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. Author manuscript; available in PMC: 2014 Jan 16.
Published in final edited form as: Cell Host Microbe. 2013 Jan 16;13(1):108–117. doi: 10.1016/j.chom.2012.11.011

Host metabolism regulates intracellular growth of Trypanosoma cruzi

Kacey L Caradonna 1, Juan C Engel 2, David Jacobi 3, Chih-Hao Lee 3, Barbara A Burleigh 1,*
PMCID: PMC3560928  NIHMSID: NIHMS430216  PMID: 23332160

SUMMARY

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.

INTRODUCTION

Chagas’ disease is a progressive and debilitating parasitic disease that develops over decades in a subset of individuals chronically infected with the obligate intracellular protozoan Trypanosoma cruzi. Current global estimates indicate that 8–11 million people are infected with T. cruzi, primarily in rural Latin America, and of these, roughly 3 million will develop life-threatening, incurable chronic Chagasic syndromes (WHO 2012). Parasite persistence in cardiac and smooth muscle, with accompanying inflammation and immune dysregulation, plays an essential role in the pathogenesis of Chagas’ disease (Gutierrez et al., 2009; Tarleton, 2001) where links to host metabolic perturbations are emerging (Garg et al., 2004; Machado et al., 2011). T. cruzi infection impacts cardiac energy metabolism (Garg et al., 2004) where attenuated mitochondrial function in the heart is associated with the development and progression of Chagasic cardiomyopathy (Wen et al., 2004). Moreover, persistent T. cruzi infection of adipose tissue in chronic Chagas’ patients (Matos Ferreira et al., 2011) predicts long-term consequences of the associated inflammation on host glucose and lipid metabolism which may influence disease progression. As the currently available drugs to treat Chagas’ disease exhibit toxicity and fail to clear intracellular parasites in the chronic stage of infection, the need to develop treatments for this neglected disease is paramount.

The vertebrate stages of T. cruzi consist of non-dividing ‘trypomastigote’ forms that actively penetrate a wide variety of host cell types to establish intracellular infection and ‘amastigote’ forms that proliferate in the host cell cytoplasm. The current model of the T. cruzi trypomastigote invasion process was derived from the collective contribution of several laboratories over the past two decades (reviewed in (Caradonna and Burleigh, 2011)). In contrast to these efforts, the cellular and metabolic processes required to fuel the growth of intracellular T. cruzi amastigotes remain largely unknown (Gutteridge and Gaborak, 1979; Inbar et al., 2012; Robello et al., 1997; Ullman and Carter, 1997). Thus, a global determination of the components, function, and consequence of host cellular pathways that are exploited by intracellular T. cruzi growth and survival will have significant implications for the understanding and treatment of chronic Chagas’ disease.

Toward this end, we have performed a genome-wide RNA interference screen targeting genes in mammalian host cells to identify cellular processes that support intracellular T. cruzi infection. Host factors associated with T. cruzi infection identified in a recent study (Genovesio et al., 2011) did not identify the primary points of action. A critical element of our screening strategy is the inclusion of secondary screens designed to discriminate host functional pathways that impact trypomastigote invasion and the pre-replication phase (<24 hr post-infection) from host processes that support intracellular T. cruzi amastigote growth (>24 – 90 hr). Consistent with our current view of the T. cruzi invasion process establishment of intracellular residence by this pathogen is largely influenced by host cell signaling molecules and cytoskeletal proteins. In contrast, the intracellular replicative phase of the T. cruzi infection cycle is supported by host metabolic networks and cellular signaling pathways, a subset of which were validated with small molecule inhibitors and biochemical rescue experiments. Thus, the ability to modulate the intracellular replicative capacity T. cruzi amastigotes by targeting host cellular and metabolic pathways offers an experimental tool that can be exploited to probe the intimate connections forged between T. cruzi and its mammalian host cell.

RESULTS

A Genome-wide RNA Interference Screen in Mammalian Host Cells Identifies Interconnected Metabolic Pathways as Key Regulators of Intracellular T. cruzi Growth

To gain functional insights into host cellular pathways exploited by T. cruzi during the establishment and maintenance of intracellular infection, a three-phase RNA interference screen was established in mammalian cells (Figure 1A). In the primary screening phase, an arrayed human siRNA library consisting of 25,586 siRNA pools (4 individual siRNAs/pool) was assayed in HeLa cells to determine the impact of host cell gene silencing on T. cruzi infection using a multiplexed end-point parasite growth assay (Figure 1B and Figure S1). This assay permitted the simultaneous measurement of β-galactosidase-expressing T. cruzi (Beta-Glo™) and host cell abundance (Cell Titer-Fluor™) as detailed in Experimental Procedures. For each well, the median absolute deviation (MAD) for the BetaGlo™/Cell Titer-Fluor™ ratios were derived and used as the first criterion for assigning hits: a MAD z-score of ≤ −1.75 or ≥ 2.5. Wells in which host cell abundance was ≤40% of non-targeting siRNA controls were excluded. Based on these criteria, the primary screen yielded 359 siRNA pools (1.69% of total) that restrict T. cruzi infection and 293 siRNA pools (1.38%) that enhance intracellular parasite burden (Figure 1C and Table S1). Focusing primarily on siRNA pools that caused an overall reduction in the intracellular burden of T. cruzi, 240 selected pools were rescreened in the parasite growth assay (Figure 1B) as 4 individual siRNAs deconvoluted from the original pool. We find that 81% of the siRNA pools contained at least one siRNA that reproduced the original effect on parasite infection (Table S1). At the rescreening stage, a few additional siRNAs that did not make the significance cut-offs in the primary screen, but which represented ‘borderline’ hits or genes involved in emerging pathways of interest were included. Genes with unknown function were not considered for further testing. Efficient gene silencing in HeLa cells was observed for the siRNA pools tested (Figure S2A).

