Trypanosoma cruzi-secreted vesicles have acid and alkaline phosphatase activities capable of increasing parasite adhesion and infection.

Trypanosoma cruzi virulence factors include molecules expressed on the cell surface as well as those secreted or shed into the extracellular medium. Phosphatase activities modulate different aspects of T. cruzi infection, although no studies to date addressed the presence and activity of phosphatases in vesicles secreted by this parasite. Here, we characterized acidic and alkaline secreted phosphatase activities of human-infective trypomastigote forms of T. cruzi from the Y strain and the CL-Brener clone. These are widely studied T. cruzi strains that represent "opposite ends of the spectrum" regarding both in vitro and in vivo behavior. Ecto-phosphatase activities were determined in live parasites, and secreted phosphatase activities were analyzed in soluble protein (SP) and vesicular membrane fractions (VFs) of parasite-conditioned medium. Our analysis using different phosphatase inhibitors strongly suggests that vesicles secreted by Y strain (VF(Y)) and CL-Brener (VF(CLB)) trypomastigotes are derived mostly from the cell surface and from exosome secretion, respectively. Importantly, our results show that the acid phosphatase activities in vesicles secreted by trypomastigotes are largely responsible for the VF-induced increase in adhesion of Y strain parasites to host cells and also for the VF-induced increase in host cell infection by CL-Brener trypomastigotes.

Endocytosis and Exocytosis in Leishmania amazonensis Are Modulated by Bromoenol Lactone.

In the protozoan pathogen Leishmania, endocytosis, and exocytosis occur mainly in the small area of the flagellar pocket membrane, which makes this parasite an interesting model of strikingly polarized internalization and secretion. Moreover, little is known about vesicle recognition and fusion mechanisms, which are essential for both endo/exocytosis in this parasite. In other cell types, vesicle fusion events require the activity of phospholipase A2 (PLA2), including Ca2+-independent iPLA2 and soluble, Ca2+-dependent sPLA2. Here, we studied the role of bromoenol lactone (BEL) inhibition of endo/exocytosis in promastigotes of Leishmania amazonensis. PLA2 activities were assayed in intact parasites, in whole conditioned media, and in soluble and extracellular vesicles (EVs) conditioned media fractions. BEL did not affect the viability of promastigotes, but reduced the differentiation into metacyclic forms. Intact parasites and EVs had BEL-sensitive iPLA2 activity. BEL treatment reduced total EVs secretion, as evidenced by reduced total protein concentration, as well as its size distribution and vesicles in the flagellar pocket of treated parasites as observed by TEM. Membrane proteins, such as acid phosphatases and GP63, became concentrated in the cytoplasm, mainly in multivesicular tubules of the endocytic pathway. BEL also prevented the endocytosis of BSA, transferrin and ConA, with the accumulation of these markers in the flagellar pocket. These results suggested that the activity inhibited by BEL, which is one of the irreversible inhibitors of iPLA2, is required for both endocytosis and exocytosis in promastigotes of L. amazonensis.

Nanomicellar Formulation of Clotrimazole Improves Its Antitumor Action toward Human Breast Cancer Cells.

BACKGROUND: Although demonstrated as a selective anticancer drug, the clinical use of clotrimazole (CTZ) is limited due to its low solubility in hydrophilic fluids. Thus, we prepared a water-soluble nanomicellar formulation of CTZ (nCTZ) and tested on the human breast cancer cell line MCF-7 biology. METHODOLOGY/PRINCIPAL FINDINGS: CTZ was nanoencapsulated in tween 80 micelles, which generated nanomicelles of, approximately, 17 nm of diameter. MCF-7 cells were treated with nCTZ and unencapsulated DMSO-solubilized drug (sCTZ) was used for comparison. After treatment, the cells were evaluated in terms of metabolism, proliferation, survival and structure. We found that nCTZ was more efficient than sCTZ at inhibiting glycolytic and other cytosolic and mitochondrial enzymes. Moreover, this increased activity was also observed for lactate production, intracellular ATP content, ROS production and antioxidant potential. As a consequence, nCTZ-treated MCF-7 cells displayed alterations to the plasma membrane, mitochondria and the nucleus. Finally, nCTZ induced both apoptosis and necrosis in MCF-7 cells. CONCLUSIONS/SIGNIFICANCE: MCF-7 cells are more sensible to nCTZ than to sCTZ. This was especially evident on regard to antioxidant potential, which is an important cell defense against drugs that affect cell metabolism. Moreover, this water-soluble formulation of CTZ strengths its potential use as an anticancer medicine.

Different secreted phosphatase activities in Leishmania amazonensis.

Leishmania has strong acid phosphatase activity on the external surface of the plasma membrane and secreted into the extracellular milieu. Secreted acid phosphatase (sAcP), which is the most abundant secreted protein of Leishmania, is also a virulence factor that plays a role in vertebrate infection and survival in sand flies. In this study, we characterized the secreted phosphatase activities in Leishmania amazonensis. Both acidic and alkaline secreted phosphatase activities were observed with ß-glycerophosphate and p-nitrophenyl phosphate (p-NPP) hydrolysis and were inhibited with sodium tartrate and sodium orthovanadate. Cytochemical labeling revealed a significant difference in the localization of the electron-dense precipitates depending on the substrate. ß-Glycerophosphate electron-dense precipitates were concentrated on both the cell surface and flagellar pocket, whereas p-NPP labeling occurred primarily within intracellular organelles. Orthovanadate-treated metacyclic promastigotes were less infective and were confined to a tight parasitophorous vacuole (PV), which is not characteristic of this Leishmania species. Based on the results, we characterized the presence of different secreted phosphatase activities in L. amazonensis, the influence of the substrate in cytochemical labeling, and the potential involvement of secreted phosphatase activity in both PV maturation and amastigote survival.