Extracellular Amastigotes Research Articles

The Intracellular Amastigote of Trypanosoma cruzi Maintains an Actively Beating Flagellum.

Throughout its complex life cycle, the uniflagellate parasitic protist, Trypanosoma cruzi, adapts to different host environments by transitioning between elongated motile extracellular stages and a nonmotile intracellular amastigote stage that replicates in the cytoplasm of mammalian host cells. Intracellular T. cruzi amastigotes retain a short flagellum that extends beyond the opening of the flagellar pocket with access to the extracellular milieu. Contrary to the long-held view that the T. cruzi amastigote flagellum is inert, we report that this organelle is motile and displays quasiperiodic beating inside mammalian host cells. Kymograph analysis determined an average flagellar beat frequency of ~0.7 Hz for intracellular amastigotes and similar beat frequencies for extracellular amastigotes following their isolation from host cells. Inhibitor studies reveal that flagellar motility in T. cruzi amastigotes is critically dependent on parasite mitochondrial oxidative phosphorylation. These novel observations reveal that flagellar motility is an intrinsic property of T. cruzi amastigotes and suggest that this organelle may play an active role in the parasite infection process. IMPORTANCE Understanding the interplay between intracellular pathogens and their hosts is vital to the development of new treatments and preventive strategies. The intracellular "amastigote" stage of the Chagas disease parasite, Trypanosoma cruzi, is a critical but understudied parasitic life stage. Previous work established that cytosolically localized T. cruzi amastigotes engage physically and selectively with host mitochondria using their short, single flagellum. The current study was initiated to examine the dynamics of the parasite flagellum-host mitochondrial interaction through live confocal imaging and led to the unexpected discovery that the T. cruzi amastigote flagellum is motile.

Modulation of STAT-1, STAT-3, and STAT-6 activities in THP-1 derived macrophages infected with two Trypanosoma cruzi strains.

Trypanosoma cruzi is the causative protozoan of Chagas' Disease, a neglected tropical disease that affects 6-7 million people worldwide. Interaction of the parasite with the host immune system is a key factor in disease progression and chronic symptoms. Although the human immune system is capable of controlling the disease, the parasite has numerous evasion mechanisms that aim to maintain intracellular persistence and survival. Due to the pronounced genetic variability of T. cruzi, co-infections or mixed infections with more than one parasite strain have been reported in the literature. The intermodulation in such cases is unclear. This study aimed to evaluate the co-infection of T. cruzi strains G and CL compared to their individual infections in human macrophages derived from THP-1 cells activated by classical or alternative pathways. Flow cytometry analysis demonstrated that trypomastigotes were more infective than extracellular amastigotes (EAs) and that strain G could infect more macrophages than strain CL. Classically activated macrophages showed lower number of infected cells and IL-4-stimulated cells displayed increased CL-infected macrophages. However, co-infection was a rare event. CL EAs decreased the production of reactive oxygen species (ROS), whereas G trypomastigotes displayed increased ROS detection in classically activated cells. Co-infection did not affect ROS production. Monoinfection by strain G or CL mainly induced an anti-inflammatory cytokine profile by decreasing inflammatory cytokines (IFN-γ, TNF-α, IL-1β) and/or increasing IL-4, IL-10, and TGF-β. Co-infection led to a predominant inflammatory milieu, with reduced IL-10 and TGF-β, and/or promotion of IFN-γ and IL-1β release. Infection by strain G reduced activation of intracellular signal transducer and activator of transcription (STAT) factors. In EAs, monoinfections impaired STAT-1 activity and promoted phosphorylation of STAT-3, both changes may prolong cell survival. Coinfected macrophages displayed pronounced activation of all STATs examined. These activations likely promoted parasite persistence and survival of infected cells. The collective results demonstrate that although macrophages respond to both strains, T. cruzi can modulate the intracellular environment, inducing different responses depending on the strain, parasite infective form, and co-infection or monoinfection. The modulation influences parasite persistence and survival of infected cells.

