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 G1 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 G1 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.

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.

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.

Aotus nancymaae, the owl monkey, provides a useful laboratory model for research to develop drugs and vaccines against human falciparum malaria; however, many Plasmodium falciparum parasites are unable to invade A. nancymaae erythrocytes, rendering the parasites noninfective to the monkeys. In previous work, we identified a key polymorphism that determined the inheritance of erythrocyte invasion in a genetic cross of two P. falciparum clones that were virulent (GB4) or noninfective (7G8) to A. nancymaae. This polymorphism, an isoleucine-to-lysine polymorphism at position 204 (I204K) of the GB4 erythrocyte binding protein PfRH5, was nevertheless not found in several other P. falciparum lines that could also invade A. nancymaae erythrocytes. Alternative PfRH5 polymorphisms occur at different positions in these virulent parasites, and additional polymorphisms are found in P. falciparum parasites that cannot infect A. nancymaae. By allelic replacement methods, we have introduced the polymorphisms of these A. nancymaae-virulent or noninfective parasites at codons 204, 347, 358, 362, 410, and 429 of the endogenous PfRH5 gene in the noninfective 7G8 line. 7G8 transformants expressing the polymorphisms of the A. nancymaae-virulent parasites show neuraminidase-sensitive (sialic acid-dependent) invasion into the monkey erythrocytes, whereas 7G8 transformants expressing the PfRH5 alleles of noninfective parasites show little or no invasion of these erythrocytes. Parasites harboring PfRH5 polymorphisms 204K or 204R are also able to invade rat erythrocytes and are differentially sensitive to the removal of surface sialic acids by neuraminidase. These studies offer insights into the PfRH5 receptor-binding domain and interactions that support the invasion of various primate and rodent erythrocytes by P. falciparum.

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The mechanisms underlying resistance of the Chagas disease parasite, Trypanosoma cruzi, to current therapies are not well understood, including the role of metabolic heterogeneity. We found that limiting exogenous glutamine protects actively dividing amastigotes from ergosterol biosynthesis inhibitors (azoles), independent of parasite growth rate. The antiparasitic properties of azoles are derived from inhibition of lanosterol 14α-demethylase (CYP51) in the endogenous sterol synthesis pathway. We find that carbons from 13C-glutamine feed into amastigote sterols and into metabolic intermediates that accumulate upon CYP51 inhibition. Incorporation of 13C-glutamine into endogenously synthesized sterols is increased with BPTES treatment, an inhibitor of host glutamine metabolism that sensitizes amastigotes to azoles. Similarly, amastigotes are re-sensitized to azoles following addition of metabolites upstream of CYP51, raising the possibility that flux through the sterol synthesis pathway is a determinant of sensitivity to azoles and highlighting the potential role for metabolic heterogeneity in recalcitrant T. cruzi infection.

Malaria, the disease caused by Plasmodium parasites, remains a major global health burden. The liver stage of Plasmodium falciparum infection is a leading target for immunological and pharmacological interventions. Therefore, novel approaches providing specific detection and isolation of live P. falciparum exoerythrocytic forms (EEFs) are warranted. Utilizing a recently generated parasite strain expressing green fluorescent protein (GFP) we established a method which, allows for detection and isolation of developing live P. falciparum liver stages by flow cytometry. Using this technique we compared the susceptibility of five immortalized human hepatocyte cell lines and primary hepatocyte cultures from three donors to infection by P. falciparum sporozoites. Here, we show that EEFs can be detected and isolated from in vitro infected cultures of the HC-04 cell line and primary human hepatocytes. We confirmed the presence of developing parasites in sorted live human hepatocytes and characterized their morphology by fluorescence microscopy. Finally, we validated the practical applications of our approach by re-examining the importance of host ligand CD81 for hepatocyte infection by P. falciparum sporozoites in vitro and assessment of the inhibitory activity of anti-sporozoite antibodies. This methodology provides us with the tools to study both, the basic biology of the P. falciparum liver stage and the effects of host-derived factors on the development of P. falciparum EEFs.

Control of parasite replication exerted by MHC class I restricted CD8+ T-cells in the liver is critical for vaccination-induced protection against malaria. While many intracellular pathogens subvert the MHC class I presentation machinery, its functionality in the course of malaria replication in hepatocytes has not been characterized. Using experimental systems based on specific identification, isolation and analysis of human hepatocytes infected with P. berghei ANKA GFP or P. falciparum 3D7 GFP sporozoites we demonstrated that molecular components of the MHC class I pathway exhibit largely unaltered expression in malaria-infected hepatocytes until very late stages of parasite development. Furthermore, infected cells showed no obvious defects in their capacity to upregulate expression of different molecular components of the MHC class I machinery in response to pro-inflammatory lymphokines or trigger direct activation of allo-specific or peptide-specific human CD8+ T-cells. We further demonstrate that ectopic expression of circumsporozoite protein does not alter expression of critical genes of the MHC class I pathway and its response to pro-inflammatory cytokines. In addition, we identified supra-cellular structures, which arose at late stages of parasite replication, possessed the characteristic morphology of merosomes and exhibited nearly complete loss of surface MHC class I expression. These data have multiple implications for our understanding of natural T-cell immunity against malaria and may promote development of novel, efficient anti-malaria vaccines overcoming immune escape of the parasite in the liver.