The Experts below are selected from a list of 273 Experts worldwide ranked by ideXlab platform
Graham H. Coombs - One of the best experts on this subject based on the ideXlab platform.
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cysteine peptidases cpa and cpb are vital for autophagy and differentiation in leishmania mexicana
Molecular Microbiology, 2006Co-Authors: Roderick A M Williams, Laurence Tetley, Jeremy C Mottram, Graham H. CoombsAbstract:In the past, ultrastructural investigations of Leishmania mexicana Amastigotes revealed structures that were tentatively identified as autophagosomes. This study has now provided definitive data that autophagy occurs in the parasite during differentiation both to metacyclic promastigotes and to Amastigotes, autophagosomes being particularly numerous during metacyclic to amastigote form transformation. Moreover, the results demonstrate that inhibiting two major lysosomal cysteine peptidases (CPA and CPB) or removing their genes not only interferes with the autophagy pathway but also prevents metacyclogenesis and transformation to Amastigotes, thus adding support to the hypothesis that autophagy is required for cell differentiation. The study suggests that L. mexicana CPA and CPB perform similar roles to the aspartic peptidase PEP4 and the serine peptidase PRB1 in Saccharomyces cerevisiae. The results also provide an explanation for why L. mexicana CPA/CPB-deficient mutants transform to Amastigotes very poorly and lack virulence in macrophages and mice.
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inhibition of lipopolysaccharide induced macrophage il 12 production by leishmania mexicana Amastigotes the role of cysteine peptidases and the nf kappab signaling pathway
Journal of Immunology, 2004Co-Authors: Pamela Cameron, Graham H. Coombs, Adrienne Mcgachy, Mary Anderson, Andrew Paul, Jeremy C Mottram, James Alexander, Robin PlevinAbstract:Infection with lesion-derived Leishmania mexicana Amastigotes inhibited LPS-induced IL-12 production by mouse bone marrow-derived macrophages. This effect was associated with expression of cysteine peptidase B (CPB) because Amastigotes of CPB deletion mutants had limited ability to inhibit IL-12 production, whereas preincubation of cells with a CPB inhibitor, cathepsin inhibitor IV, was able to suppress the effect of wild-type Amastigotes. Infection with wild-type Amastigotes resulted in a time-dependent proteolytic degradation of IκBα and IκBβ and the related protein NF-κB. This effect did not occur with Amastigotes of CPB deletion mutants or wild-type promastigotes, which do not express detectable CPB. NF-κB DNA binding was also inhibited by amastigote infection, although nuclear translocation of cleaved fragments of p65 NF-κB was still observed. Cysteine peptidase inhibitors prevented IκBα, IκBβ, and NF-κB degradation induced by Amastigotes, and recombinant CPB2.8, an amastigote-specific isoenzyme of CPB, was shown to degrade GST-IκBα in vitro. LPS-mediated IκBα and IκBβ degradation was not affected by these inhibitors, confirming that the site of degradation of IκBα, IκBβ, and NF-κB by the Amastigotes was not receptor-driven, proteosomal-mediated cleavage. Infection of bone marrow macrophages with Amastigotes resulted in cleavage of JNK and ERK, but not p38 MAPK, whereas preincubation with a cysteine peptidase inhibitor prevented degradation of these proteins, but did not result in enhanced protein kinase activation. Collectively, our results suggest that the amastigote-specific cysteine peptidases of L. mexicana are central to the ability of the parasite to modulate signaling via NF-κB and consequently inhibit IL-12 production.
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axenic cultivation and characterization of leishmania mexicana amastigote like forms
Parasitology, 1992Co-Authors: Paul A Bates, Laurence Tetley, Colin D Robertson, Graham H. CoombsAbstract:banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion Amastigotes. The axenically cultured forms grew optimally at a temperature of 32-33 °C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5-4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial Amastigotes are acidophiles.
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Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms.
Parasitology, 1992Co-Authors: Paul A Bates, Laurence Tetley, Colin D Robertson, Graham H. CoombsAbstract:A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigote-like forms in Schneider's Drosophila medium supplemented with 20% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion Amastigotes. The axenically cultured forms grew optimally at a temperature of 32-33 degrees C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5.4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial Amastigotes are acidophiles.