New insights on severe clinical manifestations and deaths from visceral leishmaniasis in free-living crab-eating foxes (Cerdocyon thous) in Brazil

Visceral leishmaniasis (VL) is a zoonotic and severe neglected tropical disease, with worldwide distribution, that still cause many deaths among dogs and humans. Brazil is the country responsible for about 97% of the cases of leishmaniasis in the Americas and the disease is still considered a serious public health concern. Among wild mammalians, studies demonstrate the involvement of Cerdocyon thous in the biological cycle of Leishmania. Nevertheless, several authors consider the clinical manifestation of the disease to be rare or mild in free-living animals. Herein, we demonstrate the occurrence of severe clinical sings and deaths caused by VL in free-living crab-eating foxes. Three specimens of foxes collected from periurban areas were diagnosed with VL. The animals presented cutaneous, ophthalmological, gastrointestinal, locomotor and hematological alterations, and died after clinical progression. We identified the presence of anti-Leishmania antibodies by immunochromatographic test in all specimens. We also observed intra and extracellular amastigotes in skin cytology and lymph node aspirate. Furthermore, Leishmania infantum DNA was identified in all samples by the polymer chain reaction technique. Additionally, we performed blood count and stool parasitological tests and observed hematological disorders common to VL, such as anemia and lymphopenia. Taken together, our data demonstrate that VL can induce clinical complications and even cause death in C. thous and corroborate that this crab-eating fox is an adequate host for L. infantum.

The cytostome-cytopharynx complex of intracellular and extracellular amastigotes of Trypanosoma cruzi exhibit structural and functional differences.

Endocytosis in Trypanosoma cruzi is mainly performed through a specialised membrane domain called cytostome-cytopharynx complex. Its ultrastructure and dynamics in endocytosis are well characterized in epimastigotes, being absent in trypomastigotes, that lack endocytic activity. Intracellular amastigotes also possess a cytostome-cytopharynx but participation in endocytosis of these forms is not clear. Extracellular amastigotes can be obtained from the supernatant of infected cells or in vitro amastigogenesis. These amastigotes share biochemical and morphological features with intracellular amastigotes but retain trypomastigote's ability to establish infection. We analysed and compared the ultrastructure of the cytostome-cytopharynx complex of intracellular amastigotes and extracellular amastigotes using high-resolution tridimensional electron microscopy techniques. We compared the endocytic ability of intracellular amastigotes, obtained through host cell lysis, with that of extracellular amastigotes. Intracellular amastigotes showed a cytostome-cytopharynx complex similar to epimastigotes'. However, after isolation, the complex undergoes ultrastructural modifications that progressively took to an impairment of endocytosis. Extracellular amastigotes do not possess a cytostome-cytopharynx complex nor the ability to endocytose. Those observations highlight morpho functional differences between intra and extracellular amastigotes regarding an important structure related to cell metabolism. TAKE AWAYS: T. cruzi intracellular amastigotes endocytose through the cytostome-cytopharynx complex. The cytostome-cytopharynx complex of intracellular amastigotes is ultrastructurally similar to the epimastigote. Intracellular amastigotes, once outside the host cell, disassembles the cytostome-cytopharynx membrane domain. Extracellular amastigotes do not possess a cytostome-cytopharynx either the ability to endocytose.

Trypanosoma cruzi extracellular amastigotes engage Rac1 and Cdc42 to invade RAW macrophages

Cell invasion by Trypanosoma cruzi extracellular amastigotes (EAs) relies significantly upon the host cell actin cytoskeleton. In past decades EAs have been established as a reliable model for phagocytosis inducer in non-phagocytic cells. Our current hypothesis is that EAs engage a phagocytosis-like mechanism in non-professional phagocytic cells; however, the molecular mechanisms in professional phagocytes still remain unexplored. In this work, we evaluated the involvement of Rac1 and Cdc42 in the actin-dependent internalization of EAs in RAW 264.7 macrophages. Kinetic assays showed similar internalization of EAs in unstimulated RAW and non-phagocytic HeLa cells but increased in LPS/IFN-γ stimulated RAW cells. However, depletion of Rac1, Cdc42 or RhoA inhibited EA internalization similarly in both unstimulated and stimulated RAW cells. Overexpression of active, but not the dominant-negative, construct of Rac1 increased EA internalization. Remarkably, for Cdc42, both the active and the inactive mutants decreased EA internalization when compared to wild type groups. Despite that, both Rac1 and Cdc42 activation mutants were similarly recruited to and colocalized with actin at the EA-macrophage contact sites when compared to their native isoforms. Altogether, these results corroborate that EAs engage phagocytic processes to invade both professional and non-professional phagocytic cells providing evidences of converging actin mediated mechanisms induced by intracellular pathogens in both cell types.