Figure 1. Genome-wide siRNA screen differentiates host processes important for Trypanosoma cruzi invasion and growth.

Figure 1

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(A) Overall strategy for the three-phase RNA interference screen. (B) Schematic for set up of T. cruzi growth assay and primary and secondary screen readouts. Note, secondary screens exploited both the multiplexed plate assay and an image-based readout in parallel with endpoints of 18 hpi and 72 hpi, to discriminate effects of host gene knockdown on intracellular parasite growth from pre-replication events in the parasite life cycle (illustrated in circular inset). (C) Ranked median average deviation (MAD) z-scores of relative infection data from primary screen. (D) Functional breakdown of secondary screen hits that impact intracellular parasite infection (18 h) and growth (72 h) from Table S3, manually curated from UniProt. See also Figures S1 and Table S1–S2.

To discriminate host factors that influence early steps in the establishment of intracellular T. cruzi infection (invasion, vacuole biogenesis, cytosolic localization) from factors regulating intracellular growth of cytosolic T. cruzi amastigotes, secondary screens were conducted to measure relative parasite infection levels at pre- and post-replication time points (18 and 72 hpi, respectively) (Figure 1B, Figure S2B). In accordance with their distinct roles in the parasite life cycle, host genes associated with T. cruzi invasion and intracellular replication (Table S2) displayed discrete patterns of functional enrichment (Figure 1D). Our results reveal that the pre-replication phase of the T. cruzi infection cycle is predominantly affected by host cell signaling pathways and cytoskeletal regulators (Table S2), in line with our current understanding of the parasite invasion process (Caradonna and Burleigh, 2011). In contrast, host factors associated with T. cruzi amastigote growth in mammalian cells were highly enriched for metabolic functions (Figure 1D and Table S2). Despite functional leads to the parasite internalization process, we concentrated on host pathways that impact the intracellular growth of T. cruzi amastigotes, for which little is currently known. Within this group, we focused on the subset with the highest enrichment and most promising pharmacological targets, specifically, Akt signaling and hits related to glucose, lipid, nucleotide and energy metabolism (Figure 2).

Figure 2. Schematic of interconnected host metabolic pathways that influence intracellular growth of T. cruzi amastigotes.

Figure 2

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Highlighted are host genes, that when silenced with specific siRNA pools, reduce (yellow) or enhance (blue) intracellular growth of T. cruzi amastigotes. Underline indicates pathways for known T. cruzi auxotrophies. See also Figure S2.

Perturbations in Host Glucose and Fatty Acid Metabolism Alter Intracellular Parasite Growth Rates

In mammalian hosts, T. cruzi replicates and persists within tissues that exhibit high rates of fatty acid metabolism (Fritz, 1961) such as cardiac muscle, smooth muscle and adipose (Brener, 1973; Combs et al., 2005; Macedo and Pena, 1998; Matos Ferreira et al., 2011). The rate of fatty acid oxidation in the mitochondria is controlled by the flux of glucose through the glycolytic pathway and the rate of conversion of pyruvate to acetyl CoA by pyruvate dehydrogenase (PDH). T. cruzi infection of cardiac tissue triggers early upregulation of host fatty acid β-oxidation genes followed by downregulation of the PDH complex (Garg et al., 2004). As a negative regulator of PDH, pyruvate dehyrogenase kinase 4 (PDK4) functions as the main gatekeeper of the balance between glucose and fatty acid oxidation (Sugden et al., 2001). Here, we find that T. cruzi growth is poorly supported in PDK4-depleted cells (Figure 3A) which display increased PDH activity (Figure 3B) and decreased fatty acid oxidation (Figure 3C). Consistent with the idea that increased flux of pyruvate through PDH contributes to a cellular environment that is restrictive for T. cruzi amastigote growth, we find that intracellular replication of T. cruzi is enhanced in primary human fibroblasts that exhibit reduced PDH activity (80% reduction; data not shown and (Constantopoulos et al., 1986)) (Figure 3D). Predicted consequences of decreased PDK4 levels and increased PDH activity include a decrease in cellular pyruvate pools and a shift in the fuel utilization balance from fatty acid oxidation toward glucose oxidation (Figure 3E) (Sugden et al., 2001). As pyruvate supplementation of the growth medium fails to rescue T. cruzi growth in PDK4-depleted cells (not shown), the interaction of intracellular T. cruzi amastigotes with host pyruvate metabolism may be more complex than the fulfillment of a nutritional requirement (NB. pyruvate uptake by T. cruzi has not been demonstrated). Instead, T. cruzi amastigotes may display a growth advantage under conditions where host cell fatty acid oxidation is favored over glucose utilization.

Figure 3. Intracellular T. cruzi growth is coupled to host fatty acid metabolism.