Barbara A Burleigh - One of the best experts on this subject based on the ideXlab platform.
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glutamine metabolism modulates azole susceptibility in trypanosoma cruzi Amastigotes
bioRxiv, 2020Co-Authors: Peter C Dumoulin, Joshua Vollrath, Jennifer X Wang, Barbara A BurleighAbstract:Abstract The mechanisms underlying resistance of the Chagas disease parasite, Trypanosoma cruzi, to current therapies are not well understood, including the potential role of metabolic heterogeneity in modulating susceptibility of intracellular Amastigotes to trypanocidal compounds. 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. Consistent with a model that decreased flux through the sterol biosynthetic pathway is protective for intracellular Amastigotes exposed to azoles, we find that Amastigotes become re-sensitized to azoles following addition of metabolites upstream of CYP51. Our results highlight the potential role of metabolic heterogeneity in recalcitrant T. cruzi infection, an avenue that is currently underexplored.
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methods for the investigation of trypanosoma cruzi amastigote proliferation in mammalian host cells
Methods of Molecular Biology, 2020Co-Authors: Peter C Dumoulin, Barbara A BurleighAbstract: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.
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stress induced proliferation and cell cycle plasticity of intracellular trypanosoma cruzi Amastigotes
Mbio, 2018Co-Authors: Peter C Dumoulin, Barbara A BurleighAbstract:ABSTRACT 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.
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modulation of host central carbon metabolism and in situ glucose uptake by intracellular trypanosoma cruzi Amastigotes
PLOS Pathogens, 2017Co-Authors: Sheena Shahsimpson, Peter C Dumoulin, Gaelle Lentini, Barbara A BurleighAbstract: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.
Vicente Larraga - One of the best experts on this subject based on the ideXlab platform.
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Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum
BMC Genomics, 2010Co-Authors: Pedro J Alcolea, Ana Alonso, Manuel J Gómez, Alicia Sánchez-gorostiaga, Mercedes Moreno-paz, Eduardo González-pastor, Alfredo Toraño, Víctor Parro, Vicente LarragaAbstract:Background The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania . Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to Amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived Amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome. Results Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings. Conclusions The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology.
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temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in leishmania infantum
BMC Genomics, 2010Co-Authors: Pedro J Alcolea, Ana Alonso, Manuel J Gómez, Alfredo Toraño, Víctor Parro, Alicia Sanchezgorostiaga, Mercedes Morenopaz, Eduardo Gonzalezpastor, Vicente LarragaAbstract:The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to Amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived Amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome. Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings. The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology.
Lynn Soong - One of the best experts on this subject based on the ideXlab platform.
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interactions between neutrophils and leishmania braziliensis Amastigotes facilitate cell activation and parasite clearance
Journal of Innate Immunity, 2015Co-Authors: Eric D Carlsen, Calvin A Henard, Zuliang Jie, Yuejin Liang, Christie Hay, Jiaren Sun, Herbert De Matos Guedes, Lynn SoongAbstract:Leishmania braziliensis and Leishmania amazonensis are both causative agents of cutaneous leishmaniasis in South America. However, patient prognosis and the host immune response differ considerably depending on the infecting parasite species. The mechanisms underlying these differences appear to be multifactorial, with both host and parasite components contributing to disease outcome. As neutrophils are a prominent component of the inflammatory infiltrate in chronic cutaneous, diffuse cutaneous and mucocutaneous lesions, we examined neutrophil activation and microbicidal activity against Amastigotes of L. amazonensis and L. braziliensis. We found that murine neutrophils internalized L. braziliensis Amastigotes with greater efficiency than did L. amazonensis Amastigotes. Additionally, L. braziliensis infection was a potent trigger for neutrophil activation, oxidative burst, degranulation and the production of interleukin (IL)-22 and IL-10, while L. amazonensis Amastigotes poorly induced these responses. Finally, neutrophils were able to kill L. braziliensis Amastigotes, especially when cells were activated with phorbol myristate acetate. L. amazonensis Amastigotes, however, were highly resistant to neutrophil microbicidal mechanisms. This study reveals, for the first time, differential neutrophil responsiveness to distinct species of Leishmania Amastigotes and highlights the complexity of neutrophil-amastigote interactions during chronic leishmaniasis.