The Potent Trypanocidal Effect of LQB303, a Novel Redox-Active Phenyl-Tert-Butyl-Nitrone Derivate That Causes Mitochondrial Collapse in Trypanosoma cruzi.

Chagas disease, which is caused by Trypanosoma cruzi, establishes lifelong infections in humans and other mammals that lead to severe cardiac and gastrointestinal complications despite the competent immune response of the hosts. Furthermore, it is a neglected disease that affects 8 million people worldwide. The scenario is even more frustrating since the main chemotherapy is based on benznidazole, a drug that presents severe side effects and low efficacy in the chronic phase of the disease. Thus, the search for new therapeutic alternatives is urgent. In the present study, we investigated the activity of a novel phenyl-tert-butyl-nitrone (PBN) derivate, LQB303, against T. cruzi. LQB303 presented trypanocidal effect against intracellular [IC50/48 h = 2.6 μM] and extracellular amastigotes [IC50/24 h = 3.3 μM] in vitro, leading to parasite lysis; however, it does not present any toxicity to host cells. Despite emerging evidence that mitochondrial metabolism is essential for amastigotes to grow inside mammalian cells, the mechanism of redox-active molecules that target T. cruzi mitochondrion is still poorly explored. Therefore, we investigated if LQB303 trypanocidal activity was related to the impairment of the mitochondrial function of amastigotes. The investigation showed there was a significant decrease compared to the baseline oxygen consumption rate (OCR) of LQB303-treated extracellular amastigotes of T. cruzi, as well as reduction of “proton leak” (the depletion of proton motive force by the inhibition of F1Fo ATP synthase) and “ETS” (maximal oxygen consumption after uncoupling) oxygen consumption rates. Interestingly, the residual respiration (“ROX”) enhanced about three times in LQB303-treated amastigotes. The spare respiratory capacity ratio (SRC: cell ability to meet new energy demands) and the ATP-linked OCR were also impaired by LQB303 treatment, correlating the trypanocidal activity of LQB303 with the impairment of mitochondrial redox metabolism of amastigotes. Flow cytometric analysis demonstrated a significant reduction of the ΔΨm of treated amastigotes. LQB303 had no significant influence on the OCR of treated mammalian cells, evidencing its specificity against T. cruzi mitochondrial metabolism. Our results suggest a promising trypanocidal activity of LQB303, associated with parasite bioenergetic inefficiency, with no influence on the host energy metabolism, a fact that may point to an attractive alternative therapy for Chagas disease.

All Roads Lead to Cytosol: Trypanosoma cruzi Multi-Strategic Approach to Invasion.

T. cruzi has a complex life cycle involving four developmental stages namely, epimastigotes, metacyclic trypomastigotes, amastigotes and bloodstream trypomastigotes. Although trypomastigotes are the infective forms, extracellular amastigotes have also shown the ability to invade host cells. Both stages can invade a broad spectrum of host tissues, in fact, almost any nucleated cell can be the target of infection. To add complexity, the parasite presents high genetic variability with differential characteristics such as infectivity. In this review, we address the several strategies T. cruzi has developed to subvert the host cell signaling machinery in order to gain access to the host cell cytoplasm. Special attention is made to the numerous parasite/host protein interactions and to the set of signaling cascades activated during the formation of a parasite-containing vesicle, the parasitophorous vacuole, from which the parasite escapes to the cytosol, where differentiation and replication take place.

