The host Rab9a/Rab32 axis is actively recruited to the Trypanosoma cruzi parasitophorous vacuole and benefits the infection cycle.

Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoan parasite that infects phagocytic and non-phagocytic mammalian cells. At early stages of infection, trypomastigotes, the infective forms of this parasite, localize in a vesicular compartment called the T. cruzi parasitophorous vacuole until the exit of parasites to the host cell cytoplasm where continue their infective cycle. Rab proteins participate in the membrane traffic's molecular machinery, functioning as central regulators of vesicle recognition and transport. In previous work, we demonstrated that endocytic Rabs are key factors of the T. cruzi infection process in non-phagocytic cells, regulating the formation and the maturation of the vacuole. In this work, we identified and characterized other molecular components of the vesicular transport pathways and their participation in the T. cruzi infection. We found that Rab9a and Rab32, two regulators of the endocytic and autophagic pathways, were actively recruited to the T. cruzi vacuoles and favored the late stages of the infective process. The recruitment was specific and dependent on T. cruzi protein synthesis. Interestingly, Rab32 association depends on the presence of Rab9a in the vacuolar membrane, while the inhibition of the cysteine-protease cruzipain, a T. cruzi virulence factor, significantly decreases both Rab9a and Rab32 association with the vacuole. In summary, this work showed for the first time that specific molecules produced and secreted by the parasite can subvert intracellular components of host cells to benefit the infection. These new data shed light on the complex map of interactions between T. cruzi and the host cell and introduce concepts that can be useful in finding new forms of intervention against this parasite in the future.

Autophagy in protists and their hosts: When, how and why?

Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.

Biological features of TcM: A new Trypanosoma cruzi isolate from Argentina classified into TcV lineage.

Trypanosoma cruzi, the etiologic agent of Chagas disease (CD) presents a wide genetic and phenotypic diversity that is classified into seven lineages or discrete typing units (DTU: TcI to TcVI and Tcbat). Although isolates and strains that belong to a particular group can share some attributes, such as geographic distribution, others like growth rate, cell tropism, and response to treatment can be highly variable. In addition, studies that test new trypanocidal drugs are frequently conducted on T. cruzi strains maintained for a long time in axenic culture, resulting in changes in parasite virulence and other important features. This work aimed to isolate and characterize a new T. cruzi strain from a chronic Chagas disease patient. The behavior of this isolate was studied by using standard in vitro assays and in vivo mice infection tests and compared with the T. cruzi Y strain (TcY), broadly used in research laboratories worldwide. Data showed that TcM behaves as a slow-growing strain in vitro that develops chronic infections in mice and displays high tropism to muscular tissues, in accordance with its clinical performance. In contrast, the Y strain behaved as an acute strain that can infect different types of cells and tissues. Interestingly, TcM, which belongs to DTU TcV, is more susceptible to benznidazole than TcY, a TcII strain considered moderately resistant to this drug. These differential properties contribute to the characterization of a TcV strain, one of the main lineages in the southern countries of South America, and open the possibility to introduce changes that improve the management of Chagas patients in the future.

Induction of Autophagy by Ursolic Acid Promotes the Elimination of Trypanosoma cruzi Amastigotes From Macrophages and Cardiac Cells.

Chagas disease, caused by the parasite Trypanosoma cruzi, is an infectious illness endemic to Latin America and still lacks an effective treatment for the chronic stage. In a previous study in our laboratory, we established the protective role of host autophagy in vivo during T. cruzi infection in mice and proposed this process as one of the mechanisms involved in the innate immune response against this parasite. In the search for an autophagy inducer that increases the anti-T. cruzi response in the host, we found ursolic acid (UA), a natural pentacyclic triterpene with many biological actions including autophagy induction. The aim of this work was to study the effect of UA on T. cruzi infection in vitro in the late infection stage, when the nests of intracellular parasites are forming, in both macrophages and cardiac cells. To test this effect, the cells were infected with T. cruzi for 24 h and then treated with UA (5-10 µM). The data showed that UA significantly decreased the number of amastigotes found in infected cells in comparison with non-treated cells. UA also induced the autophagy response in both macrophages and cardiac cells under the studied conditions, and the inhibition of this pathway during UA treatment restored the level of infection. Interestingly, LC3 protein, the main marker of autophagy, was recruited around amastigotes and the acidic probe LysoTracker localized with them, two key features of xenophagy. A direct cytotoxic effect of UA was also found on trypomastigotes of T. cruzi, whereas epimastigotes and amastigotes displayed more resistance to this drug at the studied concentrations. Taken together, these data showed that this natural compound reduces T. cruzi infection in the later stages by promoting parasite damage through the induction of autophagy. This action, in addition to the effect of this compound on trypomastigotes, points to UA as an interesting lead for Chagas disease treatment in the future.