Figure 3

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(A) Western blot of knockdown (inset) and distribution data for the number of intracellular parasites in HeLa cells transfected with non-targeting siRNA (siControl) or siPDK4 at 72 hpi. Bar graph values derived from image-based analysis of secondary screening data (Table S3). (B) PDH activity and (C) β-oxidation measured in HeLa cells transfected with non-targeting (siControl) or siPDK4 (n=6). Error bars indicate standard error (**p<0.01, ***p<0.001). (D) Primary human fibroblast deficient in pyruvate dehydrogenase (PDH) or acyl-CoA dehydrogenase (very long chain) ACADVL were infected with T. cruzi and counted 48 hpi. Scatter plots of numbers of parasites/infected cell for 100 infected cells (per cell line) with median highlighted (black line). Significance was determined by Kruskal-Wallis test followed by Dunn’s Multiple Comparison test to directly compare infection of metabolically deficient cells to healthy control (***p<0.001). Experiment was repeated three times with similar results. (E) Schematic of balance between PDH regulation and fatty oxidation and its effects on parasite growth.

In mammals, β-oxidation occurs in the peroxisomes and the mitochondria (Poulos, 1995). Very long chain fatty acids (VLCFAs; ≥C22) are transported into peroxisomes and oxidized to produce shorter chain fatty acids that are shuttled to the mitochondria for continued oxidation by a number of acyl-CoA dehydrogenases (ACADs) to ultimately produce acetyl CoA (Ghisla and Thorpe, 2004). Consistent with the notion that T. cruzi amastigotes flourish in an environment which favors fatty acid oxidation, peroxisomal proteins involved in activation/transport of VLCFAs (SLC27A2) and β-oxidation (ACAA1, IDH1) were identified in our screen as positively associated with intracellular T. cruzi growth (Figure 2). In addition, primary human fibroblasts deficient in mitochondrial ACADVL (Fang et al., 2000) which accommodates substrate acyl chains of (≥C16), sustain significantly lower levels of intracellular T. cruzi growth (Figure 3D). In contrast, parasite growth restriction was not observed in cells deficient for medium or short chain ACADs (data not shown). Combined, these data identify long chain fatty acid oxidation as a key process in mammalian host cells associated with intracellular T. cruzi growth. The upregulation of fatty acid utilization pathways in axenically-derived T. cruzi amastigotes (Atwood et al., 2005) indicates the likelihood that fatty acid metabolism in the intracellular parasite is coupled with that of their mammalian hosts. Whether T. cruzi amastigotes avail of fatty acid intermediates generated in host peroxisomal or mitochondrial oxidative pathways or whether they benefit indirectly from the production of energy and reductive intermediates formed in these catabolic processes remains to be determined.

Parasite Growth is Dependent on the Availability of Host-derived Cofactors and Metabolic Products

In mammalian cells, electrons generated via fatty acid β-oxidation and amino acid catabolism in the mitochondria are transferred to Coenzyme Q10 (CoQ10) via the electron transfer flavoprotein dehydrogenase ETFDH (Figure 2). Limiting CoQ10 production in host cells by silencing the biosynthetic enzyme trans-prenyltransferase (TPRT) restricts intracellular amastigote growth (Figure 4A). The parasite growth defect is completely restored with CoQ10 supplementation of the culture medium (Figure 4B). In addition to its role in mitochondrial energy production, CoQ10 is critical for de novo pyrimidine synthesis (Lopez-Martin et al., 2007) and exhibits antioxidant properties (Jeya et al., 2010). T. cruzi growth in TPRT-knockdown cells is partially restored with the addition of uridine but not following supplementation with the antioxidant N-acetylcysteine (NAC) (Figure 4B). These data indicate that CoQ10-deficiency in the host restricts T. cruzi growth by limiting the availability of pyrimidines. While T. cruzi has pyrimidine synthetic capability (Gutteridge and Gaborak, 1979) our host functional data imply that intracellular amastigotes are reliant on host pyrimidine pools to support their growth. Consistent with this observation, limiting pyrimidine nucleoside and/or nucleobase generation downstream of the host cytosolic nucleotidase, NT5C3, is suboptimal for intracellular T. cruzi growth (Figure 2).

Figure 4. Modulation of perturbed pathways with metabolite supplementation restores parasite growth.

Figure 4

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(A) Distribution data for the number of intracellular T. cruzi in HeLa cells transfected with non-targeting siRNA (siControl) or siRNAs targeting trans-prenyltransferase TPRT (siTPRT) which is required for CoenzymeQ10 (CoQ10) synthesis. (B) Scatter plots of intracellular parasites in infected cells transfected with non-targeting control (siControl) or siTPRT supplemented with CoQ10 (5 µM), uridine (10 µM), or N-acetylcysteine (NAC; 1 mM). (C) Distribution data for the number of intracellular parasites in HeLa cells transfected with non-targeting siRNA (siControl) or siRNAs targeting GTP cyclohydrolase 1 (GCH1), the first enzyme in the biopterin synthesis pathway (D) Supplementation of growth medium with 7,8 dihydro-L-biopterin rescues T. cruzi growth defect in GCH1-depleted cells. (B,D) Black line indicated count median for each treatment. Significance was determined between control unsupplemented TPRT or GCH1-deficient cells (blue) and other conditions (green) by Kruskal-Wallis test followed by Dunn’s Multiple comparison test (***p<0.001). Scatter plot distributions shown are representative of three independent experiments.