Dipeptidyl nitrile derivatives suppress the Trypanosoma cruzi in vitro infection

Chagas disease affects several countries around the world with health and sanitation problems. Cysteine proteases are essential for the virulence and replication of the Trypanosoma cruzi, being modulated by dipeptidyl nitriles and derivatives. Here, four dipeptidyl nitrile derivatives were assayed in three T. cruzi morphologies and two strains (Tulahuen and Y) using a set of assays: (i) analysis of the inhibitory activity against cysteine proteases; (ii) determination of the cytotoxic activity and selectivity index; (iii) verification of the inhibition of the trypomastigote invasion in the host cell. These compounds could inhibit the activity of cysteine proteases using the selective substrate Z-FR-MCA for the trypomastigote lysate and extracellular amastigotes. Interestingly, these compounds did not present relevant enzymatic inhibition for the epimastigote lysate. Most of the substances were also cytotoxic and selective against the trypomastigotes and intracellular amastigotes. The best compound of the series (Neq0662) could reduce the enzymatic activity of the cysteine proteases for the trypomastigotes and amastigotes. It was equipotent to the benznidazole drug in the cytotoxic studies using these two parasite forms. Neq0662 was also selective for the parasite, and it inhibited the invasion of the mammalian host cell in all conditions tested at 10 μM. The stereochemistry of the trifluoromethyl group was an important factor for the bioactivity when the two diastereomers (Neq0662 and Neq0663) were compared. All-in-all, these results indicate that these compounds could move further in the drug development stage because of its promising bioactive profile.

Evaluation of synergy between host and pathogen-directed therapies against intracellular Leishmania donovani

Visceral leishmaniasis (VL) is associated with treatment complications due to the continued growth of resistant parasites toward currently available pathogen-directed therapeutics. To limit the emergence and combat resistant parasites there is a need to develop new anti-leishmanial drugs and alternative treatment approaches, such as host-directed therapeutics (HDTs). Discovery of new anti-leishmanial drugs including HDTs requires suitable in vitro assay systems. Herein, we modified and evaluated a series of resazurin assays against different life-stages of the VL causing parasite, Leishmania donovani to identify novel HDTs. We further analyzed the synergy of combinatorial interactions between traditionally used pathogen-directed drugs and HDTs for clearance of intracellular L. donovani. The inhibitory concentration at 50% (IC50) of the five evaluated therapies [amphotericin B (AMB), miltefosine, paromomycin, DNER-4, and AR-12 (OSU-03012)] was determined against promastigotes, extracellular amastigotes, and intracellular amastigotes of L. donovani via a resazurin-based assay and compared to image-based microscopy. Using the resazurin-based assay, all evaluated therapies showed reproducible anti-leishmanial activity against the parasite's different life-stages. These results were consistent to the traditional image-based technique. The gold standard of therapy, AMB, showed the highest potency against intracellular L. donovani, and was further evaluated for combinatorial effects with the HDTs. Among the combinations analyzed, pathogen-directed AMB and host-directed AR-12 showed a synergistic reduction of intracellular L. donovani compared to individual treatments. The modified resazurin assay used in this study demonstrated a useful technique to measure new anti-leishmanial drugs against both intracellular and extracellular parasites. The synergistic interactions between pathogen-directed AMB and host-directed AR-12 showed a great promise to combat VL, with the potential to reduce the emergence of drug-resistant strains.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System.

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas' disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Trypanosoma cruzi extracellular amastigotes selectively trigger the PI3K/Akt and Erk pathways during HeLa cell invasion

Cell invasion by Trypanosoma cruzi extracellular amastigotes involves different signaling pathways to induce phagocytosis-like mechanisms. Previous works indicated that PI3K/Akt, Src and Erk might be involved in EA invasion; however, participation of these molecules in this process remains elusive. Here, we observed that EA activated Akt, Erk but not Src. Interference of EA invasion with specific inhibitors corroborated this observation. Our results show that EA is capable of selectively triggering complex signaling pathways. Activation of PI3K/Akt and Erk, kinases related to actin cytoskeleton rearrangement and phagocytosis, reinforces the idea that T. cruzi EA subverts the phagocytic machinery during invasion.

Utilization of proliferable extracellular amastigotes for transient gene expression, drug sensitivity assay, and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi.

Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.

