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Abstract Background The requirements for growth and survival of the intracellular pathogen Trypanosoma cruzi within mammalian host cells are poorly understood. Transcriptional profiling of the host cell response to infection serves as a rapid read-out for perturbation of host physiology that, in part, reflects adaptation to the infective process. Using Affymetrix oligonucleotide array analysis we identified common and disparate host cell responses triggered by T. cruzi infection of phenotypically diverse human cell types. Results We report significant changes in transcript abundance in T. cruzi-infected fibroblasts, endothelial cells and smooth muscle cells (2852, 2155 and 531 genes respectively; fold-change ≥ 2, p-value < 0.01) 24 hours post-invasion. A prominent type I interferon response was observed in each cell type, reflecting a secondary response to secreted cytokine in infected cultures. To identify a core cytokine-independent response in T. cruzi-infected fibroblasts and endothelial cells transwell plates were used to distinguish cytokine-dependent and -independent gene expression profiles. This approach revealed the induction of metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding as common themes in T. cruzi-infected cells. In addition, the downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection may impede host cell cycle progression. The observation of impaired cytokinesis in T. cruzi-infected cells, following nuclear replication, confirmed this prediction. Conclusion Metabolic pathways and cellular processes were identified as significantly altered at the transcriptional level in response to T. cruzi infection in a cytokine-independent manner. Several of these alterations are supported by previous studies of T. cruzi metabolic requirements or effects on the host. However, our methods also revealed a T. cruzi-dependent block in the host cell cycle, at the level of cytokinesis, previously unrecognized for this pathogen-host cell interaction.

Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions., Competing Interests: The authors declare no conflicts of interest.

The flagellated kinetoplastid protozoan and causative agent of human Chagas disease, Trypanosoma cruzi, inhabits both invertebrate and mammalian hosts over the course of its complex life cycle. In these disparate environments, T. cruzi uses its single flagellum to propel motile life stages and, in some instances, to establish intimate contact with the host. Beyond its role in motility, the functional capabilities of the T. cruzi flagellum have not been defined. Moreover, the lack of proteomic information for this organelle, in any parasite life stage, has limited functional investigation. In this study, we employed a proximity-dependent biotinylation approach based on the differential targeting of the biotin ligase TurboID to the flagellum or cytosol in replicative stages of T. cruzi to identify proteins that are enriched in the flagellum by mass spectrometry. Proteomic analysis of the resulting biotinylated protein fractions yielded 218 candidate flagellar proteins in T. cruzi epimastigotes (insect stage) and 99 proteins in intracellular amastigotes (mammalian stage). Forty of these enriched flagellar proteins were common to both parasite life stages and included orthologs of known flagellar proteins in other trypanosomatid species, proteins specific to the T. cruzi lineage and hypothetical proteins. With the validation of flagellar localization for several of the identified candidates, our results demonstrate that TurboID-based proximity proteomics is an effective tool for probing subcellular compartments in T. cruzi. The proteomic data sets generated in this work offer a valuable resource to facilitate functional investigation of the understudied T. cruzi flagellum. IMPORTANCE Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, which causes substantial morbidity and mortality in South and Central America. Throughout its life cycle, T. cruzi interacts with insect and mammalian hosts via its single flagellum, establishing intimate contact with host membranes. Currently, few flagellar proteins have been identified in T. cruzi that could provide insight into the mechanisms involved in mediating physical and biochemical interactions with the host. Here, we set out to identify flagellar proteins in the main replicative stages of T. cruzi using a proximity-labeling approach coupled with mass spectrometry. The >200 candidate flagellar proteins identified represent the first large-scale identification of candidate flagellar proteins in T. cruzi with preliminary validation. These data offer new avenues to investigate the biology of T. cruzi-host interactions, a promising area for development of new strategies aimed at the control of this pathogen., Competing Interests: The authors declare no conflict of interest.

Trypanosomatids are a diverse group of uniflagellate protozoan parasites that include globally relevant pathogens such as Trypanosoma cruzi, the causative agent of Chagas disease. Trypanosomes lack the fatty acid synthase system typically used for de novo fatty acid (FA) synthesis in other eukaryotes. Instead, these microbes have evolved a modular FA elongase (ELO) system comprised of individual ELO enzymes (ELO1-4) that can operate processively to generate long chain- and very long chain-FAs. The importance of ELO's for maintaining lipid homeostasis in trypanosomatids is currently unclear, given their ability to take up and utilize exogenous FAs for lipid synthesis. To assess ELO function in T. cruzi, we generated individual KO lines, Δelo1, Δelo2, and Δelo3, in which the genes encoding ELO1-3 were functionally disrupted in the parasite insect stage (epimastigote). Using unbiased lipidomic and metabolomic analyses, in combination with metabolic tracing and biochemical approaches, we demonstrate that ELO2 and ELO3 are required for global lipid homeostasis, whereas ELO1 is dispensable for this function. Instead, ELO1 activity is needed to sustain mitochondrial activity and normal growth in T. cruzi epimastigotes. The cross-talk between microsomal ELO1 and the mitochondrion is a novel finding that, we propose, merits further examination of the trypanosomatid ELO pathway as critical for central metabolism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

In addition to scavenging exogenous cholesterol, the parasitic kinetoplastid Trypanosoma cruzi can endogenously synthesize sterols. Similar to fungal species, T. cruzi synthesizes ergostane type sterols and is sensitive to a class of azole inhibitors of ergosterol biosynthesis that target the enzyme lanosterol 14α-demethylase (CYP51). In the related kinetoplastid parasite Leishmania donovani , CYP51 is essential, yet in Leishmania major , the cognate enzyme is dispensable for growth; but not heat resistance. The essentiality of CYP51 and the specific role of ergostane-type sterol products in T. cruzi has not been established. To better understand the importance of this pathway, we have disrupted the CYP51 gene in T. cruzi epimastigotes ( ΔCYP51 ). Disruption of CYP51 leads to accumulation of 14-methylated sterols and a concurrent absence of the final sterol product ergosterol. While ΔCYP51 epimastigotes have slowed proliferation compared to wild type parasites, the enzyme is not required for growth; however, ΔCYP51 epimastigotes exhibit sensitivity to elevated temperature, an elevated mitochondrial membrane potential and fail to establish growth as intracellular amastigotes in vitro . Further genetic disruption of squalene epoxidase ( ΔSQLE ) results in the absence of all endogenous sterols and sterol auxotrophy, yet failed to rescue tolerance to stress in ΔCYP51 parasites, suggesting the loss of ergosterol and not accumulation of 14-methylated sterols modulates stress tolerance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dumoulin, Vollrath, Won, Wang and Burleigh.)