Purines and pteridine auxotrophy (Gutteridge and Gaborak, 1979; Robello et al., 1997) predicts that intracellular T. cruzi amastigotes will scavenge these essential nutrients from the host cell cytosol. As biochemical data for the amastigote forms of T. cruzi are limited (Gutteridge and Gaborak, 1979; Nakajima-Shimada et al., 1996), the functional links to host purine and pteridine metabolic pathways uncovered in our unbiased RNAi screen were particularly valuable (Figure 2). Limiting tetrahydrobiopterin production in host cells by silencing a key biosynthetic enzyme GTP cyclohydrolase 1 (GCH1) restricts intracellular T. cruzi growth (Figure 4C) where the parasite growth defect is rescued following biochemical supplementation of GCH1-depleted cells with 7,8 dihydro-L-biopterin (Figure 4D). These findings provide functional evidence that intracellular T. cruzi amastigotes are tightly coupled to the biopterin biosynthetic capabilities of the host cell. Purines are acquired by trypanosomatids from the environment via members of parasite-encoded equilibrative nucleoside transporters (Landfear, 2008) (http://tritrypdb.org/tritrypdb/). Silencing of purine nucleoside phosphorylase (PNP) in host cells has the predicted consequence of limiting purine nucleoside to nucleobase conversion. As such, the associated increase in T. cruzi amastigote growth in PNP-depleted cells was striking (Figure 2) and leads to the testable hypothesis that intracellular stages of T. cruzi preferentially transport purine nucleosides. Combined with functional evidence that host pyrimidines are critical for fueling intracellular amastigote growth (Figure 4B), despite the capacity for de novo synthesis (Gutteridge and Gaborak, 1979), data from our RNA interference screen provide functional evidence for the reliance of T. cruzi amastigotes on host purine and pyrimidine pools as suggested by inhibitor studies (Nakajima-Shimada et al., 1996). These findings may have important implications considering purine and pyrimidine salvage pathways in intracellular T. cruzi amastigotes as potential therapeutic targets.

Homeostatic mechanisms that maintain high ATP/ADP ratios are critical for cell viability and growth. As such, preservation of cellular nucleotide pools is likely to present a fundamental challenge for T. cruzi-infected host cells. The majority of cellular ATP is generated in the mitochondria in a reaction that is coupled to oxidative phosphorylation and the transfer of electrons generated via glucose, fatty acid, and amino acid catabolism. Host genes participating in these processes emerged in our secondary screen as strongly associated with intracellular T. cruzi amastigote growth (Figures 1D and 2, Table S2) including a catalytic subunit of the mitochondrial ATP synthase, ATP5B. Limiting the conversion of cytosolic ATP to other nucleoside triphosphates via transphosphorylation reactions catalyzed by nucleoside diphosphate kinases, such as NME3 and NME4, significantly boosts intracellular T. cruzi growth (Figure 2 and Table S1) suggesting that maintenance of cellular ATP/ADP ratios provides a distinct advantage for the parasite which could include keeping the activity of AMPK in check (as demonstrated in Figure 5H, acute silencing of AMPK catalytic or regulatory subunits provides a more favorable growth environment for intracellular T. cruzi). Viewed within the larger context of nucleotide metabolism, these data highlight critical nodes in host bioenergetic and nutrient generating pathways to which cytosolically-localized T. cruzi amastigotes couple to meet metabolic needs.

Figure 5. Host cell Akt regulates intracellular T. cruzi growth via mTORC1-dependent and independent mechanisms.

Figure 5

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(A) Western blot of Akt1 and Akt2 expression levels in HeLa cells as compared to β-actin, following siRNA-mediated silencing of Akt1, 2 and 3 isoforms singly and in combination. (B) Silencing of Akt1 in HeLa cells inhibits intracellular T. cruzi growth (72 hpi) but not the ability to establish intracellular infection (18 hpi) as determined with the multiplexed plate assay (detailed in Methods) and compared to non-targeting control siRNA. Data represented as the mean +/− SD (n≥3). (C) Distribution plots of intracellular T. cruzi amastigotes (# parasites / infected cell) in HeLa transfected with non-targeting control siRNA (siControl) or following acute silencing of Akt1 (siAkt). (D) Relative phospho-AktSer473 levels in HeLa cells determined by western blot in mock- or T. cruzi-infected or insulin-stimulated (Ins) cells 72 hpi in 0.5% serum containing media. AktVIIIi was added to cells at 18hpi. (E) Dose-dependent inhibition of T. cruzi growth in HeLa cells treated with AktVIIIi at 18 hpi and relative infection measured at 72 hpi using the multiplexed plate assay. Values represent the mean +/− SD (n≥3). (F) T. cruzi growth in Akt1/2-deficient MEFs measured at 72 hpi is significantly lower than in WT controls and refractory to 1 µM AktVIIIi added at 18 hpi. (G) Intracellular CFSE-labeled T. cruzi amastigotes measured by flow cytometry reveal a uniform undivided population at 18 hpi (generation=0, red line) with overlapping intensity to extracellular CFSE-labeled parasites fixed at time 0 (blue) (left panel) and 5 distinct generations at 48 hpi in untreated HFF (middle panel). AktVIIIi (1µM) added at 18 hpi inhibits T. cruzi proliferation (right panel). (H) Acute silencing of PRKAB1, the regulatory subunit of AMPK, enhances intracellular growth of T. cruzi as confirmed in secondary screens. (I) Constitutive mTORC1 activation in stable TSC2 knockdown HeLa cells (shTSC2) enhances T. cruzi growth (measured at 48 hpi) as compared to control cells expressing shRNA to firefly luciferase (shLUC) and is sensitive to rapamycin (500nM) and AktVIIIi (2 µM) added at 18 hpi. (F,I) Line indicates parasite/cell median. Significance was determined by Kruskal-Wallis and Dunn’s Mulitple Comparison tests (***p<0.001). Experiments shown are representative of 2–3 independent observations. (J) Proposed interaction of T. cruzi with Akt-activated mTORC1-dependent and –independent signaling pathways. siRNA-mediated depletion of host gene resulting in decreased intracellular T. cruzi growth (yellow) or increased growth (blue) is shown. See also Figure S3.

