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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.
Anil K. Rastogi - One of the best experts on this subject based on the ideXlab platform.
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In vitro cultivation and characterization of axenic Amastigotes of Leishmania.
Trends in parasitology, 2001Co-Authors: Nidhi Gupta, Neena Goyal, Anil K. RastogiAbstract:The establishment of axenic cultures of the Amastigote stage of Leishmania is important to understand the mechanisms regulating the differentiation, survival and pathogenicity of the parasite with a view to develop and identify molecular and chemotherapeutic targets. Recent developments in axenic culture and the characterization of Amastigotes of different species of Leishmania are discussed.
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Characterization of intracellular metabolites of axenic Amastigotes of Leishmania donovani by 1H NMR spectroscopy.
Acta tropica, 1999Co-Authors: Nidhi Gupta, Neena Goyal, U.k. Singha, Vinod Bhakuni, Raja Roy, Anil K. RastogiAbstract:The intracellular metabolites of long-term in vitro cultured axenic Amastigotes of Leishmania donovani (strain Dd8) were determined and compared with those of promastigotes and intracellular Amastigotes, employing proton NMR spectroscopy. The presence of two new metabolites, i.e. betaine and beta-hydroxybutyrate were reported. Betaine was detected in all the three stages being highest in the promastigotes while beta-hydroxybutyrate could be detected only in promastigotes and axenic Amastigotes. Among other metabolites, succinate and valine were found in higher quantities in intracellular Amastigotes and axenic Amastigotes than in promastigotes. Acetoacetate was present only in axenic and intracellular Amastigotes. The comparative metabolite profile of different parasite forms reveals that axenic Amastigotes seem to represent an intermediate stage between promastigotes and intracellular Amastigotes in spite of their strong resemblance to intracellular Amastigotes in morphology, infectivity, biochemical studies and even in the manifestation of Amastigote specific A2 protein.
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Membrane characterization of Amastigote-like forms of Leishmania donovani.
Tropical medicine & international health : TM & IH, 1996Co-Authors: Nidhi Gupta, Neena Goyal, Raj Kumar, Ashok K. Agrawal, Prahlad K. Seth, Anil K. RastogiAbstract:During in vitro transformation, Leishmania donovani promastigotes converted into Amastigote-like forms and underwent several changes in membrane parameters. They exhibited significantly increased microviscosity comparable to true Amastigotes. Activities of several functionally important membrane bound enzymes were also altered, thereby indicating a change in their orientation. Peanut agglutinin was found to be specific for agglutination of stationary phase promastigotes whereas wheat-germ agglutinin was specific for the Amastigote-like forms as well as for pure Amastigotes, implying the presence of specific glycoconjugates on the parasite surface.
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.
-
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.
Pedro J Alcolea - 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.