The objective of this study was to provide information on Trypanosoma cruzi genetic diversity among isolates obtained from different biological sources circulating in endemic areas of Panama. Initial discrete typing units (DTUs) assignment was performed evaluating three single locus molecular markers (mini-exon, heat shock protein 60 and glucose-6-phosphate isomerase genes). Further diversity within TcI lineages was explored using a multi-locus sequence typing approach with six maxicircle genes. Haplotype network analysis and evolutionary divergency estimations were conducted to investigate the genetic relatedness between Panamanian TcI isolates and isolates from different endemic regions in the Americas. Our molecular approach validated that TcI is the predominant DTU circulating in Panama across different hosts and vector species, but also confirmed the presence of TcIII and TcVI circulating in the country. The phylogenetic tree topography for most Panamanian TcI isolates displayed a high level of genetic homogeneity between them. The haplotype network analysis inferred a higher genetic diversity within Panamanian TcI isolates, displaying eight different haplotypes circulating in endemic regions of the country, and revealed geographical structuring among TcI from different endemic regions in the Americas. This study adds novelty on the genetic diversity of T. cruzi circulating in Panama and complements regional phylogeographic studies regarding intra-TcI variations.

Throughout their life cycle, parasitic organisms experience a variety of environmental conditions. To ensure persistence and transmission, some protozoan parasites are capable of adjusting their replication or converting to distinct life cycle stages. Trypanosoma cruzi is a 'generalist' parasite that is competent to infect various insect (triatomine) vectors and mammalian hosts. Within the mammalian host, T. cruzi replicates intracellularly as amastigotes and can persist for the lifetime of the host. The persistence of the parasites in tissues can lead to the development of Chagas disease. Recent work has identified growth plasticity and metabolic flexibility as aspects of amastigote biology that are important determinants of persistence in varied growth conditions and under drug pressure. A better understanding of the link between amastigote and host/tissue metabolism will aid in the development of new drugs or therapies that can limit disease pathology., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chagas' disease arises as a direct consequence of the lytic cycle of Trypanosoma cruzi in the mammalian host. While invasion is well studied for this pathogen, study of egress has been largely neglected. Here, we provide the first description of T. cruzi egress documenting a coordinated mechanism by which T. cruzi engineers its escape from host cells in which it has proliferated and which is essential for maintenance of infection and pathogenesis. Our results indicate that this parasite egress is a sudden event involving coordinated remodeling of host cell cytoskeleton and subsequent rupture of host cell plasma membrane. We document that host cells maintain plasma membrane integrity until immediately prior to parasite release and report the sequential transformation of the host cell's actin cytoskeleton from normal meshwork in noninfected cells to spheroidal cages-a process initiated shortly after amastigogenesis. Quantification revealed gradual reduction in F-actin over the course of infection, and using cytoskeletal preparations and electron microscopy, we were able to observe disruption of the F-actin proximal to intracellular trypomastigotes. Finally, Western blotting experiments suggest actin degradation driven by parasite proteases, suggesting that degradation of cytoskeleton is a principal component controlling the initiation of egress. Our results provide the first description of the cellular mechanism that regulates the lytic component of the T. cruzi lytic cycle. We show graphically how it is possible to preserve the envelope of host cell plasma membrane during intracellular proliferation of the parasite and how, in cells packed with amastigotes, differentiation into trypomastigotes may trigger sudden egress. IMPORTANCE Understanding how Trypanosoma cruzi interacts with host cells has been transformed by high-quality studies that have examined in detail the mechanisms of T. cruzi host cell invasion. In contrast, little is known about the latter stages of the parasite's lytic cycle: how parasites egress and thereby sustain round after round of infection. Our results show that once in the host cell cytosol and having undergone amastigogenesis, T. cruzi begins to alter the host cell cytoskeleton, remodeling normal F-actin meshworks into encapsulating spheroidal cages. Filamentous actin diminishes over the course of the lytic cycle, and just prior to egress, the filaments comprising the cages are severely degraded where adjacent to the parasites. We conclude that sudden egress follows breach of the containment afforded by the actin cytoskeleton and subsequent plasma membrane rupture-a process that when understood in molecular detail may serve as a target for future novel therapeutic interventions.

Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that has a heterogeneous population composed of a pool of strains with distinct characteristics, including variable levels of virulence. In previous work, transcriptome analyses of parasite genes after infection of human foreskin fibroblasts (HFF) with virulent (CL Brener) and non-virulent (CL-14) clones derived from the CL strain, revealed a reduced expression of genes encoding parasite surface proteins in CL-14 compared to CL Brener during the final steps of the intracellular differentiation from amastigotes to trypomastigotes. Here we analyzed changes in the expression of host genes during in vitro infection of HFF cells with the CL Brener and CL-14 strains by analyzing total RNA extracted from cells at 60 and 96 hours post-infection (hpi) with each strain, as well as from uninfected cells. Similar transcriptome profiles were observed at 60 hpi with both strains compared to uninfected samples. However, at 96 hpi, significant differences in the number and expression levels of several genes, particularly those involved with immune response and cytoskeleton organization, were observed. Further analyses confirmed the difference in the chemokine/cytokine signaling involved with the recruitment and activation of immune cells such as neutrophils upon T. cruzi infection. These findings suggest that infection with the virulent CL Brener strain induces a more robust inflammatory response when compared with the non-virulent CL-14 strain. Importantly, the RNA-Seq data also exposed an unexplored role of fibroblasts as sentinel cells that may act by recruiting neutrophils to the initial site of infection. This role for fibroblasts in the regulation of the inflammatory response during infection by T. cruzi was corroborated by measurements of levels of different chemokines/cytokines during in vitro infection and in plasma from Chagas disease patients as well as by neutrophil activation and migration assays., Competing Interests: The authors have declared that no competing interests exist.

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. 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.