Genetic and Pharmacological Targeting of Host Akt Signaling Inhibits Intracellular T. cruzi Replication

The serine/threonine kinase, Akt, participates in an extensive cellular network that controls multiple pathways including pro-survival responses, glucose metabolism and nutrient sensing via the mTORC1 pathway (Huang and Manning, 2009; Weichhart, 2012). Akt is activated in a variety of mammalian host cells during the T. cruzi invasion process (Chuenkova et al., 2001; Woolsey et al., 2003) prompting a pro-survival response in infected host cells that is sustained throughout the intracellular parasite growth cycle (Chuenkova and PereiraPerrin, 2009). A previously unrecognized function for host Akt as a regulator of intracellular T. cruzi growth was uncovered in our genome-wide RNA interference screen. Acute silencing of Akt1 restricts intracellular T. cruzi growth in HeLa cells without discernable effects on pre-replication events in the parasite life cycle (Figure 5A,B,C). Whereas isoform-specificity for Akt1 was observed in HeLa cells, silencing of both Akt1 and Akt2 in mouse embryonic fibroblasts (MEF) and human dermal fibroblasts (HFF) was necessary to observe comparable effects on T. cruzi growth (Figure S3A,B). The availability of highly selective inhibitors of Akt1/2 isoforms (Green et al., 2008) permits chemical validation of Akt signaling as a key host cell pathway regulating intracellular T. cruzi replication. Pharmacological targeting of host Akt1/2 with the allosteric inhibitor AktVIIIi restricts intracellular parasite growth and signaling (Figure 5D,E) in a range of mammalian cell types (Figure S3C), with the exception of Akt1/2-deficient MEF in which residual T. cruzi growth is refractory to AktVIIIi (Figure 5F). To determine if targeting of host Akt inhibits the replicative capacity of the parasite, we established a proliferation assay for intracellular T. cruzi amastigotes based on flow cytometric analysis of CFSE stained parasites (Figure 5G). In untreated cells, five generations of intracellular T. cruzi amastigotes can be resolved by 48 hours post infection. Treatment of infected fibroblasts with AktVIIIi at 18 hpi (immediately prior to the first division of intracellular parasites) resulted in significant inhibition of parasite proliferation following an initial round of parasite cell division (Figure 5G). Thus, pharmacological targeting of host Akt demonstrates the proof-of-principle concept that unbiased functional screens are valuable tools for the identification of cellular targets in mammalian host cells that, when inhibited pharmacologically, restrict intracellular T. cruzi infection. This principle can be applied to any druggable molecular target that emerges from this or similar screening approaches.

T. cruzi Growth is Enhanced by Manipulation of Anabolic Growth Pathways

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an Akt-dependent pathway that senses cellular nutrient, energy, and redox status and controls protein synthesis (Huang and Manning, 2009; Weichhart, 2012). mTORC1 signaling is inhibited by nutrient and growth factor deficiency or by treatment with the small molecule inhibitor rapamycin. Perturbations in the host mTORC1 pathway in T. cruzi-infected cells revealed in transcriptomic studies (Costales et al., 2009) suggest that parasite intersects this pathway, directly or indirectly. Consistent with this suggestion, acute silencing of genes encoding catalytic (PRKAA1) and non-catalytic (PRKAB1) subunits of AMP kinase (AMPK), a negative regulator of mTORC1, significantly enhance intracellular growth of T. cruzi (Figure 5H and Tables S1 and S2). To better assess the impact of mTORC1 on parasite replication, we exploited a HeLa cell line stably expressing shRNA targeting tuberous sclerosis protein 2 (TSC2), an Akt substrate and a negative regulator of mTORC1 for infection studies. We find that T. cruzi growth is insensitive to rapamycin in control cells, however, constitutive activation of mTORC1 in shTSC2 cells (Huang et al., 2008) fueled increased intracellular T. cruzi growth in a rapamycin-sensitive manner (Figure 5I). The observation that both basal and mTORC1-stimulated T. cruzi growth are sensitive to AktVIIIi, whereas only the accelerated parasite growth in TSC2-depleted cells is sensitive to rapamycin (Figure 5I), exposes the plasticity of parasite populations with respect to their ability to access mTORC1-dependent and independent pathways downstream of Akt to support replication in mammalian cells (Figure 5J). The ability of T. cruzi to integrate its metabolic needs with different host cellular pathways would offer a level of flexibility to facilitate parasite survival in the face of changing environmental conditions encountered as the parasite adapts to life in multiple hosts and cell types in nature.

