 function, we sought to directly compare key biochemical features of hGal and mGal-1. Thus, we performed crystallization and structure determination assays of mGal-1, and determined the carbohydrate binding specificy of mGal-1 and hGal-1 using a glycan array and using hemagglutination assay. We also evaluated the ability of both Gal-1 to induce exposure of phosphatidylserine (PS) in activated neutrophils from the bone marrow of normal or ?-2 integrin (Mac-1) deficient mice, in order to investigate the involvement of Gal-1/Mac-1 interaction in this process. To accomplish this, homogeneous and active preparations of hGal-1 and mGal-1 were used in the study. mGal-1 crystals were obtained in 20% polyethylene glycol 3350 and 0.2 M ammonium fluoride. Data from X-ray diffraction were collected and processed, yielding a structure with a final resolution of 2.4 Å. The amino acid substitutions found between mGal-1 and hGaI-1 are detected on the solvent-exposed surfaces where the CRDs are located and not on the proteins dimerization surfaces. A comparative structural analysis between mGal-1 and hGal-1 shows that these amino acid substitutions confer to mGal-1 a greater number of ionizable residues, polar character, appearance of the acid regions clustered, and a slight increase of volume distribution. In hemagglutination assays, twice the concentration of mGal-1 was required to cause equivalent agglutination of human, sheep or rabbit erythrocytes as hGal-1. Glycan array analysis demonstrated that both galectins have affinity for branched glycans containing terminal galactose residues. However, hGal-1 appeared to display higher levels of binding that mGal-1. Preparations of mGal-1 and hGal-1 induced similar levels of PS exposure on normal or Mac-1 deficient neutrophils, suggesting that the interaction Gal-1/Mac-1 is not involved in this process. Thus, hGal-1 and mGal-1 appear to possess considerable differences in glycan recognition that likely reflects subtle difference in amino acid sequence. Furthermore, the interaction Gal-1/Mac-1 do not appear to participate in this PS exposure process, which suggest that other Gal-1 receptors are likely important in this process.

O mecanismo responsável pela exposição da fosfatidilserina (PS) nas membranas celulares não está bem definido. Uma atividade dependente de ATP está envolvida, provavelmente uma ATPase tipo-P. ATPases tipo P são uma família de proteínas transmembranares envolvidas no transporte de metais, íons e fosfolipídios através da membrana plasmática. As P4 ATPases translocam aminofosfolipidios (APTLs) como a PS durante a apoptose. No entanto, o sentido do transporte de PS pela APLT não está claramente definido. Os macrófagos reconhecem a PS exposta na superfície das células apoptóticas, o que inibe sua capacidade microbicida. Formas promastigotas e amastigotas de Leishmania ssp. sofrem apoptose, porém a exposição de PS na superfície dos promastigotas sempre leva à morte, enquanto que nos amastigotas não está necessariamente associada à morte e permite a internalização desses protozoários e sua sobrevivência no macrófago. Esse trabalho teve como objetivo a caracterização molecular da APLT de L. (L.) amazonensis e a avaliação de seu papel na exposição de PS nesse parasita. / The mechanism responsible for phosphatidylserine (PS) exposure in biological membranes is still an open subject. An ATP-dependent activity is involved, probably a Type P- ATPase. Type P ATPases are a family of transmembrane proteins involved in the transport of metals, ions and phospholipids across plasma membrane. P4 ATPases mediate phospholipid transport (APLT) as PS during the process of cell death by apoptosis. However, the direction (inwards or outwards) of this translocation has not been defined. Macrophages recognize exposed PS on the surface of apoptotic cells, what inhibits their microbicidal capacity. Promastigotes and amastigotes of Leishmania ssp. die by apoptosis, but PS exposure on promastigotes always leads to apoptosis, whereas PS exposure by amastigotes is not necessarily associated to death and allows their internalization and survival in the macrophage. This work aimed to characterize APLT from L. (L.) amazonensis and to evaluate its role in PS exposure in this parasite.