The mammalian stages of the parasite Trypanosoma cruzi , the causative agent of Chagas disease, exhibit a wide host species range and extensive within-host tissue distribution. These features, coupled with the ability of the parasites to persist for the lifetime of the host, suggest an inherent capacity to tolerate changing environments. To examine this potential, we studied proliferation and cell cycle dynamics of intracellular T. cruzi amastigotes experiencing transient metabolic perturbation or drug pressure in the context of an infected mammalian host cell. Parasite growth plasticity was evident and characterized by rapid and reversible suppression of amastigote proliferation in response to exogenous nutrient restriction or exposure to metabolic inhibitors that target glucose metabolism or mitochondrial respiration. In most instances, reduced parasite proliferation was accompanied by the accumulation of amastigote populations in the G 1 phase of the cell cycle, in a manner that was rapidly and fully reversible upon release from the metabolic block. Acute amastigote cell cycle changes at the G 1 stage were similarly observed following exposure to sublethal concentrations of the first-line therapy drug, benznidazole, and yet, unlike the results seen with inhibitors of metabolism, recovery from exposure occurred at rates inversely proportional to the concentration of benznidazole. Our results show that T. cruzi amastigote growth plasticity is an important aspect of parasite adaptation to stress, including drug pressure, and is an important consideration for growth-based drug screening. IMPORTANCE Infection with the intracellular parasite Trypanosoma cruzi can cause debilitating and potentially life-threatening Chagas disease, where long-term parasite persistence is a critical determinant of clinical disease progression. Such tissue-resident T. cruzi amastigotes are refractory to immune-mediated clearance and to drug treatment, suggesting that in addition to exploiting immune avoidance mechanisms, amastigotes can facilitate their survival by adapting flexibly to diverse environmental stressors. We discovered that T. cruzi intracellular amastigotes exhibit growth plasticity as a strategy to adapt to and rebound from environmental stressors, including metabolic blockades, nutrient starvation, and sublethal exposure to the first-line therapy drug benznidazole. These findings have important implications for understanding parasite persistence, informing drug development, and interpreting drug efficacy., (Copyright © 2018 Dumoulin and Burleigh.)

Trypanosoma cruzi is the kinetoplastid protozoan parasite that causes human Chagas disease, a chronic disease with complex outcomes including severe cardiomyopathy and sudden death. In mammalian hosts, T. cruzi colonises a wide range of tissues and cell types where it replicates within the host cell cytoplasm. Like all intracellular pathogens, T. cruzi amastigotes must interact with its immediate host cell environment in a manner that facilitates access to nutrients and promotes a suitable niche for replication and survival. Although potentially exploitable to devise strategies for pathogen control, fundamental knowledge of the host pathways co-opted by T. cruzi during infection is currently lacking. Here, we report that intracellular T. cruzi amastigotes establish close contact with host mitochondria via their single flagellum. Given the key bioenergetic and homeostatic roles of mitochondria, this striking finding suggests a functional role for host mitochondria in the infection process and points to the T. cruzi amastigote flagellum as an active participant in pathogenesis. Our study establishes the basis for future investigation of the molecular and functional consequences of this intriguing host-parasite interaction., (© 2017 John Wiley & Sons Ltd.)

Intracellular infection and multi-organ colonization by the protozoan parasite, Trypanosoma cruzi, underlie the complex etiology of human Chagas disease. While T. cruzi can establish cytosolic residence in a broad range of mammalian cell types, the molecular mechanisms governing this process remain poorly understood. Despite the anticipated capacity for fatty acid synthesis in this parasite, recent observations suggest that intracellular T. cruzi amastigotes may rely on host fatty acid metabolism to support infection. To investigate this prediction, it was necessary to establish baseline lipidome information for the mammalian-infective stages of T. cruzi and their mammalian host cells. An unbiased, quantitative mass spectrometric analysis of lipid fractions was performed with the identification of 1079 lipids within 30 classes. From these profiles we deduced that T. cruzi amastigotes maintain an overall lipid identity that is distinguishable from mammalian host cells. A deeper analysis of the fatty acid moiety distributions within each lipid subclass facilitated the high confidence assignment of host- and parasite-like lipid signatures. This analysis unexpectedly revealed a strong host lipid signature in the parasite lipidome, most notably within its glycerolipid fraction. The near complete overlap of fatty acid moiety distributions observed for host and parasite triacylglycerols suggested that T. cruzi amastigotes acquired a significant portion of their lipidome from host triacylglycerol pools. Metabolic tracer studies confirmed long-chain fatty acid scavenging by intracellular T. cruzi amastigotes, a capacity that was significantly diminished in host cells deficient for de novo triacylglycerol synthesis via the diacylglycerol acyltransferases (DGAT1/2). Reduced T. cruzi amastigote proliferation in DGAT1/2-deficient fibroblasts further underscored the importance of parasite coupling to host triacylglycerol pools during the intracellular infection cycle. Thus, our comprehensive lipidomic dataset provides a substantially enhanced view of T. cruzi infection biology highlighting the interplay between host and parasite lipid metabolism with potential bearing on future therapeutic intervention strategies.

Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving several morphologically and biochemically distinct stages that establish intricate interactions with various insect and mammalian hosts. It has also a heterogeneous population structure comprising strains with distinct properties such as virulence, sensitivity to drugs, antigenic profile and tissue tropism. We present a comparative transcriptome analysis of two cloned T. cruzi strains that display contrasting virulence phenotypes in animal models of infection: CL Brener is a virulent clone and CL-14 is a clone that is neither infective nor pathogenic in in vivo models of infection. Gene expression analysis of trypomastigotes and intracellular amastigotes harvested at 60 and 96 hours post-infection (hpi) of human fibroblasts revealed large differences that reflect the parasite's adaptation to distinct environments during the infection of mammalian cells, including changes in energy sources, oxidative stress responses, cell cycle control and cell surface components. While extensive transcriptome remodeling was observed when trypomastigotes of both strains were compared to 60 hpi amastigotes, differences in gene expression were much less pronounced when 96 hpi amastigotes and trypomastigotes of CL Brener were compared. In contrast, the differentiation of the avirulent CL-14 from 96 hpi amastigotes to extracellular trypomastigotes was associated with considerable changes in gene expression, particularly in gene families encoding surface proteins such as trans-sialidases, mucins and the mucin associated surface proteins (MASPs). Thus, our comparative transcriptome analysis indicates that the avirulent phenotype of CL-14 may be due, at least in part, to a reduced or delayed expression of genes encoding surface proteins that are associated with the transition of amastigotes to trypomastigotes, an essential step in the establishment of the infection in the mammalian host. Confirming the role of members of the trans-sialidase family of surface proteins for parasite differentiation, transfected CL-14 constitutively expressing a trans-sialidase gene displayed faster kinetics of trypomastigote release in the supernatant of infected cells compared to wild type CL-14.

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.