Amastigote Synapse: The Tricks of Trypanosoma cruzi Extracellular Amastigotes

To complete its life cycle within the mammalian host, Trypanosoma cruzi, the agent of Chagas’ disease, must enter cells. Trypomastigotes originating from the insect vector (metacyclic) or from infected cells (bloodstream/tissue culture-derived) are the classical infective forms of the parasite and enter mammalian cells in an actin-independent manner. By contrast, amastigotes originating from the premature rupture of infected cells or transformed from swimming trypomastigotes (designated extracellular amastigotes, EAs) require functional intact microfilaments to invade non-phagocytic host cells. Earlier work disclosed the key features of EA-HeLa cell interplay: actin-rich protrusions called ‘cups’ are formed at EA invasion sites on the host cell membrane that are also enriched in actin-binding proteins, integrins and extracellular matrix elements. In the past decades we described the participation of membrane components and secreted factors from EAs as well as the actin-regulating proteins of host cells involved in what we propose to be a phagocytic-like mechanism of parasite uptake. Thus, regarding this new perspective herein we present previously described EA-induced ‘cups’ as parasitic synapse since they can play a role beyond its architecture function. In this review, we focus on recent findings that shed light on the intricate interaction between extracellular amastigotes and non-phagocytic HeLa cells.

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes.

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease.

Clinical and diagnostic aspects of feline cutaneous leishmaniosis in Venezuela

Venezuela is an endemic area for human and canine leishmaniosis due to Leishmania infantum and parasites of the Leishmania braziliensis and L. mexicana complexes. Limited data are available on feline leishmaniosis (FeL) in this region. The aim of this study was to describe clinical and diagnostic aspects of FeL in Venezuela. Thirty-one domestic cats from urban areas of Lara State in Venezuela were enrolled. Twenty-five were healthy. Six other cats had solitary or multiple nodular lesions, which were located on the nose, ears, tail and lower limbs. Cutaneous lesions were characterized by diffuse pyogranulomatous infiltrate in all sick cats with numerous intracellular and extracellular amastigotes, and immunohistochemistry was positive for Leishmania in five sick cats. All healthy cats were seronegative for L. infantum and L. braziliensis antigens by ELISA. Two out of five sick cats yielded a positive ELISA result to both Leishmania antigens with higher antibody levels to L. braziliensis compared to L. infantum. Significantly higher antibody levels by ELISA as well as a higher number of bands by Western blot (WB) were found for L. braziliensis when compared to L. infantum antigens in all sera from Venezuelan sick and healthy cats. All healthy cats were blood Leishmania spp. qPCR negative while three out of six sick cats were blood qPCR positive. All paraffin-embedded skin biopsies (n = 4) as well as cutaneous cytology (n = 3) were positive by Leishmania spp. qPCR in sick cats. Leishmania speciation was obtained only from the cutaneous lesion samples from cytological preparations of two out of three sick cats which were identified as infected with L. mexicana or a closely related specie. Feline leishmaniosis should be included in the differential diagnosis list of nodular-ulcerative lesions. The most reliable diagnostic technique in sick cats is cytological or histopathological examination along with immunohistochemistry, since blood PCR and serology by ELISA might be negative. WB appears to be more sensitive in detecting infection. Cats with leishmaniosis from Venezuela are most likely infected with species of L. mexicana or a closely related species or the L. braziliensis species complex and not with L. infantum.

Rac1/WAVE2 and Cdc42/N-WASP Participation in Actin-Dependent Host Cell Invasion by Extracellular Amastigotes of Trypanosoma cruzi.

This study evaluated the participation of host cell Rho-family GTPases and their effector proteins in the actin-dependent invasion by Trypanosoma cruzi extracellular amastigotes (EAs). We observed that all proteins were recruited and colocalized with actin at EA invasion sites in live or fixed cells. EA internalization was inhibited in cells depleted in Rac1, N-WASP, and WAVE2. Time-lapse experiments with Rac1, N-WASP and WAVE2 depleted cells revealed that EA internalization kinetics is delayed even though no differences were observed in the proportion of EA-induced actin recruitment in these groups. Overexpression of constitutively active constructs of Rac1 and RhoA altered the morphology of actin recruitments to EA invasion sites. Additionally, EA internalization was increased in cells overexpressing CA-Rac1 but inhibited in cells overexpressing CA-RhoA. WT-Cdc42 expression increased EA internalization, but curiously, CA-Cdc42 inhibited it. Altogether, these results corroborate the hypothesis of EA internalization in non-phagocytic cells by a phagocytosis-like mechanism and present Rac1 as the key Rho-family GTPase in this process.