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leishmania amazonensis Amastigotes trigger neutrophil activation but resist neutrophil microbicidal mechanisms
Infection and Immunity, 2013Co-Authors: Eric D Carlsen, Calvin A Henard, Nisha Jain Garg, Vsevolod L. Popov, Lynn SoongAbstract:Neutrophils are thefirst cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensismetacyclic promastigotes and Amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-), uptake of Amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived Amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Ourfindings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis.
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enhanced replication of leishmania amazonensis Amastigotes in gamma interferon stimulated murine macrophages implications for the pathogenesis of cutaneous leishmaniasis
Infection and Immunity, 2004Co-Authors: Hai Qi, Jiaxiang Ji, Nanchaya Wanasen, Lynn SoongAbstract:During Leishmania major infection in mice, gamma interferon (IFN-γ) plays an essential role in controlling parasite growth and disease progression. In studies designed to ascertain the role of IFN-γ in Leishmania amazonensis infection, we were surprised to find that IFN-γ could promote L. amazonensis amastigote replication in macrophages (MΦs), although it activated MΦs to kill promastigotes. The replication-promoting effect of IFN-γ on Amastigotes was independent of the source and genetic background of MΦs, was apparently not affected by surface opsonization of Amastigotes, was not mediated by interleukin-10 or transforming growth factor β, and was observed at different temperatures. Consistent with the different fates of promastigotes and Amastigotes in IFN-γ-stimulated MΦs, L. amazonensis-specific Th1 transfer helped recipient mice control L. amazonensis infection established by promastigotes but not L. amazonensis infection established by Amastigotes. On the other hand, IFN-γ could stimulate MΦs to limit amastigote replication when it was coupled with lipopolysaccharides but not when it was coupled with tumor necrosis factor alpha. Thus, IFN-γ may play a bidirectional role at the level of parasite-MΦ interactions; when it is optimally coupled with other factors, it has a protective effect against infection, and in the absence of such synergy it promotes amastigote growth. These results reveal a quite unexpected aspect of the L. amazonensis parasite and have important implications for understanding the pathogenesis of the disease and for developing vaccines and immunotherapies.
Peter C Dumoulin - One of the best experts on this subject based on the ideXlab platform.
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glutamine metabolism modulates azole susceptibility in trypanosoma cruzi Amastigotes
bioRxiv, 2020Co-Authors: Peter C Dumoulin, Joshua Vollrath, Jennifer X Wang, Barbara A BurleighAbstract:Abstract The mechanisms underlying resistance of the Chagas disease parasite, Trypanosoma cruzi, to current therapies are not well understood, including the potential role of metabolic heterogeneity in modulating susceptibility of intracellular Amastigotes to trypanocidal compounds. 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. Consistent with a model that decreased flux through the sterol biosynthetic pathway is protective for intracellular Amastigotes exposed to azoles, we find that Amastigotes become re-sensitized to azoles following addition of metabolites upstream of CYP51. Our results highlight the potential role of metabolic heterogeneity in recalcitrant T. cruzi infection, an avenue that is currently underexplored.
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methods for the investigation of trypanosoma cruzi amastigote proliferation in mammalian host cells
Methods of Molecular Biology, 2020Co-Authors: Peter C Dumoulin, Barbara A BurleighAbstract: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.
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stress induced proliferation and cell cycle plasticity of intracellular trypanosoma cruzi Amastigotes
Mbio, 2018Co-Authors: Peter C Dumoulin, Barbara A BurleighAbstract:ABSTRACT 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.
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modulation of host central carbon metabolism and in situ glucose uptake by intracellular trypanosoma cruzi Amastigotes
PLOS Pathogens, 2017Co-Authors: Sheena Shahsimpson, Peter C Dumoulin, Gaelle Lentini, Barbara A BurleighAbstract: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.