Energy metabolism is an attractive target for the development of new therapeutics against protozoan pathogens, including Trypanosoma cruzi, the causative agent of human Chagas disease. Despite emerging evidence that mitochondrial electron transport is essential for the growth of intracellular T. cruzi amastigotes in mammalian cells, fundamental knowledge of mitochondrial energy metabolism in this parasite life stage remains incomplete. The Clark-type electrode, which measures the rate of oxygen consumption, has served as the traditional tool to study mitochondrial energetics and has contributed to our understanding of it in T. cruzi. Here, we evaluate the Seahorse XF(e)24 extracellular flux platform as an alternative method to assess mitochondrial bioenergetics in isolated T. cruzi parasites. We report optimized assay conditions used to perform mitochondrial stress tests with replicative life cycle stages of T. cruzi using the XF(e)24 instrument, and discuss the advantages and potential limitations of this methodology, as applied to T. cruzi and other trypanosomatids., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our comprehensive, high resolution transcriptomic dataset provides a substantially more detailed interpretation of T. cruzi infection biology and offers a basis for future drug and vaccine discovery efforts.

Protozoan infections are a serious global health problem. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents--granzyme (Gzm) proteases and the pore-forming perforin (PFN)--into the infected cell. However, these cytotoxic molecules do not kill intracellular parasites. CD8(+) CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes, and GNLY, PFN and Gzms rapidly kill intracellular bacteria. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell-mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.

Background: Trypanosoma cruzi, causative agent of Chagas disease, displays high intraspecific genetic diversity: six genetic lineages or discrete typing units (DTUs) are currently recognized, termed TcI through TcVI. Each DTU presents a particular distribution pattern across the Americas, and is loosely associated with different transmission cycles and hosts. Several DTUs are known to circulate in Central America. It has been previously suggested that TcI infection is benign and does not lead to chronic chagasic cardiomyopathy (CCC)., Findings: In this study, we genotyped T. cruzi parasites circulating in the blood and from explanted cardiac tissue of an El Salvadorian patient who developed reactivation Chagas disease while on immunosuppressive medications after undergoing heart transplant in the U.S. as treatment for end-stage CCC. Parasite typing was performed through molecular methods (restriction fragment length polymorphism of polymerase reaction chain amplified products, microsatellite typing, maxicircle sequence typing and low-stringency single primer PCR, [LSSP-PCR]) as well as lineage-specific serology. We show that the parasites infecting the patient belong to the TcI DTU exclusively. Our data indicate that the parasites isolated from the patient belong to a genotype frequently associated with human infection throughout the Americas (TcIDOM)., Conclusions: Our results constitute compelling evidence in support of TcI DTU's ability to cause end-stage CCC and help dispel any residual bias that infection with this lineage is benign, pointing to the need for increased surveillance for dissemination of this genotype in endemic regions, the USA and globally.

The capacity for rapid localization of epitope-tagged or fluorescent fusion proteins in cells is an important tool for biological discovery and functional analysis. For Trypanosoma cruzi, the protozoan parasite that causes human Chagas disease, visualization of ectopically-expressed proteins in the clinically-relevant mammalian stages is hindered by the necessity to first perform transfection and lengthy selection procedures in the insect vector form of the parasite. Here, we demonstrate the ability to by-pass the insect stage with the delivery of plasmid DNA to non-dividing, tissue culture trypomastigotes such that upon host cell infection, transgenes are expressed and rapidly localized in intracellular T. cruzi amastigotes. The inclusion of a sorting step prior to host cell infection by trypomastigotes greatly enriches (>90%) the number of transgene-expressing amastigotes observed in mammalian host cells. This is a significant methodological advance that has the potential to accelerate the pace of discovery in the Chagas disease field., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nucleotide sugar transporters (NSTs) are indispensible for the biosynthesis of glycoproteins by providing the nucleotide sugars needed for glycosylation in the lumen of the Golgi apparatus. Mutations in NST genes cause human and cattle diseases and impaired cell walls of yeast and fungi. Information regarding their function in the protozoan parasite, Trypanosoma brucei, a causative agent of African trypanosomiasis, is unknown. Here, we characterized the substrate specificities of four NSTs, TbNST1-4, which are expressed in both the insect procyclic form (PCF) and mammalian bloodstream form (BSF) stages. TbNST1/2 transports UDP-Gal/UDP-GlcNAc, TbNST3 transports GDP-Man, and TbNST4 transports UDP-GlcNAc, UDP-GalNAc, and GDP-Man. TbNST4 is the first NST shown to transport both pyrimidine and purine nucleotide sugars and is demonstrated here to be localized at the Golgi apparatus. RNAi-mediated silencing of TbNST4 in the procyclic form caused underglycosylated surface glycoprotein EP-procyclin. Similarly, defective glycosylation of the variant surface glycoprotein (VSG221) as well as the lysosomal membrane protein p67 was observed in Δtbnst4 BSF T. brucei. Relative infectivity analysis showed that defects in glycosylation of the surface coat resulting from tbnst4 deletion were insufficient to impact the ability of this parasite to infect mice. Notably, the fact that inactivation of a single NST gene results in measurable defects in surface glycoproteins in different life cycle stages of the parasite highlights the essential role of NST(s) in glycosylation of T. brucei. Thus, results presented in this study provide a framework for conducting functional analyses of other NSTs identified in T. brucei.

Mammalian cell invasion by the protozoan parasite Trypanosoma cruzi involves host cell microtubule dynamics. Microtubules support kinesin-dependent anterograde trafficking of host lysosomes to the cell periphery where targeted lysosome exocytosis elicits remodelling of the plasma membrane and parasite invasion. Here, a novel role for microtubule plus-end tracking proteins (+TIPs) in the co-ordination of T. cruzi trypomastigote internalization and post-entry events is reported. Acute silencing of CLASP1, a +TIP that participates in microtubule stabilization at the cell periphery, impairs trypomastigote internalization without diminishing the capacity for calcium-regulated lysosome exocytosis. Subsequent fusion of the T. cruzi vacuole with host lysosomes and its juxtanuclear positioning are also delayed in CLASP1-depleted cells. These post-entry phenotypes correlate with a generalized impairment of minus-end directed transport of lysosomes in CLASP1 knock-down cells and mimic the effects of dynactin disruption. Consistent with GSK3β acting as a negative regulator of CLASP function, inhibition of GSK3β activity enhances T. cruzi entry in a CLASP1-dependent manner and expression of constitutively active GSK3β dampens infection. This study provides novel molecular insights into the T. cruzi infection process, emphasizing functional links between parasite-elicited signalling, host microtubule plus-end tracking proteins and dynein-based retrograde transport. Highlighted in this work is a previously unrecognized role for CLASPs in dynamic lysosome positioning, an important aspect of the nutrient sensing response in mammalian cells., (© 2012 Blackwell Publishing Ltd.)

Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas' disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite's replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Trypanosoma cruzi infection leads to development of chronic Chagas disease. In this article, we provide an update on the current knowledge of the mechanisms employed by the parasite to gain entry into the host cells and establish persistent infection despite activation of a potent immune response by the host. Recent studies point to a number of T. cruzi molecules that interact with host cell receptors to promote parasite invasion of the diverse host cells. T. cruzi expresses an antioxidant system and thromboxane A(2) to evade phagosomal oxidative assault and suppress the host's ability to clear parasites. Additional studies suggest that besides cardiac and smooth muscle cells that are the major target of T. cruzi infection, adipocytes and adipose tissue serve as reservoirs from where T. cruzi can recrudesce and cause disease decades later. Further, T. cruzi employs at least four strategies to maintain a symbiotic-like relationship with the host, and ensure consistent supply of nutrients for its own survival and long-term persistence. Ongoing and future research will continue to help refining the models of T. cruzi invasion and persistence in diverse tissues and organs in the host., (© 2012 Blackwell Publishing Ltd.)

Novel technologies that include recombinant pathogens and rapid detection methods are contributing to the development of drugs for neglected diseases. Recently, the results from the first high throughput screening (HTS) to test compounds for activity against Trypanosoma cruzi trypomastigote infection of host cells were reported. We have selected 23 compounds from the hits of this HTS, which were reported to have high anti-trypanosomal activity and low toxicity to host cells. These compounds were highly purified and their structures confirmed by HPLC/mass spectrometry. The compounds were tested in vitro, where about half of them confirmed the anti-T. cruzi activity reported in the HTS, with IC50 values lower than 5 µM. We have also adapted a rapid assay to test anti-T. cruzi compounds in vivo using mice infected with transgenic T. cruzi expressing luciferase as a model for acute infection. The compounds that were active in vitro were also tested in vivo using this assay, where we found two related compounds with a similar structure and low in vitro IC50 values (0.11 and 0.07 µM) that reduce T. cruzi infection in the mouse model more than 90% after five days of treatment. Our findings evidence the benefits of novel technologies, such as HTS, for the drug discovery pathway of neglected diseases, but also caution about the need to confirm the results in vitro. We also show how rapid methods of in vivo screening based in luciferase-expressing parasites can be very useful to prioritize compounds early in the chain of development.

Trypanosoma cruzi, the protozoan parasite that causes human Chagas' disease, induces a type I interferon (IFN) (IFN-α/β) response during acute experimental infection in mice and in isolated primary cell types. To examine the potential impact of the type I IFN response in shaping outcomes in experimental T. cruzi infection, groups of wild-type (WT) and type I IFN receptor-deficient (IFNAR(-/-)) 129sv/ev mice were infected with two different T. cruzi strains under lethal and sublethal conditions and several parameters were measured during the acute stage of infection. The results demonstrate that type I IFNs are not required for early host protection against T. cruzi. In contrast, under conditions of lethal T. cruzi challenge, WT mice succumbed to infection whereas IFNAR(-/-) mice were ultimately able to control parasite growth and survive. T. cruzi clearance in and survival of IFNAR(-/-) mice were accompanied by higher levels of IFN-γ production by isolated splenocytes in response to parasite antigen. The suppression of IFN-γ in splenocytes from WT mice was independent of IL-10 levels. While the impact of type I IFNs on the production of IFN-γ and other cytokines/chemokines remains to be fully determined in the context of T. cruzi infection, our data suggest that, under conditions of high parasite burden, type I IFNs negatively impact IFN-γ production, initiating a detrimental cycle that contributes to the ultimate failure to control infection. These findings are consistent with a growing theme in the microbial pathogenesis field in which type I IFNs can be detrimental to the host in a variety of nonviral pathogen infection models.

The protozoan parasite Trypanosoma cruzi, which causes human Chagas' disease, exerts a variety of effects on host extracellular matrix (ECM) including proteolytic degradation of collagens and dampening of ECM gene expression. Exposure of primary human dermal fibroblasts to live infective T. cruzi trypomastigotes or their shed/secreted products results in a rapid down-regulation of the fibrogenic genes collagenIα1, fibronectin and connective tissue growth factor (CTGF/CCN2). Here we demonstrate the ability of a secreted/released T. cruzi factor to antagonize ctgf/ccn2 expression in dermal fibroblasts in response to TGF-ß, lysophosphatidic acid or serum, where agonist-induced phosphorylation of the mitogen-activated protein (MAP) kinases Erk1/2, p38 and JNK was also inhibited. Global analysis of gene expression in dermal fibroblasts identified a discrete subset of TGF-ß-inducible genes involved in cell proliferation, wound repair, and immune regulation that are inhibited by T. cruzi secreted/released factors, where the genes exhibiting the highest sensitivity to T. cruzi are known to be regulated by MAP kinase-activated transcription factors. Consistent with this observation, the Ets-family transcription factor binding site in the proximal promoter region of the ctgf/ccn2 gene (-91 bp to -84 bp) was shown to be required for T. cruzi-mediated down-regulation of ctgf/ccn2 reporter expression. The cumulative data suggest a model in which T. cruzi-derived molecules secreted/released early in the infective process dampen MAP kinase signaling and the activation of transcription factors that regulate expression of fibroblast genes involved in wound repair and tissue remodelling, including ctgf/ccn2. These findings have broader implications for local modulation of ECM synthesis/remodelling by T. cruzi during the early establishment of infection in the mammalian host and highlight the potential for pathogen-derived molecules to be exploited as tools to modulate the fibrogenic response.