DISCUSSION

At the outset of the present study, we had little prior knowledge of host cellular pathways that function to support intracellular infection by the obligate intracellular parasite, Trypanosoma cruzi. As a result of the unbiased functional screen and accompanying experimental validation reported here, we are now able to place the predicted metabolic dependencies of T. cruzi (Gutteridge and Gaborak, 1979; Inbar et al., 2012; Ullman and Carter, 1997) within a larger interconnected framework encompassing nucleotide metabolism, glucose / fatty acid metabolism, host energetics, and Akt signaling. The identification of biochemical pathways in our screen that yield products known to be required for T. cruzi growth and replication (i.e. purine and biopterin) provides a high level of confidence in the functional associations that have emerged in the screen.

The functional link exposed by our unbiased screen between host CoenzymeQ10 production, pyrimidine biosynthesis and T. cruzi amastigote growth was previously unrecognized. Acute silencing of host TPRT, which is required for CoQ10 biosynthesis, dramatically inhibited the growth of intracellular amastigotes without exerting a detrimental effect on the host cell. In biochemical supplementation experiments we were able to show that addition of CoQ10 was sufficient to rescue parasite growth. The ability to restore parasite growth with uridine supplementation as well suggests that the main requirement for CoQ10 is associated with its activity as a cofactor for the pyrimidine biosynthetic enzyme, dihydroorotate dehydrogenase. Considering that T. cruzi has the capacity to synthesize pyrimidines de novo, the inferred reliance on host pyrimidine pools was not expected. Collectively, these findings highlight the utility of a functional genomics approach to identify critical regulators of the dynamic T. cruzi-host interaction.

The emergence of the host serine/threonine kinase Akt as a regulator of T. cruzi amastigote replication was unexpected considering the extensive focus on Akt as a key mediator of the pro-survival response triggered by T. cruzi (Chuenkova and PereiraPerrin, 2009). The coupling of T. cruzi amastigote replication to a critical regulator of host cell survival suggests a sophisticated strategy for establishment of long-term infection in the host. It should be noted that T. cruzi is not unique in its exploitation of host Akt-signaling pathways (Kuijl et al., 2007; Kumar et al., 2010) where its influence on host phagolysosomal fusion events is manipulated by intracellular bacteria (Kuijl et al., 2007). The mechanistic basis for the influence of host Akt on T. cruzi replication remains to be determined. However, given its wide functional reach in mammalian cells, including a prominent role in glucose and lipid metabolism (Hay, 2011), it is likely that the influence of host Akt on intracellular T. cruzi replication is complex.

Several observations suggest that intracellular T. cruzi amastigotes benefit from a host cell metabolic environment that favors fatty acid oxidation over glucose oxidation. Acute silencing of PDK4, the main gatekeeper of the balance between glucose and fatty acid utilization, inhibits amastigote growth, whereas parasites exhibit a growth advantage in cells with PDH deficiency. Host genes involved in very long chain fatty acid oxidation in host peroxisomes were identified in the screen as influencing parasite growth and cells with defective mitochondrial fatty acid oxidation were less competent to support intracellular T. cruzi growth. Proteomic evidence shows that the amastigote form of the T. cruzi life cycle upregulates the capacity for fatty acid uptake and oxidization (Atwood et al., 2005) suggesting the natural coupling of T. cruzi growth to fatty acid metabolism in the host.

Along side functional insights emerging from our study, our observations suggest a level of plasticity in T. cruzi amastigotes populations in which intracellular parasite growth rates are responsive to environmental cues. While this is a highly intuitive concept – ie restricting ‘nutrient’ pools should slow intracellular parasite growth – our study provides experimental evidence that intracellular T. cruzi growth can be significantly altered (↑ or ↓) simply by perturbing host cellular functions. This flexibility is further exemplified by the differential ‘usage’ of host Akt-dependent pathways, both mTORC1-dependent and –independent, to fuel intracellular T. cruzi amastigote growth, exposing layers of redundancy with respect to host pathway utilization by the parasite. The capacity for adaptation would be especially relevant in the context of a dynamic natural infection in the mammalian host. Plasticity within parasite populations, reflected in an ability to alter growth rates to match particular metabolic microenvironments, may be a critical mechanism underlying tissue tropism and persistent infection. Selective persistence of T. cruzi in muscle and adipose tissue presents the possibility that cellular metabolism characteristic to these tissues, such as an increased reliance on fatty acid metabolism for energy production (Fritz, 1961), offers a selective advantage to the parasite enabling it to survive in the face of an otherwise punishing immune response.

