, 1997). In the symbiosis of trypanosomatids, intense metabolic exchanges occur between both partners: the bacterium contains essential enzymes that complete important biosynthetic pathways of the protozoa and in exchange receives suitable physical conditions and energy supply from the host (reviewed by Motta, 2010). As in most prokaryotes, sterols are absent in symbiont membranes that have cardiolipin (CL) as the major phospholipid, followed by similar amounts of PC and PE and a minor quantity of PI (Palmié-Peixoto et al., 2006). The endosymbiont enhances the protozoan phospholipid production and depends in part on its host cell to obtain PC (Azevedo-Martins et al.,

2007). Organelles of symbiotic origin play Ibrutinib manufacturer important roles in the eukaryotic cell lipid biosynthesis. Significant levels of phospholipid production occur in mitochondria that synthesize phosphatidic acid (PA) and phosphatidylglycerol, which is used to produce CL, a lipid that is mainly found in prokaryotes and mitochondria, as well as PE (Van Meer et al., 2008). In this work, we tested the effect of miltefosine Selleck Silmitasertib on cell proliferation, ultrastructure, and phospholipid biosynthesis of A. deanei. The main proposal of miltefosine treatment on this nonpathogenic trypanosomatid species is to evaluate, if once the protozoan

phospholipid production is affected, how does it influence the symbiotic bacterium and mitochondrion composition. Thus, it is worth considering that both structures have symbiotic origin and are related to the protozoan phospholipid metabolism. Angomonas deanei was grown BCKDHA at 28 °C for 24 h in Warren’s culture medium (Warren, 1960) supplemented with 10% fetal calf serum. Miltefosine (Cayman Chemical) was used after dilutions of a 100 mM stock solution dissolved in absolute methanol. Cells (1.0 × 106 mL−1) were inoculated in culture medium and after 12 h (exponential growth phase) were submitted to different drug concentrations: 10, 25, 50, 75, and 100 μM. Protozoa were collected from the culture at 12 h intervals until 72 h of growth;

then, part of the cells were counted in a Neubauer chamber, and the remainder was processed for transmission electron microscopy as described below. To verify the effect of miltefosine on phospholipid biosynthesis, cells were grown for 24, 36, and 48 h in Warren medium containing 4 μCi of 32Pi, whereas for cell fractioning assays, protozoa were cultivated for 24 h in the presence of 10 μCi of 32Pi. Isolated symbionts and mitochondria were obtained by cell fractioning as established by Alfieri & Camargo (1978), with some modifications. Cells were disrupted using an ultrasonic disruptor GEX-600 (three series of 15-s pulses at 10% amplitude). The homogenate passed throws differential centrifugations and sucrose gradient, to obtain a rich fraction of endosymbionts and mitochondria. Then, fractions were resuspended in 1 mL of Tris-HCl 20 mM and sucrose 0.