One of the more accepted concepts in our understanding of the biology of early Trypanosoma cruzi-host cell interactions is that the mammalian-infective trypomastigote forms of the parasite must transit the host cell lysosomal compartment in order to establish a productive intracellular infection. The acidic environment of the lysosome provides the appropriate conditions for parasite-mediated disruption of the parasitophorous vacuole and release of T. cruzi into the host cell cytosol, where replication of intracellular amastigotes occurs. Recent findings indicate a level of redundancy in the lysosome-targeting process where T. cruzi trypomastigotes exploit different cellular pathways to access host cell lysosomes in non-professional phagocytic cells. In addition, the reversible nature of the host cell penetration process was recently demonstrated when conditions for fusion of the nascent parasite vacuole with the host endosomal-lysosomal system were not met. Thus, the concept of parasite retention as a critical component of the T. cruzi invasion process was introduced. Although it is clear that host cell recognition, attachment and signalling are required to initiate invasion, integration of this knowledge with our understanding of the different routes of parasite entry is largely lacking. In this chapter, we focus on current knowledge of the cellular pathways exploited by T. cruzi trypomastigotes to invade non-professional phagocytic cells and to gain access to the host cell lysosome compartment., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.

Background: The requirements for growth and survival of the intracellular pathogen Trypanosoma cruzi within mammalian host cells are poorly understood. Transcriptional profiling of the host cell response to infection serves as a rapid read-out for perturbation of host physiology that, in part, reflects adaptation to the infective process. Using Affymetrix oligonucleotide array analysis we identified common and disparate host cell responses triggered by T. cruzi infection of phenotypically diverse human cell types., Results: We report significant changes in transcript abundance in T. cruzi-infected fibroblasts, endothelial cells and smooth muscle cells (2852, 2155 and 531 genes respectively; fold-change > or = 2, p-value < 0.01) 24 hours post-invasion. A prominent type I interferon response was observed in each cell type, reflecting a secondary response to secreted cytokine in infected cultures. To identify a core cytokine-independent response in T. cruzi-infected fibroblasts and endothelial cells transwell plates were used to distinguish cytokine-dependent and -independent gene expression profiles. This approach revealed the induction of metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding as common themes in T. cruzi-infected cells. In addition, the downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection may impede host cell cycle progression. The observation of impaired cytokinesis in T. cruzi-infected cells, following nuclear replication, confirmed this prediction., Conclusion: Metabolic pathways and cellular processes were identified as significantly altered at the transcriptional level in response to T. cruzi infection in a cytokine-independent manner. Several of these alterations are supported by previous studies of T. cruzi metabolic requirements or effects on the host. However, our methods also revealed a T. cruzi-dependent block in the host cell cycle, at the level of cytokinesis, previously unrecognized for this pathogen-host cell interaction.

Surface adhesion proteins are essential for Trypanosoma cruzi invasion of mammalian cells. Here we show that Dispersed Gene Family-1 (DGF-1) members, previously identified as nuclear repeated sequences present in several chromosomes and comprising the third largest T. cruzi specific gene family, have conserved adhesin motifs including four segments with significant similarity to human beta 7 integrin. Flow cytometry and biotinylation assays with anti-DGF-1 antibodies indicated that, as expected, DGF-1 members are expressed on the trypomastigote surface. The DGF-1 genealogy, inferred using T. cruzi Genome Project data and network phylogeny algorithms, suggests that this gene family is separated in at least three groups with differential distribution of functional domains. To identify which members of this gene family are expressed we used a combined approach of RT-PCR and codon usage profiles, showing that expressed members have a very biased codon usage favoring GC, whereas non-expressed members have a homogeneous distribution. Shannon information entropy was used to measure sequence variability and revealed four major high entropy segments in the extracellular domain of DGF-1 overlapping with important putative functional modules of the predicted proteins. Testing for natural selection, however, indicated that these high entropy segments were not under positive selection, which contradicts the notion that positive selection is the cause of high variability in specific domains of a protein relative to other less variable regions in the same molecule. We conjectured that members of the DGF-1 family might be associated with the ability of T. cruzi to bind extracellular matrix proteins, such as fibronectin and laminin, and speculated on mechanisms that would be generating the localized diversity in these molecules in the absence of selection.

Early interactions between the protozoan parasite Trypanosoma cruzi and mammalian hosts at primary sites of infection (skin and mucosal membranes) are predicted to be critical determinants of parasite survival and dissemination in the host. To investigate the early host response triggered by three different strains of T. cruzi at a local infection site, changes in host gene expression were monitored in a murine intradermal infection model using Affymetrix oligonucleotide arrays. Robust induction of IFN-stimulated genes was observed in excised skin 24 h postinfection where the level of IFN-stimulated gene induction was parasite strain-dependent, with the least virulent strain triggering a muted IFN response. Infection of mice immunodepleted of IFN-gamma-producing cells or infection of IFN-gamma-deficient mice had minimal impact on the IFN response generated in T. cruzi-infected mice. In contrast, infection of mice lacking the type I IFN receptor demonstrated that type I IFNs are largely responsible for the IFN response generated at the site of infection. These data highlight type I IFNs as important components of the innate immune response to T. cruzi at the site of inoculation and their role in shaping the early transcriptional response to this pathogen.

To investigate the effects of Trypanosoma cruzi on the mechanical properties of infected host cells, cytoskeletal stiffness and remodeling dynamics were measured in parasite-infected fibroblasts. We find that cell stiffness decreases in a time-dependent fashion in T. cruzi-infected human foreskin fibroblasts without a significant change in the dynamics of cytoskeletal remodeling. In contrast, cells exposed to T. cruzi secreted/released components become significantly stiffer within 2 h of exposure and exhibit increased remodeling dynamics. These findings represent the first direct mechanical data to suggest a physical picture in which an intact, stiff, and rapidly remodeling cytoskeleton facilitates early stages of T. cruzi invasion and parasite retention, followed by subsequent softening and disassembly of the cytoskeleton to accommodate intracellular replication of parasites. We further suggest that these changes occur through protein kinase A and inhibition of the Rho/Rho kinase signaling pathway. In the context of tissue infection, changes in host cell mechanics could adversely affect the function of the infected organs, and may play an important role on the pathophysiology of Chagas' disease., ((c) 2008 Wiley-Liss, Inc.)

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.

Innate immune recognition of intracellular pathogens involves both extracellular and cytosolic surveillance mechanisms. The intracellular protozoan parasite Trypanosoma cruzi triggers a robust type I IFN response in both immune and nonimmune cell types. In this study, we report that signaling through TBK1 and IFN regulatory factor 3 is required for T. cruzi-mediated expression of IFN-beta. The TLR adaptors MyD88 and TRIF, as well as TLR4 and TLR3, were found to be dispensable, demonstrating that T. cruzi induces IFN-beta expression in a TLR-independent manner. The potential role for cytosolic dsRNA sensing pathways acting through RIG-I and MDA5 was ruled out because T. cruzi was shown to trigger robust expression of IFN-beta in macrophages lacking the MAVS/IPS1/VISA/CARDif adaptor protein. The failure of T. cruzi to activate HEK293-IFN-beta-luciferase cells, which are highly sensitive to cytosolic triggers of IFN-beta expression including Listeria, Sendai virus, and transfected dsRNA and dsDNA, further indicates that the parasite does not engage currently recognized cytosolic surveillance pathways. Together, these findings identify the existence of a novel TLR-independent pathogen-sensing mechanism in immune and nonimmune cells that converges on TBK1 and IFN regulatory factor 3 for activation of IFN-beta gene expression.

