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.

Comparative analysis of humoral immune response upon the three first vaccines applied in Argentina: IgG production and neutralizing capacity against SARS-CoV-2.

The population that has not received a SARS-CoV-2 vaccine is at high risk for infection whereas vaccination prevents COVID-19 severe disease, hospitalization, and death. In Argentina, to date, more than 50 million doses of vaccines against SARS-CoV-2 have been administered. The three main vaccines applied are Sputnik V, Oxford-AstraZeneca, and Sinopharm. In this study, we have compared the antibody response of voluntary individuals at day 0 (first dose vaccination day) and at 21-25 days post first and second dose. Our results indicate that at 21-25 days after the administration of the first doses of Sputnik V the large majority of the people vaccinated 80% (n = 15) presented high humoral responses as determined by the measurement of IgG against the Spike protein and the Receptor Binding Domain (RBD). In the case of those vaccinated with AstraZeneca, the percentage was 80% (n = 15) whereas this value was reduced to only 25% (n = 16) in persons that received Sinopharm. However, after the second doses, most of the recipients had significant levels of antibodies. The virus neutralizing capacity of the antibodies generated was evaluated using a pseudotyped VSV-SARS-CoV2 Spike expressing eGFP and the data was analyzed by fluorescence microscopy and flow cytometry. The results indicate that a good correlation exists between the levels of IgG and the neutralizing capacity of the antibodies against the recombinant virus. Our results stand out the importance of applying the second dose of Sinopharm. Thus, the present report provides data that will contribute to decisions making about the vaccine implementation plans of action for, not only our region but our country to support the fight against the COVID-19 global pandemic.

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.

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.

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.

The superfamily keeps growing: Identification in trypanosomatids of RibJ, the first riboflavin transporter family in protists.

BACKGROUND: Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. CONCLUSIONS/SIGNIFICANCE: The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target.

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.

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity.

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.

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.