Despite suggestions of flexibility and adaptation to changing environmental conditions, T. cruzi growth in mammalian cells is highly susceptible to targeted perturbations in host metabolic and signaling functions. As such, pharmacological targeting host metabolism to uncouple intracellular T. cruzi from its nutritional dependencies may prove an effective strategy for controlling pathogen spread. The concept of host-targeted therapies is not new for virologists (Ikeda and Kato, 2007; Schols, 2004) and is gaining traction for non-viral pathogens (Jayaswal et al., 2010; Kuijl et al., 2007). Drug discovery efforts for Chagas’ disease have focused primarily on the identification of target pathways in the parasite (Buckner and Navabi, 2010). However, with inherent heterogeneity in drug sensitivity among T. cruzi isolates/strains (eg. (Bustamante and Tarleton, 2011)), the consideration of host-targeted therapies for Chagas’ disease as an alternative to, or in combination with, anti-trypanosomal compounds, is clearly warranted. This study opens an opportunity in the Chagas’ disease field to exploit host genetics and small molecules to gain fundamental insights into the T. cruzi infection process with a view toward the identification of pharmacological targets in the parasite and the host.

EXPERIMENTAL PROCEDURES

siRNA Screen

HeLa cells were reverse transfected with arrayed siRNA SMARTpools (50nM) (Thermo Fisher Scientific, Lafayette, CO) complexed in Oligofectamine reagent (Invitrogen). At 48h post-transfection, cells were infected with T. cruzi m.o.i. 5 for 2h, followed by two washes with PBS, and incubation in DMEM containing 2%FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, and 2 mM L-glutamine (D2) (phenol-free) for a total of 72h. Each assay plate also included ketoconazole (15 µM) as a positive control for inhibition of T. cruzi growth, siRNA to polo-like kinase1 (PLK1) as a control for transfection and cell death, and non-targeting siControl (Dharmacon siRNA#2). The screen was performed in triplicate or duplicate according to plate content ie. library plates enriched in pseudogenes or genes of unknown function were screened in duplicate.

Multiplexed T. cruzi infection assay

Trypanosoma cruzi Tulahúen strain stably expressing β-galactosidase (generously provided by Fred Buckner, U. Washington) was maintained by weekly passage in LLcMK2 cells (ATCC) as described (Woolsey et al. 2003). Relative intracellular T. cruzi-β-galactosidase infection and host cell viability were measured in a multiplexed plate assay using CellTiterFluor™ (Cell Viability Assay; Promega, Madison, WI) and Beta-Glo™ reagent (Promega), per manufacturer’s protocol, measured on the EnVision Plate Reader (Perkin Elmer). The median absolute deviation (MAD) for the ratio of BetaGlo™/Cell Titer-Fluor™ (LUM/INTENS) values measured in each well for each screening plate was calculated. The MAD z-score [z = (x − m)/(MAD * K)] was calculated for each SMARTpool on the plate, where x is the LUM/INTENS for each well, m is the median LUM/INTENS of the plate, MAD is the median absolute deviation of the LUM/INTENS ratios on the plate, and K is the scale factor 1.4826. A SMARTpool was further considered if the MAD z-score was ≤ −1.75 or ≥2.5 and ≥40% host cell viability [%v = ((x − background)/ (plate ave − background))] of each well where x is the intensity value for the well, background is the average PLK1 intensity for the plate, and ‘plate ave’ is the average intensity for the plate. In assays where the plate components were not pinned at random, as in the cherry picked and secondary assays, the LUM/INTENS was compared to the LUM/INTENS of control siRNA2. A SMARTpool was considered a hit if the relative ratio ((LUM/INTENS)target siRNA/(LUM/INTENS)control siRNA) scored ≤ 0.66 and fulfilled the ≥40% cell viability cut-off established above.

Secondary Screens

For secondary screens, custom plates containing selected siRNA pools (purchased from Dharmacon; Thermo Fisher Scientific, Lafayette, CO) targeting annotated genes in pathways that emerged as hits in the primary or rescreening phases of the genome-wide screen. HeLa cells were transfected as above, with the exception that 25nM (final) siRNA was assayed per well and each siRNA pool was assayed in three independent T. cruzi infection experiment with ≥ two biological replicates per experiment. Mean LUM/INTENS values were determined across experiments for each siRNA SMARTpool which was considered a candidate ‘hit’ if the relative ratio scored ≤ 0.66 (comparing target to control siRNA, as above) and fulfilled the ≥40% host cell viability cutoff at 18 or 72 hpi. In addition to the multiplexed plate assay, a parallel group of plates was fixed in 4% para-formaldehyde/PBS, DAPI stained and image-based detection of intracellular parasites was collected using the In Cell 2000 High Content Analyzer (GE Healthcare). For the siRNA pools confirmed in the multiplexed plate readout as having a significant impact on intracellular T. cruzi growth, images were used to obtain data for the relative number of intracellular parasites/infected cell with manual confirmation. These distributions were compared to the non-targeting siRNA control group using the Mann-Whitney test. In some instances, binned distributions were plotted for clarity.

Pyruvate Dehydrogenase Activity

HeLa cells were reverse transfected with 25 nM siRNA in a 6 well plate. At 24h post-transfection media was exchanged for D2, and at 48h exchanged again for D2 (without pyruvate and phenol indicator). At 72h post-transfection cells were washed with PBS, and lysates collected in 80 µl of provided sample buffer containing Complete Mini – EDTA free protease inhibitor cocktail (Roche) on ice. Protein concentration in clarified lysates (3,000 rpm spin for 10 min) was measured using the DC Protein Assay (BioRad) and adjusted to the same concentration in 50 µl. Pyruvate dehydrogenase activity was measured in each sample with a PDH activity assay kit (Abcam) according to manufacturer’s instructions. Assays were read on an Epson photo quality scanner, and densitometry for each band was calculated using Alpha View software version 3.1.1.0 (Alpha Innotech).

