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. In-depth knowledge of the functional processes governing host colonization and transmission of pathogenic microorganisms is essential for the advancement of effective intervention strategies. This study focuses on Trypanosoma cruzi, the vector-borne protozoan parasite responsible for human Chagas disease and the leading cause of infectious myocarditis worldwide. To gain global insights into the biology of this parasite and its interaction with mammalian host cells, we have exploited a deep-sequencing approach to generate comprehensive, high-resolution transcriptomic maps for mammalian-infective stages of T. cruzi with the simultaneous interrogation of the human host cell transcriptome across an infection time course. We demonstrate that the establishment of intracellular T. cruzi infection in mammalian host cells is accompanied by extensive remodeling of the parasite and host cell transcriptomes. Despite the lack of transcriptional control mechanisms in trypanosomatids, our analyses identified functionally-enriched processes within sets of developmentally-regulated transcripts in T. cruzi that align with known or predicted biological features of the parasite. The novel insights into the biology of intracellular T. cruzi infection and the regulation of amastigote development gained in this study establish a unique foundation for functional network analyses that will be instrumental in providing functional links between parasite dependencies and host functional pathways that have the potential to be exploited for intervention.

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 t...

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. The kinetoplastid protozoan, Trypanosoma cruzi, is a highly successful parasite with a broad mammalian host range and the capacity to colonize a variety of tissues within a given host to establish life-long infection. T. cruzi infection causes Chagas disease in humans, characterized by severe cardiomyopathy and gastrointestinal motility disorders, with limited treatment options. Despite the critical role of T. cruzi amastigotes in sustaining mammalian infection, little is known about their metabolic requirements or the range of nutrients available to these parasites in the host cell cytoplasm. Here, we demonstrate that T. cruzi infection triggers a host response in infected cells that includes increased mitochondrial respiration and biogenesis and increased glucose uptake into infected cells. We show that exogenous glucose supports optimal intracellular parasite replication and that cytosolic T. cruzi amastigotes access glucose in situ, potentially via a facilitated transport process characterized here. These findings expand our view of the range of carbons available to the intracellular parasite and suggest even greater metabolic flexibility of the tissue-infective stages of T. cruzi than previously appreciated, a capability predicted to contribute to successful host colonization.

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.

Chagas' disease, the leading cause of heart failure in Latin America, results from infection with the intracellular protozoan parasiteTrypanosoma 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 toT. cruziinfection. TheT. cruzi-elicited hypertrophic response is characterized by increased expression of genes encoding the contractile proteins MyHCβ and MyHCα, followed by an approximately twofold increase in cell size. Hypertrophy was observed in both parasite-containing and noninfected cell populations represented inT. cruzi-infected cultures, indicating the involvement of a soluble mediator in this process. Conditioned medium harvested fromT. cruzi-infected cultures, which contained significant levels of interleukin-1β (IL-1β) 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 byT. cruzi. These novel findings indicate that IL-1β, which is rapidly induced in response toT. cruzi, promotes cardiomyocyte hypertrophy early in the infective process and may contribute to maintenance of cardiomyocyte function during establishment ofT. cruziinfection in the heart.