The cell-invasive, trypomastigote form of Trypanosoma cruzi exhibits a unique relationship with lysosomes in target host cells. In contrast to many intracellular pathogens that are adept at avoiding contact with lysosomes, T. cruzi requires transient residence within this acidic organelle for productive infection. The low pH environment of lysosomes facilitates parasite egress from the vacuole and delivery into the host cytosol, a critical step in the T. cruzi developmental program. Recent studies also suggest that early lysosome fusion with invading or recently internalized parasites is critical for cellular retention of parasites. To ensure targeting to host cell lysosomes, T. cruzi trypomastigotes exploit two distinct modes of invasion that rapidly converge in the cell. In this chapter, we summarize the recent progress and changing views regarding the role of host cell lysosomes in the T. cruzi infection process where our discussion is limited to invasion of nonprofessional phagocytic cells.

Cardiac hypertrophy is a common response to injury and hemodynamic stress and an important harbinger of heart failure and death. Herein, we identify the Kruppel-like factor 15 (KLF15) as an inhibitor of cardiac hypertrophy. Myocardial expression of KLF15 is reduced in rodent models of hypertrophy and in biopsy samples from patients with pressure-overload induced by chronic valvular aortic stenosis. Overexpression of KLF15 in neonatal rat ventricular cardiomyocytes inhibits cell size, protein synthesis and hypertrophic gene expression. KLF15-null mice are viable but, in response to pressure overload, develop an eccentric form of cardiac hypertrophy characterized by increased heart weight, exaggerated expression of hypertrophic genes, left ventricular cavity dilatation with increased myocyte size, and reduced left ventricular systolic function. Mechanistically, a combination of promoter analyses and gel-shift studies suggest that KLF15 can inhibit GATA4 and myocyte enhancer factor 2 function. These studies identify KLF15 as part of a heretofore unrecognized pathway regulating the cardiac response to hemodynamic stress.

Studies of cardiac pathology and heart failure have implicated cardiomyocyte apoptosis as a critical determinant of disease. Recent evidence indicates that the intracellular protozoan parasite Trypanosoma cruzi, which causes heart disease in chronically infected individuals, impinges on host apoptotic pathways in a cell type-dependent manner. T. cruzi infection of isolated neuronal cells and cardiomyocytes protects against apoptotic cell death, whereas apoptosis is triggered in T cells in T. cruzi-infected animals. In this study, we demonstrate that the ability of T. cruzi to protect cardiac cells in vitro from apoptosis triggered by a combination of tumor necrosis factor alpha and serum reduction correlates with the presence of intracellular parasites and involves activation of host cell NF-kappaB. We further demonstrate that the apoptotic block diminishes activation of caspase 3. The ability of T. cruzi to prevent apoptosis of infected cardiomyocytes is likely to play an important role in establishment of persistent infection in the heart while minimizing potential damage and remodeling that is associated with cardiomyocyte apoptosis in cardiovascular disease.

Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagasic cardiomyopathy, elicits a robust hypertrophic response in isolated cardiomyocytes. Previous studies established that T. cruzi-elicited cardiomyocyte hypertrophy is mediated by interleukin-1beta produced by infected cardiomyocyte cultures. Here, we define key upstream signaling events leading to cardiomyocyte hypertrophy in response to T. cruzi infection, to be dependent on Toll-like receptor 2 and NF-kappaB. Furthermore, we demonstrate that cardiomyocyte hypertrophy, which is initiated by live infective T. cruzi trypomastigotes or stimulation of isolated myocytes with secreted/released trypomastigote molecules, is a common outcome of the cardiomyocyte recognition of pathogen-associated molecular patterns by intrinsic Toll-like receptors. This study is the first to link pathogen recognition by intrinsic Toll-like receptors to cardiomyocyte hypertrophy.

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease in humans, is capable of invading and replicating within a wide variety of nucleated mammalian cell types. Host cell invasion by infective T. cruzi trypomastigotes is governed by parasite-triggered activation of host cell signaling pathways. Recent studies highlighting a role for host cell phosphatidylinositol 3-kinases (PI3Ks) in the T. cruzi invasion process have revealed surprising new insights into the mechanism of host cell invasion by this pathogen. In this Perspective, we discuss these findings and propose alternative models of T. cruzi invasion that incorporate this new information.

Connective tissue growth factor (CTGF) is a secreted cytokine that plays a fundamental role in the development of tissue fibrosis by mediating many of the profibrotic effects of TGF-beta. We present the novel finding that the intracellular pathogen Trypanosoma cruzi elicits immediate and sustained repression of basal CTGF expression in dermal fibroblasts, followed by down-regulation of the extracellular matrix proteins, fibronectin, and collagen I alpha1. To address mechanisms underlying this response, the major CTGF-regulating pathways were investigated. We report that both T. cruzi trypomastigotes and secreted parasite factor(s) antagonize TGF-beta-dependent induction of CTGF in fibroblasts. Of the TGF-beta-dependent signaling pathways required for CTGF expression, we demonstrate that T. cruzi interferes with cellular Erk1/2 phosphorylation but not Smad3 signaling. While increased stimulation of Erk phosphorylation alone was insufficient to override the parasite-mediated repression of CTGF, stimulation of fibroblasts with increased concentrations of TGF-beta, which activates both Smad3 and Erk1/2, completely abrogated this inhibition. Together with the finding that T. cruzi-mediated down-regulation of CTGF expression requires de novo host cell protein synthesis, our data indicate that the unique ability of T. cruzi to interfere with the host fibrogenic response is a complex process requiring input from multiple host cell signaling pathways.

