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Due to its unique biology the mitochondrion of Trypanosoma brucei has attracted a lot of interest since many decades, making it arguably the best studied mitochondrion outside yeast and mammals. Here we describe a method allowing purification of mitochondria from procyclic trypanosomes that yields highly enriched and functional organelles. The method is based on isotonic lysis of cells by nitrogen cavitation, DNase I digestion, differential centrifugation and Nycodenz gradient centrifugation. The method is scalable and can be adapted to culture volumes a small as 100 mL or as large as 24 L.This protocol describes the use of heavy water (2H2O) labeling to determine the growth rate and metabolic state of Leishmania parasites in culture and in infected animals. In vitro labeling studies are undertaken by cultivating defined parasite developmental stages in standard medium supplemented with 5% 2H2O, resulting in the incorporation of deuterium (2H) into a range of metabolic precursors used in macromolecule (DNA, RNA, protein, lipid, and glycan) synthesis. The rate of turnover of different parasite macromolecules can subsequently be determined by analysis of deuterium enrichment in the different constituents of these macromolecules by gas chromatography-mass spectrometry (GC-MS). To measure the growth rate and physiological state of parasite stages in lesion tissue, infected mice were provided with 9% 2H2O in their drinking water for various periods of time and 2H-enrichment in the macromolecular constituents of isolated lesion-derived parasite stages determined by GC-MS. This protocol provides quantitative information on key cellular processes, such as replication (DNA turnover), transcription (RNA turnover), translation (protein turnover), membrane biogenesis (lipid turnover), and central carbon metabolism (glycan turnover) that define the growth state and phenome of different parasite stages in vitro and in vivo. This approach can be used to assess the impact of host immune responses on parasite growth and physiology (using different Leishmania strains/species, mouse lines), characterize different parasite populations during chronic and acute infections, and assess parasite responses to drug treatments. It is also broadly applicable to other microbial pathogens.2D gel electrophoresis enables resolution of intact proteins in complex mixtures and is thus useful for comparative proteomic analysis, particularly of posttranslationally modified proteoforms that might not be distinguished by shotgun proteomic analysis of peptides. 2D gel electrophoresis is a multistep procedure that can require sample-specific optimization. We present a comprehensive protocol that is effective for 2D electrophoretic analysis of proteins from Leishmania promastigotes and may also be employed for Leishmania amastigotes and for trypanosomes.Exosomes, a class of extracellular vesicles, are released by eukaryotes, bacteria, and archaea, as evident from both in vitro and in vivo studies. These nano-sized double-membraned vesicles play an important role in cell-to-cell communication, dysregulation of the immune system, and pathogenesis in a number of diseases, including leishmaniasis. Leishmania is a genus of obligate intracellular parasites, which infect host macrophages, are transmitted through the bite of a sandfly, and are shown to secrete exosomes with immunomodulatory activities. Given the importance of these vesicles in Leishmania spp. virulence, it is necessary to perform appropriate isolation and characterization in order to further study their relevance in the parasite's infectious life cycle. In this chapter, we describe four methods for the isolation of extracellular vesicles derived from Leishmania species including ultracentrifugation, polyethylene glycol-based precipitation, size-exclusion chromatography, and sucrose-gradient fractionation. Further, we describe the preparation of isolated samples for characterization by nanoparticle tracking analysis, transmission electron microscopy, and proteomic profiling.In its mammalian host, the kinetoplastid protozoan parasite, Trypanosoma cruzi, is obliged to establish intracellular residence in order to replicate. This parasite can infect and replicate within a diverse array of cell and tissue types across many mammalian host species. The establishment of quantitative assays to assess the replicative capacity of intracellular T. cruzi amastigotes under different conditions is a critical facet to understanding this host-pathogen interaction. Telaglenastat price Several complementary methods are outlined here. Their strengths and deficiencies in quantifying intracellular amastigote growth and death are discussed. We describe three assays to assess growth/replication. (1) A high throughput multiplexed plate-based assay that quantifies both host cell and parasite abundance. This method allows for the rapid and simultaneous screening of many conditions (e.g., small molecule inhibitors, the impact of host gene knockdown or of altered environmental parameters). (2) Simple fluorescence microscopy-based enumeration of amastigotes within host cells and (3) flow cytometry-based quantification of amastigote proliferation following isolation from host cells. Each approach has advantages but none of these can assess lethal outcomes in a quantitative manner. For this, we describe a clonal outgrowth assay that identifies the proportion of parasites that succumb to a defined exposure. Even using these assays, it can be challenging to differentiate between direct (targeting the parasite) and/or indirect (targeting the host) effects of a given treatment on amastigote growth. Therefore, we also outline a method of purification of intracellular amastigotes that allows for downstream biochemical and metabolic investigations specifically on the isolated amastigote.Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Most of the different PDE variants play specific physiological functions; in fact, PDEs can associate with other proteins allowing them to be strategically anchored throughout the cell. In this regard, precise cellular expression and compartmentalization of these enzymes produce the specific control of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) gradients in cells and enable their integration with other signaling pathways.In trypanosomatids, some PDEs are essential for their survival and play fundamental roles in the adaptation of these parasites to different environmental stresses, as well as in the differentiation between their different life cycle forms. Given that these enzymes not only are similar to human PDEs but also have differential biochemical properties, and due to the great knowledge of drugs that target human PDEs, trypanosomatid PDEs could be postulated as important therapeutic targets through the repositioning of drugs.
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