Repurposing Carvedilol as a Novel Inhibitor of the Trypanosoma cruzi Autophagy Flux That Affects Parasite Replication and Survival.

T. cruzi, the causal agent of Chagas disease, is a parasite able to infect different types of host cells and to persist chronically in the tissues of human and animal hosts. These qualities and the lack of an effective treatment for the chronic stage of the disease have contributed to the durability and the spread of the disease around the world. There is an urgent necessity to find new therapies for Chagas disease. Drug repurposing is a promising and cost-saving strategy for finding new drugs for different illnesses. In this work we describe the effect of carvedilol on T. cruzi. This compound, selected by virtual screening, increased the accumulation of immature autophagosomes characterized by lower acidity and hydrolytic properties. As a consequence of this action, the survival of trypomastigotes and the replication of epimastigotes and amastigotes were impaired, resulting in a significant reduction of infection and parasite load. Furthermore, carvedilol reduced the whole-body parasite burden peak in infected mice. In summary, in this work we present a repurposed drug with a significant in vitro and in vivo activity against T. cruzi. These data in addition to other pharmacological properties make carvedilol an attractive lead for Chagas disease treatment.

Induction of autophagy increases the proteolytic activity of reservosomes during Trypanosoma cruzi metacyclogenesis.

Cruzipain, the major cysteine protease of the pathogenic protozoa Trypanosoma cruzi, is an important virulence factor that plays a key role in the parasite nutrition, differentiation and host cell infection. Cruzipain is synthesized as a zymogen, matured, and delivered to reservosomes. These organelles that store proteins and lipids ingested by endocytosis undergo a dramatic decrease in number during the metacyclogenesis of T. cruzi. Autophagy is a process that digests the own cell components to supply energy under starvation or different stress situations. This pathway is important during cell growth, differentiation and death. Previously, we showed that the autophagy pathway of T. cruzi is induced during metacyclogenesis. This work aimed to evaluate the participation of macroautophagy/autophagy in the distribution and function of reservosomes and cruzipain during this process. We found that parasite starvation promotes the cruzipain delivery to reservosomes. Enhanced autophagy increases acidity and hydrolytic activity in these compartments resulting in cruzipain enzymatic activation and self- processing. Inhibition of autophagy similarly impairs cruzipain traffic and activity than protease inhibitors, whereas mutant parasites that exhibit increased basal autophagy, also display increased cruzipain processing under control conditions. Further experiments showed that autophagy induced cruzipain activation and self-processing promote T. cruzi differentiation and host cell infection. These findings highlight the key role of T. cruzi autophagy in these processes and reveal a potential new target for Chagas disease therapy.Abbreviations: Baf: bafilomycin A1; CTE: C-terminal extension; Cz: cruzipain; IIF: indirect immunofluorescence; K777: vinyl sulfone with specific Cz inhibitory activity; Prot Inh: broad-spectrum protease inhibitor; Spa1: spautin-1; Wort: wortmannin.

Endocytic Rabs Are Recruited to the Trypanosoma cruzi Parasitophorous Vacuole and Contribute to the Process of Infection in Non-professional Phagocytic Cells.

Trypanosoma cruzi is the parasite causative of Chagas disease, a highly disseminated illness endemic in Latin-American countries. T. cruzi has a complex life cycle that involves mammalian hosts and insect vectors both of which exhibits different parasitic forms. Trypomastigotes are the infective forms capable to invade several types of host cells from mammals. T. cruzi infection process comprises two sequential steps, the formation and the maturation of the Trypanosoma cruzi parasitophorous vacuole. Host Rab GTPases are proteins that control the intracellular vesicular traffic by regulating budding, transport, docking, and tethering of vesicles. From over 70 Rab GTPases identified in mammalian cells only two, Rab5 and Rab7 have been found in the T. cruzi vacuole to date. In this work, we have characterized the role of the endocytic, recycling, and secretory routes in the T. cruzi infection process in CHO cells, by studying the most representative Rabs of these pathways. We found that endocytic Rabs are selectively recruited to the vacuole of T. cruzi, among them Rab22a, Rab5, and Rab21 right away after the infection followed by Rab7 and Rab39a at later times. However, neither recycling nor secretory Rabs were present in the vacuole membrane at the times studied. Interestingly loss of function of endocytic Rabs by the use of their dominant-negative mutant forms significantly decreases T. cruzi infection. These data highlight the contribution of these proteins and the endosomal route in the process of T. cruzi infection.