One of the hallmarks of Trypanosoma cruzi invasion of non-professional phagocytes is facilitation of the process by host cell actin depolymerization. Host cell entry by invasive T. cruzi trypomastigotes is accomplished by exploiting a cellular wound repair process involving Ca(2+)-regulated lysosome exocytosis (i.e. lysosome-dependent) or by engaging a recently recognized lysosome-independent pathway. It was originally postulated that cortical actin microfilaments present a barrier to lysosome-plasma membrane fusion and that transient actin depolymerization enhances T. cruzi entry by increasing access to the plasma membrane for lysosome fusion. Here we demonstrate that cytochalasin D treatment of host cells inhibits early lysosome association with invading T. cruzi trypomastigotes by uncoupling the cell penetration step from lysosome recruitment and/or fusion. These findings provide the first indication that lysosome-dependent T. cruzi entry is initiated by plasma membrane invagination similar to that observed for lysosome-independent entry. Furthermore, prolonged disruption of host cell actin microfilaments results in significant loss of internalized parasites from infected host cells. Thus, the ability of internalized trypomastigotes to remain cell-associated and to fuse with host cell lysosomes is critically dependent upon host cell actin reassembly, revealing an unanticipated role for cellular actin remodelling in the T. cruzi invasion process.

The application of genome-scale approaches to study Trypanosoma cruzi-host interactions at different stages of the infective process is becoming possible with sequencing and assembly of the T. cruzi genome nearing completion and sequence information available for both human and mouse genomes. Investigators have recently begun to exploit DNA microarray technology to analyze host transcriptional responses to T. cruzi infection and dissect developmental processes in the complex T. cruzi life-cycle. Collectively, information generated from these and future studies will provide valuable insights into the molecular requirements for establishment of T. cruzi infection in the host and highlight the molecular events coinciding with disease progression. While the field is in its infancy, the availability of genomic information and increased accessibility to relatively high-throughput technologies represents a significant advancement toward identification of novel drug targets and vaccine candidates for the treatment and prevention of Chagas' disease.

Mammalian cell invasion by the protozoan parasite, Trypanosoma cruzi, is facilitated by the activation of host cell phosphatidylinositol 3 (PI 3)-kinases. We demonstrate that the well-characterized Ca2+-regulated lysosome-mediated parasite entry pathway is abolished by wortmannin pretreatment. In addition, we have characterized a novel route of T. cruzi invasion unexpectedly revealed in the course of this study. For over a decade, targeted exocytosis of lysosomes at the host cell plasma membrane was considered as the primary mechanism for T. cruzi entry into non-professional phagocytic cells. We now provide evidence that a significant fraction (50% or greater) of invading T. cruzi trypomastigotes exploit an alternate actin-independent entry pathway that involves formation of a tightly associated host cell plasma membrane-derived vacuole enriched in the lipid products of class I PI 3-kinases, PtdInsP3/PtdIns(3,4)P2. Initially devoid of lysosomal markers, the resultant parasite-containing vacuoles gradually acquire lysosome associated membrane protein 1 (lamp-1) and fluid phase endocytic tracer from the lysosomal compartment. In striking contrast to latex bead phagosomes, few T. cruzi vacuoles associate with the early endosomal marker, EEA1 and the 'maturation' process becomes refractory to PI 3-kinase inhibition immediately following parasite internalization. Jointly, these data provide a new paradigm for T. cruzi invasion of non-professional phagocytic cells and reveal a novel vacuole maturation process that appears to bypass the requirement for EEA1.

Chagas' disease, the leading cause of heart failure in Latin America, results from infection with the intracellular protozoan parasite Trypanosoma cruzi. Host cell responses elicited in the myocardium early in the infective process are thought to be critical for establishment of infection by this pathogen; however, these changes have not been well characterized. We report here that primary cardiomyocytes undergo hypertrophy as an early response to T. cruzi infection. The T. cruzi-elicited hypertrophic response is characterized by increased expression of genes encoding the contractile proteins MyHC beta and MyHC alpha, followed by an approximately twofold increase in cell size. Hypertrophy was observed in both parasite-containing and noninfected cell populations represented in T. cruzi-infected cultures, indicating the involvement of a soluble mediator in this process. Conditioned medium harvested from T. cruzi-infected cultures, which contained significant levels of interleukin-1 beta (IL-1 beta) but not endothelin-1 or tumor necrosis factor alpha, was sufficient to induce hypertrophy in isolated cardiomyocytes. Addition of a high-affinity receptor chimera, IL-1 trap, to cardiomyocyte cultures blocked the overall increase in cell size elicited by T. cruzi. These novel findings indicate that IL-1 beta, which is rapidly induced in response to T. cruzi, promotes cardiomyocyte hypertrophy early in the infective process and may contribute to maintenance of cardiomyocyte function during establishment of T. cruzi infection in the heart.

Mammalian cell invasion by the protozoan pathogen Trypanosoma cruzi is critical to its survival in the host. To promote its entry into a wide variety of non-professional phagocytic cells, infective trypomastigotes exploit an arsenal of heterogenous surface glycoproteins, secreted proteases and signalling agonists to actively manipulate multiple host cell signalling pathways. Signals initiated in the parasite upon contact with mammalian cells also function as critical regulators of the invasion process. Whereas the full spectrum of cellular responses modulated by T. cruzi is not yet known, mounting evidence suggests that these pathways impinge on a number of cellular processes, in particular the ubiquitous wound-repair mechanism exploited for lysosome-mediated parasite entry. Furthermore, differential engagement of host cell signalling pathways in a cell type-specific manner and modulation of host cell gene expression by T. cruzi are becoming recognized as essential determinants of infectivity and intracellular survival by this pathogen.

Host cell infection by the intracellular pathogen, Trypanosoma cruzi, involves activation of signaling pathways, cytoskeletal reorganization, and targeted recruitment of host cell lysosomes. To determine the consequences of T. cruzi invasion on host cell gene expression, high density microarrays consisting of approximately 27,000 human cDNAs were hybridized with fluorescent probes generated from T. cruzi-infected human fibroblasts (HFF) at early time points following infection (2-24 h). Surprisingly, no genes were induced > or =2-fold in HFF between 2 and 6 h post-infection (hpi) in repeated experiments while immediate repression of six host cell transcripts was observed. A significant increase in transcript abundance for 106 host cell genes was observed at 24 hpi. Among the most highly induced is a set of interferon-stimulated genes, indicative of a type I interferon (IFN) response to T. cruzi. In support of this, T. cruzi-infected fibroblasts begin to secrete IFNbeta at 18 hpi following the induction of IFNbeta transcripts. As compared with global transcriptional responses evoked by other intracellular pathogens, T. cruzi is a stealth parasite that elicits few changes in host cell transcription during the initiation of infection.