Modulation of host central carbon metabolism and in situ glucose uptake by intracellular Trypanosoma cruzi amastigotes.

Obligate intracellular pathogens satisfy their nutrient requirements by coupling to host metabolic processes, often modulating these pathways to facilitate access to key metabolites. Such metabolic dependencies represent potential targets for pathogen control, but remain largely uncharacterized for the intracellular protozoan parasite and causative agent of Chagas disease, Trypanosoma cruzi. Perturbations in host central carbon and energy metabolism have been reported in mammalian T. cruzi infection, with no information regarding the impact of host metabolic changes on the intracellular amastigote life stage. Here, we performed cell-based studies to elucidate the interplay between infection with intracellular T. cruzi amastigotes and host cellular energy metabolism. T. cruzi infection of non-phagocytic cells was characterized by increased glucose uptake into infected cells and increased mitochondrial respiration and mitochondrial biogenesis. While intracellular amastigote growth was unaffected by decreased host respiratory capacity, restriction of extracellular glucose impaired amastigote proliferation and sensitized parasites to further growth inhibition by 2-deoxyglucose. These observations led us to consider whether intracellular T. cruzi amastigotes utilize glucose directly as a substrate to fuel metabolism. Consistent with this prediction, isolated T. cruzi amastigotes transport extracellular glucose with kinetics similar to trypomastigotes, with subsequent metabolism as demonstrated in 13C-glucose labeling and substrate utilization assays. Metabolic labeling of T. cruzi-infected cells further demonstrated the ability of intracellular parasites to access host hexose pools in situ. These findings are consistent with a model in which intracellular T. cruzi amastigotes capitalize on the host metabolic response to parasite infection, including the increase in glucose uptake, to fuel their own metabolism and replication in the host cytosol. Our findings enrich current views regarding available carbon sources for intracellular T. cruzi amastigotes and underscore the metabolic flexibility of this pathogen, a feature predicted to underlie successful colonization of tissues with distinct metabolic profiles in the mammalian host.

ERM Proteins Play Distinct Roles in Cell Invasion by Extracellular Amastigotes of Trypanosoma cruzi.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas' disease. In mammalian hosts, T. cruzi alternates between trypomastigote and amastigote forms. Additionally, trypomastigotes can differentiate into amastigotes in the extracellular environment generating infective extracellular amastigotes (EAs). Ezrin-radixin-moesin (ERM) are key proteins linking plasma membrane to actin filaments, the major host cell component responsible for EA internalization. Our results revealed that depletion of host ezrin and radixin but not moesin inhibited EAs invasion in HeLa cells. ERM are recruited and colocalize with F-actin at EA invasion sites as shown by confocal microscopy. Invasion assays performed with cells overexpressing ERM showed increased EAs invasion in ezrin and radixin but not moesin overexpressing cells. Finally, time-lapse experiments have shown altered actin dynamics leading to delayed EA internalization in ezrin and radixin depleted cells when compared to control or moesin depleted cells. Altogether, these findings show distinct roles of ERM during EAs invasion, possibly regulating F-actin dynamics and plasma membrane interplay.

Unique behavior of Trypanosoma cruzi mevalonate kinase: A conserved glycosomal enzyme involved in host cell invasion and signaling.

Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals, and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway components and proteins related to actin cytoskeleton modifications. We hypothesized that TcMVK is a bifunctional enzyme that in addition to playing a classical role in isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for T. cruzi invasion.

Revealing Annexin A2 and ARF-6 enrollment during Trypanosoma cruzi extracellular amastigote-host cell interaction.

BackgroundInvasion of host cells by Trypanosoma cruzi extracellular amastigotes is host actin polymerization-dependent. However, the role of proteins related to actin dynamics during invasion by amastigotes remains to be investigated. Here we describe the role of Annexin A2 and ARF-6 during extracellular amastigote-mammalian cell interactions.FindingsOur results showed ARF-6 accumulation in the amastigote-containing parasitophorous vacuole containing amastigote forms; demonstrated ARF-6 and Annexin A2 critical impact over parasite cell invasion and revealed the effect of Annexin A2 expression on intracellular parasite multiplication.ConclusionARF-6 and Annexin A2 are involved in invasion of mammalian cells by T. cruzi amastigotes.