Autophagy plays a protective role against Trypanosoma cruzi infection in mice.

Autophagy is a catabolic pathway required for cellular and organism homeostasis. Autophagy participates in the innate and adaptive immune responses at different levels. Xenophagy is a class of selective autophagy that involves the elimination of intracellular pathogens. Trypanosoma cruzi is the causative agent of Chagas, a disease that affects 8 million individuals worldwide. Previously, our group has demonstrated that autophagy participates in the invasion of T. cruzi in non-phagocytic cells. In this work we have studied the involvement of autophagy in the development of T. cruzi infection in mice. Beclin-1 is a protein essential for autophagy, required for autophagosome biogenesis and maturation. We have performed an acute model of infection on the autophagic deficient Beclin-1 heterozygous knock-out mice (Bcln±) and compared to control Bcln+/+ animals. In addition, we have analyzed the infection process in both peritoneal cells and RAW macrophages. Our results have shown that the infection was more aggressive in the autophagy-deficient mice, which displayed higher numbers of parasitemia, heart´s parasitic nests and mortality rates. We have also found that peritoneal cells derived from Bcln± animals and RAW macrophages treated with autophagy inhibitors displayed higher levels of infection compared to controls. Interestingly, free cytosolic parasites recruited LC3 protein and other markers of xenophagy in control compared to autophagy-deficient cells. Taken together, these data suggest that autophagy plays a protective role against T. cruzi infection in mice, xenophagy being one of the processes activated as part of the repertoire of immune responses generated by the host.

Autophagy: A necessary process during the Trypanosoma cruzi life-cycle.

Autophagy is a well-conserved process of self-digestion of intracellular components. T. cruzi is a protozoan parasite with a complex life-cycle that involves insect vectors and mammalian hosts. Like other eukaryotic organisms, T. cruzi possesses an autophagic pathway that is activated during metacyclogenesis, the process that generates the infective forms of parasites. In addition, it has been demonstrated that mammalian autophagy has a role during host cell invasion by T. cruzi, and that T. cruzi can modulate this process to its own benefit. This review describes the latest findings concerning the participation of autophagy in both the T. cruzi differentiation processes and during the interaction of parasites within the host cells. Data to date suggest parasite autophagy is important for parasite survival and differentiation, which offers interesting prospects for therapeutic strategies. Additionally, the interruption of mammalian autophagy reduces the parasite infectivity, interfering with the intracellular cycle of T. cruzi inside the host. However, the impact on other stages of development, such as the intracellular replication of parasites is still not clearly understood. Further studies in this matter are necessaries to define the integral effect of autophagy on T. cruzi infection with both in vitro and in vivo approaches.

The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis.

Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients) to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development.

Neglected Tropical Protozoan Diseases: Drug Repositioning as a Rational Option.

Neglected tropical diseases represent a major sanitary problem and a huge economic burden to endemic countries, and are currently expanding to non-endemic countries owing to migration currents. Though long abandoned in the past, recent research on novel therapeutics has already started to show results. Drug repositioning is one of the prominent, more successful strategies to approach the development of new treatments for these diseases. Here we present an overview on the limitations of the current available medications to treat African trypanosomiasis, Chagas disease and Leishmaniasis, along with a review on drug candidates presently undergoing clinical trials and drug candidates identified through drug repositioning initiatives.

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay.

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells.

Trypanosoma cruzi Coexpressing Ornithine Decarboxylase and Green Fluorescence Proteins as a Tool to Study the Role of Polyamines in Chagas Disease Pathology.

Polyamines are essential for Trypanosoma cruzi, the causative agent of Chagas disease. As T. cruzi behaves as a natural auxotrophic organism, it relies on host polyamines biosynthesis. In this paper we obtained a double-transfected T. cruzi parasite that expresses the green fluorescent protein (GFP) and a heterologous ornithine decarboxylase (ODC), used itself as a novel selectable marker. These autotrophic and fluorescent parasites were characterized; the ODC presented an apparent Km for ornithine of 0.51 ± 0.16 mM and an estimated V(max) value of 476.2 nmoles/h/mg of protein. These expressing ODC parasites showed higher metacyclogenesis capacity than the auxotrophic counterpart, supporting the idea that polyamines are engaged in this process. This double-transfected T. cruzi parasite results in a powerful tool-easy to follow by its fluorescence-to study the role of polyamines in Chagas disease pathology and in related processes such as parasite survival, invasion, proliferation, metacyclogenesis, and tissue spreading.