Beta-Oxidation Assay

Transfected cells (as above) were loaded with 2 µCi 3H-palmitate (Perkin Elmer) for 2h in media containing 2% fatty acid-free BSA, 0.25 mM carnitine, and low glucose DMEM. Resulting 3H2O released from beta-oxidation into the supernatant was cleared of residual labeled palmitate/BSA through sequential extractions with 10% TCA, 5% TCA/ 10% BSA, and chloroform:methanol. Supernatant was collected, added to 5 ml of Ecolume scintillation fluid (MP Biomedicals), and radioactivity measured. All activity measurements were normalized to total protein quantified from cell lysates.

Biochemical supplementation

HeLa cells (20,000) were reverse transfected, as indicated above, with 25nM of control, TPRT, or GCH1 targeting siRNA (Dharmacon/Thermo Fisher Scientific, Lafayette, CO) and seeded onto 10 mm, round, coverslips (Electron Microscopy Sciences, Hatfield, PA). At 18h post-transfection, media was exchanged for D2 supplemented with CoenzymeQ10 (Sigma-Aldrich, St Louis, MO) (solubilized in ethanol to 5mM, diluted in FCS, then in serum-free DMEM to final concentrations of 5 µM CoenzymeQ10 and 2% FCS), 10 µM Uridine (Sigma-Aldrich, St Louis, MO), 1 mM N-acetylcysteine (NAC) (Sigma-Aldrich, St Louis, MO), 7,8 dihydro-L-biopterin (Caymen Chemicals, Ann Arbor, MI), or vehicle controls. At 48h post-transfection cells were infected with T. cruzi (m.o.i.=5) for 2h, washed twice to remove unattached parasites, and left in supplemented media. At 72h post-infection cells were rinsed with sterile PBS and fixed in 2% para-formaldehyde/PBS. Parasite and host nuclei were DAPI stained and parasites per infected cell were counted by fluorescent microscopy using an Eclipse TE300 (Nikon Instruments Inc., Melville, NY) under 60× magnification.

T. cruzi growth inhibition

Parasite-infected cells were treated at 18 hpi with AktVIIIi (EMD Chemicals, Cambridge, MA) or rapamycin (Tocris, Minneapolis, MN) and relative infection determined in a multiplexed plate assay as described above. Intracellular T. cruzi / infected cell was determined for a minimum of 100 cells following visualization/ counting of parasite and host cell nuclei with DAPI. The median number of intracellular parasites was compared to the corresponding untreated control using Kruskal-Wallis test followed by Dunn’s Multiple Comparison test.

T. cruzi amastigote proliferation assay

Freshly isolated T. cruzi trypomastigotes were labeled with 1.5 µM CFSE from the CellTrace™ CFSE Cell Proliferation Kit (Life technologies) per manufacturer’s protocol. Subconfluent monolayers of HFF were infected with CFSE-labeled parasites to achieve 1 parasite/infected cell for most infected cells prior to the first parasite replication event. Monolayers were washed extensively and cells harvested by scraping and 5 passages through a 26-guage needle to liberate the intracellular amastigotes at both 18 hpi (pre-replication) and 48 hpi (post-replication). Cellular debris was removed by centrifugation. Relative CFSE fluorescence was measured for ~10,000 parasites by LSRFortessa (BD Biosciences) and proliferation was plotted and analyzed by FloJo Data Analysis Software (Tree Star, Inc.).

Supplementary Material

Table S1
Table S2
Supplemental Material

HIGHLIGHTS.

  • Intracellular Trypanosoma cruzi couples its growth to host metabolism

  • Host energetics, pteridine, nucleoside and fatty acid metabolism fuel T. cruzi growth

  • Pharmacological targeting of host Akt blocks T. cruzi replication

ACKNOWLEDGEMENTS

We are indebted to the staff at the Institute of Chemistry and Cell Biology-Longwood Caroline Shamu, Jennifer Smith, David Wrobel, and Jennifer Nale for discussions and help with data analysis, and Stewart Rudnicki, Sean Johnston, and Doug Flood for assistance with library screening. We thank David Ndegwa (HSPH) for technical support, Jemila Kester (HSPH) for assistance with TPRT experiments, Yaneve Fonge (HSPH) for assistance with the Akt experiments and Steven Chen (UCSF-SMDC) for assistance with the IN Cell. We thank B. Manning and A. Toker for cell lines and helpful discussions and D. Wirth, B. Bloom, C. Tschudi, L. Passador and J. Gunawardena for critical reading of the manuscript. This work was supported by a Harvard Medical School Milton Fund award and National Institutes of Health grants AI090366 and AI099689 to B.A.B., NS067590 to J.C.E and DK075046 to C.-H.L. K.L.C. was supported by the Bayer Fund for Scholars in Infectious Diseases.

Footnotes

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SUPPLEMENTAL INFORMATION

Supplemental information including Extended Experimental Procedures, three figures, and two Tables can be found with this article online.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1
NIHMS430216-supplement-01.xlsx (3.6MB, xlsx)
Table S2
NIHMS430216-supplement-02.xls (5.1MB, xls)
Supplemental Material
NIHMS430216-supplement-Supplemental_Information.doc (1MB, doc)

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