Promastigotas de Leishmania são encontradas no trato digestivo do inseto vetor e podem ser cultivados em alguns meios de cultura, onde ocorre a diferenciação regulada geneticamente entre procíclicos e metacíclicos infectivos. Amastigotas são intracelulares e irão se estabelecer e se multiplicar dentro dos vacúolos parasitóforos dos macrófagos. Os metacíclicos e os amastigotas possuem capacidade de evasão do sistema de defesa do hospedeiro vertebrado e conseguem estabelecer uma relação parasita/hospedeiro bem-sucedida. A apoptose, em organismos multicelulares, é uma forma controlada geneticamente de suicídio celular. A eliminação das células apoptóticas é feita pelos fagócitos sem que seja induzida uma resposta inflamatória. Em Leishmania, os eventos da apoptose parecem seguir os mesmos dos encontrados em metazoários, tais como: queda do potencial da mitocôndria, ativação de caspases, clivagem de substratos naturais de caspases, além da externalização de fosfatidilserina (PS) e seu papel na modulação da fagocitose. Nossos dados demonstraram que Leishmania é capaz de mimetizar e utilizar um dos fenômenos observados na apoptose em metazoários, a exposição de PS, como um mecanismo adaptativo para se estabelecer como um parasita de mamíferos. / Promastigotes of Leishmania are found in the midgut environment of the vector insect and can be grown in some culture medium. In culture the genetically regulated differentiation from procyclic to infective metacyclic can be observed. Amastigotes are intracellular parasites and which multiply inside phagolysosomes of macrophages. Metacyclics and amastigotes are endowed with the capacity to evade from the innate and adaptive immune system of the vertebrate host and to establish successful infections. Apoptosis, in multicellular organisms, is a form genetically controlled cellular suicide. The elimination of apoptotic cells is made by phagocytes without initiating an inflammatory event. In Leishmania, the events of apoptosis are similar to those observed in multicellular organisms such as: decrease in mitochondrial transmembrane potential, caspase activation, cleavage of caspase substrates besides exposure of phosphatidylserine (PS) and its role in the modulation of phagocytosis. Our data showed that Leishmania is capable of mimicking one of the features of apoptosis, mainly PS exposure, and to use as an adaptive mechanism for survival in mammalian hosts.

No description available.

<p>  </p> <p>Ebola virus (EBOV) infection remains to be a challenge to human health by its high mortality rate. Though it has been discovered for almost 50 years, there are only two antibody-based therapies approved today, and the mortality rate is still greater than 30% with the treatment. Authentic EBOV studies are strictly limited to biosafety level-4 (BSL-4) labs, which slows the development of treatment. While more simple and safer systems have been developed to understand different stages of EBOV infection, such as the matrix protein (VP40) virus-like particle (VLP) and minigenome systems, we still lack a systematical view of EBOV infection. On the other hand, mathematical modeling has been used to assist biological and medical studies for many years, as it has the advantage of integrating data and providing quantitative insight to a biosystem. In our study, we took advantage of mathematical modeling and build the primary ordinary differential equation-based (ODE-based) model of EBOV at subcellular level step by step. We built the budding pathway of EBOV VP40 first, calibrated and validated our model with experimental data. We proposed that phosphatidylserine (PS) can directly influence the stability of VP40 filaments and the budding process of VLPs. Also, the oligomerization of VP40 filaments may follow the nucleation-elongation process. Next, we conducted in-silico simulation to evaluate the treatment efficiency of fendiline, a drug lowering cell membrane PS level, in treating EBOV. We found that while in general, fendiline can decrease VLP production, there can be fendiline-induced VLP production increases at certain time points due to slow filament growth or fast VLP budding rates. Also, we concluded that fendiline is relatively more effective when applied in the budding stage of EBOV life cycle. Moreover, fendiline efficacy may increase when applied with a VLP budding step targeted treatment. Finally, we integrated nucleoprotein (NP) into our model. We reproduced the two-stage interaction between NP and VP40 and predict that NP increases VLP production through influencing filament oligomerization and VLP budding steps. Also, the dual-effect of NP on VLP production may exist, as a too high NP/VP40 production ratio can decrease VLP production. From the aspect of protein expression time, we found that a bit earlier NP production than VP40 production is beneficial for both inclusion body-containing (IB-containing) VLP production and prevention in energy waste on production of VLPs without IBs. Overall, we have built a solid foundation towards a mathematical EBOV model and demonstrated the value of models in assisting experimental EBOV studies.</p>

No description available.

