From the cell biology to the development of new chemotherapeutic approaches against trypanosomatids: dreams and reality.

Members of the Trypanosomatidae family comprise a large number of species that are causative agents of important diseases such as sleeping sickness, Chagas' disease and Leishmaniasis. These organisms are also of biological interest since they are able to change the morphology according to the environment where they live, through a process of reversible cell transformation, and possess structures and organelles that are not found in mammalian cells. This review analyses the process of transformation, which takes place during the life cycle of Trypanosoma cruzi in the vertebrate and invertebrate hosts. Special attention is given to the interaction of the parasite with vertebrate cells. In addition, the present knowledge of structures and organelles such as the nucleus, the plasma membrane, the sub-pellicular microtubules, the flagellum, the kinetoplast-mitochondrion complex, the peroxisome (glycosome), the acidocalcisome and the structures and organelles involved in the endocytic pathway, is reviewed from a cell biology perspective. The possible use of available data for the development of new anti parasite drugs is also discussed

Morphological and Functional Aspects of Cytoskeleton of Trypanosomatids

Trypanosomatidae are protozoans that include monogenetic parasites, such as the Blastocrithidia and Herpetomonas genera, as well as digenetic parasites, such as the Trypanosoma and Leishmania genera. Their life cycles alternate between insect vectors and mammalian hosts. The parasite’s life cycle involves symmetrical division and different transitional developmental stages. In trypanosomatids, the cytoskeleton is composed of subpellicular microtubules organized in a highly ordered array of stable microtubules located beneath the plasma membrane, the paraflagellar rod, which is a lattice-like structure attached alongside the flagellar axoneme and a cytostome-cytopharynx. The complex life cycle, the extremely precise cytoskeletal organization and the single copy structures present in trypanosomatids provide interesting models for cell biology studies. The introduction of molecular biology, FIB/SEM (focused ion beam scanning electron microscopy) and electron microscopy tomography approaches and classical methods, such as negative staining, chemical fixation and ultrafast cryofixation have led to the determination of the three-dimensional (3D) structural organization of the cells. In this chapter, we highlight the recent findings on Trypanosomatidae cytoskeleton emphasizing their structural organization and the functional role of proteins involved in the biogenesis and duplication of cytoskeletal structures. The principal finding of this review is that all approaches listed above enhance our knowledge of trypanosomatids biology showing that cytoskeleton elements are essential to several important events throughout the protozoan life cycle

Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs

Sterols are constituents of the cellular membranes that are essential for their normal structure and function. In mammalian cells, cholesterol is the main sterol found in the various membranes. However, other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. It is now well established that an important metabolic pathway in fungi and in members of the Trypanosomatidae family is one that produces a special class of sterols, including ergosterol, and other 24-methyl sterols, which are required for parasitic growth and viability, but are absent from mammalian host cells. Currently, there are several drugs that interfere with sterol biosynthesis (SB) that are in use to treat diseases such as high cholesterol in humans and fungal infections. In this review, we analyze the effects of drugs such as (a) statins, which act on the mevalonate pathway by inhibiting HMG-CoA reductase, (b) bisphosphonates, which interfere with the isoprenoid pathway in the step catalyzed by farnesyl diphosphate synthase, (c) zaragozic acids and quinuclidines, inhibitors of squalene synthase (SQS), which catalyzes the first committed step in sterol biosynthesis, (d) allylamines, inhibitors of squalene epoxidase, (e) azoles, which inhibit C14α-demethylase, and (f) azasterols, which inhibit Δ24(25)-sterol methyltransferase (SMT). Inhibition of this last step appears to have high selectivity for fungi and trypanosomatids, since this enzyme is not found in mammalian cells. We review here the IC50 values of these various inhibitors, their effects on the growth of trypanosomatids (both in axenic cultures and in cell cultures), and their effects on protozoan structural organization (as evaluted by light and electron microscopy) and lipid composition. The results show that the mitochondrial membrane as well as the membrane lining the protozoan cell body and flagellum are the main targets. Probably as a consequence of these primary effects, other important changes take place in the organization of the kinetoplast DNA network and on the protozoan cell cycle. In addition, apoptosis-like and autophagic processes induced by several of the inhibitors tested led to parasite death

The Endomembrane System of Giardia intestinalis

Giardia intestinalis is a protozoan that colonizes the small intestine of virtually all mammals, adhering to the mucosal epithelial cells. It is a cosmopolitan parasite and agent of giardiasis, which can lead to human diarrheal diseases. The Giardia life cycle presents two forms—the trophozoite and the cyst—which are responsible for infection and transmission, respectively. This cell has been considered an excellent model for evolutionary studies, even though there are controversial hypotheses as to whether this parasite is an early eukaryote or not. G. intestinalis has a unique and very basic endomembrane system. The trophozoite gathers a very small pack of membrane-bounded structures: nuclei, endoplasmic reticulum (ER), peripheral vesicles (PV) and mitosomes. These organelles are involved in many functions from regulatory aspects in gene expression as well as membrane traffic events. Two functional nuclei are observed in the parasite; they are always located symmetrically in the anterior region of the trophozoite. The ER and PV commonly share and accumulate functions in the secretory pathway, they are responsible for endocytosis and digestion processes. The mitosome is a mitochondria-related organelle that does not produce ATP and lacks several mitochondrial characteristics. During the parasite differentiation into cyst, different types of vesicles appear into the cell body: the encystation specific vesicles (ESVs) and the encystation carbohydrate-positive vesicles (ECVs). These vesicles work together to form the parasite’s cyst wall in order to ensure that the cell reaches the cyst stage. Interestingly, Giardia does not present a morphologically recognized Golgi apparatus. It has been claimed that during the encystation process, the ESVs could represent a Golgi-like structure, because this organelle presents some characteristics of that high eukaryotic Golgi apparatus. In this book chapter, we highlight the G. intestinalis endomembrane system, emphasizing their morphology, proteins involved in its organization as well as their functional role

Monitoring of dynamin during the Toxoplasma gondii cell cycle

The obligate intracellular protozoan parasite Toxoplasma gondii actively invades virtually all warm-blooded nucleated cells. This process results in a non-fusogenic vacuole, inside which the parasites replicate continuously until egress signaling is triggered. In this work, we investigated the role of the large GTPase dynamin in the interaction of T. gondii with the host cell by using laser and electron microscopy during three key stages: invasion, development and egress. The detection of dynamin during invasion indicates the occurrence of endocytosis, while T. gondii egress appeared to be independent of dynamin participation. However, the presence of dynamin during T. gondii development suggests that this molecule plays undescribed roles in the tachyzoite's cell cycle