Miria G. Pereira

Subcellular proteomics of Trypanosoma cruzi reservosomes

Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and LC‐MS/MS analysis to identify reservosome‐resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC‐MS/MS analysis identified in total 709 T. cruzi‐specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins, and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles.

Reservosomes: multipurpose organelles?

Reservosomes are endocytic organelles from Trypanosoma cruzi epimastigotes that store proteins and lipids for future use. The lack of molecular markers for the compartments of this parasite makes it difficult to clarify all reservosome functions, as they present characteristics of pre-lysosomes, lysosomes and recycling compartments.

New insights into the morphology of Trypanosoma cruzi reservosome

Reservosomes are late endosomes present only in members of the Schizotrypanum subgenus of the Trypanosoma genus and are defined as the site of storage of endocytosed macromolecules and lysosomal enzymes. They have been extensively described in Trypanosoma cruzi epimastigote: are bounded by a membrane unit, present an electron‐dense protein matrix with electron‐lucent lipid inclusions, being devoid of inner membranes. Here we performed a detailed ultrastructural analysis of these organelles using a variety of electron microscopy techniques, including ultrathin sectioning, uranyl acetate stained preparations, and freeze fracture, either in intact epimastigotes or in isolated reservosomes. New informations were obtained. First, both isolated and in situ reservosomes presented small profiles of inner membranes that are morphologically similar to the membrane surrounding the organelle. In uranyl acetate stained preparations, internal membrane profiles turned out to be longer than they appeared in ultrathin section images and traversed the organelle diameter. Internal vesicles were also found. Second, endocytosed cargo are not associated with internal vesicles and reach reservosomes on board of vesicles that fuse with the boundary membrane, delivering cargo directly into reservosome lumen. Third, electron‐lucent bodies with saturated lipid core surrounded by a membrane monolayer and with unusual rectangular shape were also observed. Fourth, it was possible to demonstrate the presence of intramembranous particles on the E face of both internal vesicles and the surrounding membrane. Collectively, these results indicate that reservosomes have a complex internal structure, which may correlate with their multiple functions. Microsc. Res. Tech., 2008.

Trypanosoma cruzi Epimastigotes Are Able to Store and Mobilize High Amounts of Cholesterol in Reservosome Lipid Inclusions

Background Reservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells. Methodology/Principal Findings Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones. Conclusions/Significance Taken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation.

LDL uptake by Leishmania amazonensis: Involvement of membrane lipid microdomains

Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-β-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.

Trypanosoma cruzi Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions.

Trypanosoma cruzi epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes.

Endocytosis in Trypanosoma cruzi

Endocytic activity is particularly intense in Trypanosoma cruzi epimastigotes, while in amastigotes and trypo- mastigotes it is untraceable. Cargo molecules enters through the cytostome or flagellar pocket at the parasite anterior re- gion, goes along a branched early endosomal network of tubules and vesicles spread from nuclear periphery to the pos- terior pole, until delivery to reservosomes, the final compartment. Reservosomes are acid compartments that store protein and lipid cargo and also accumulate lysosomal hydrolases, modulating digestive activity. Although T. cruzi infective forms are unable to uptake molecules, its lysosome related organelles represent a potential targets for anti-parasitic chemotherapy.

An evaluation of lipid metabolism in the insect trypanosomatid Herpetomonas muscarum uncovers a pathway for the uptake of extracellular insect lipoproteins

Lipid uptake and metabolism by trypanosomatid parasites from vertebrate host blood have been well established in the literature. However, there is a lack of knowledge regarding the same aspects concerning the parasites that cross the hemolymph of their invertebrate hosts. We have investigated the lipid composition and metabolism of the insect trypanosomatid Herpetomonas muscarum by 3H- palmitic acid and phosphate (32Pi) and the parasite interaction with Lipophorin (Lp) the main lipid carrying protein of insect hemolymph. Gas chromatography-mass spectrometry (GC-MS) analyses were used to identify the fatty acids and sterols composition of H.muscarum. Furthermore, we investigated the Lp binding site in the plasma membrane of parasite by Immunolocalization. We showed that H. muscarum incorporated 3H-palmitic acid and inorganic phosphate (32Pi) which were readily used as precursor molecules of lipid biosynthetic pathways. Furthermore, H. muscarum was able to take up both protein and lipid moieties of Lp which could be used as nutrient sources. Moreover, we have also demonstrated for the first time the presence of a Lp binding site in the membrane of a parasite. Such results point out the role of describing the metabolic pathways of trypanosomatids in order to provide a better understanding of parasite-host interaction peculiarities. Such studies may enhance the potential form the identification of novel chemotherapeutic targets in harmful parasites.

Lipophorin Drives Lipid Incorporation and Metabolism in Insect Trypanosomatids

Insect trypanosomatids are inhabitants of the insect digestive tract. These parasites can be either monoxenous or dixenous. Plant trypanosomatids are known as insect trypanosomatids once they and are transmitted by phytophagous insects. Such parasites can be found in latex, phloem, fruits and seeds of many plant families. Infections caused by these pathogens are a major cause of serious economic losses. Studies by independent groups have demonstrated the metabolic flow of lipids from the vertebrate host to trypanosomatids. This mechanism is usually present when parasites possess an incomplete de novo lipid biosynthesis pathway. Here, we show that both insect trypanosomatids Phytomonas françai and Leptomonas wallacei incorporate (3)H-palmitic acid and inorganic phosphate. These molecules are used for lipid biosynthesis. Moreover, we have isolated the main hemolymphatic lipoprotein, Lipophorin (Lp) from Oncopeltus fasciatus, the natural insect vector of such parasites. Both parasites were able to incorporate Lp to be utilized both as a lipid and protein source for their metabolism. Also, we have observed the presence of Lp binding sites in the membrane of a parasite. In conclusion, we believe that the elucidation of trypanosomatid metabolic pathways will lead to a better understanding of parasite-host interactions and the identification of novel potential chemotherapy targets.

Reservosomes of Trypanosoma cruzi

Reservosomes are lysosome-related organelles (LROs) of Trypanosoma cruzi with the special capacity of nutrient storage and hydrolase accumulation. They represent the final compartment of epimastigote endocytic pathway and the site of activity of cruzipain, the major T. cruzi protease. They are essential for epimastigote differentiation into trypomastigote infective forms. Trypomastigotes, as well as amastigotes, present related LROs that do not have storage capacity. Typical epimastigote reservosomes have an electrondense protein matrix in which planar membrane units, rare vesicles, and lipid inclusions are immersed. More than 700 proteins are identified in reservosome proteome, confirming the known organelle functions and indicating its participation in other still unexplored pathways.