No description available.

In großen Teilen der Welt verursachen intrazelluläre Parasiten der Spezies Leishmania schwerwiegende Infektionen beim Menschen. Die Exposition eines Phospholipids (Phosphatidylserin, PS) steht unter Verdacht Fresszellen zur Aufnahme der Parasiten zu stimulieren. Bisher ist die Regulation der Phospholipidverteilung in der Plasmamembran dieser Parasiten kaum erforscht. In der vorliegenden Arbeit wurde ein lipidtransportierender Proteinkomplex identifiziert, der einen wesentlichen Beitrag zur asymmetrischen Lipidverteilung in der Plasmamembran von Leishmania donovani leistet. Die Zerstörung des Komplexes führte zum Verlust des einwärts gerichteten Lipidtransports und zur Anreicherung von Phosphatidylethanolamin (PE) auf der Zelloberfläche des Parasiten. Diese veränderte Lipidasymmetrie hatte jedoch keinen Einfluss auf die Phagozytose durch Makrophagen. Darüber hinaus brachte die Untersuchung des Insektenstadiums (Promastigote) verschiedener Leishmania Spezies zu Tage, dass die Menge an PS unterhalb des Detektionslimits modernster Nachweisverfahren liegt. Des Weiteren konnte gezeigt werden, dass der Parasit über einen Scramblase-Mechanismus verfügt, der durch intrazelluläres Kalzium stimulierbar ist. Die Scramblase-Aktivität ist, im Gegensatz zu dem zuvor beschriebenen einwärts gerichteten Lipidtransport, energieunabhängig und ermöglicht die bidirektionale Translokation von fluoreszenzmarkiertem Phosphatidylcholin (PC), PE, PS und Sphingomyelin (SM). Dementsprechend konnte nach Kalziumstimulierung endogenes PE auch in der äußeren Lipidschicht der Plasmamembran detektiert werden, wobei deren Barrierefunktion nicht beeinträchtigt wurde. Diese Ergebnisse geben neue Einblicke in die dynamische Regulation der Lipidverteilung über die Plasmamembran des Parasiten und verdeutlichen, dass die Exposition von PS und PE nicht essentiell für das Eindringen der Leishmanien in die Wirtszellen ist. / The protozoan parasite Leishmania causes severe infections in humans throughout the world. Following the transmission via sand flies to its mammalian host the extracellular parasite has to gain entry into phagocytic cells to initiate a successful infection. Specific surface exposed phospholipids have been implicated in Leishmania macrophage-interaction, but the mecha-nisms controlling and regulating the plasma membrane lipid distribution remains to be eluci-dated. In the present work a lipid transporting protein complex was identified in Leishmania dono-vani which plays an essential role in maintaining an asymmetric lipid distribution across the plasma membrane. Loss of the protein complex abolishes the inward-directed lipid transport and thus e.g. to an increased cell surface exposure of phosphatidylethanolamine (PE). In spite of this altered lipid asymmetry the uptake by macrophages is unaffected. Moreover, Leishma-nia promastigotes of different species lack detectable amounts of phosphatidylserine (PS) although being infective. Furthermore, a scramblase activity following a cytosolic calcium signal was demonstrated. This scramblase mechanism facilitated, in contrast to the previous described inward directed lipid transport, the bidirectional movement of fluorescent lipid analogues of PC, PE, PS and SM in an energy-independent manner. In accordance with these findings endogenous PE was exposed to the outer plasma membrane leaflet following the Ca2+-signal, while the plasma membrane itself remained intact. These results provide novel insight into the dynamic regulation of the transbilayer lipid distri-bution across the parasite plasma membrane and reveal that exposure of PS and PE is not cru-cial for invasion of the host cell by Leishmania donovani promastigotes.