Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 30
Filtrar
Mais filtros

País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35074914

RESUMO

Catabolism of sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose), the ubiquitous sulfosugar produced by photosynthetic organisms, is an important component of the biogeochemical carbon and sulfur cycles. Here, we describe a pathway for SQ degradation that involves oxidative desulfurization to release sulfite and enable utilization of the entire carbon skeleton of the sugar to support the growth of the plant pathogen Agrobacterium tumefaciens SQ or its glycoside sulfoquinovosyl glycerol are imported into the cell by an ATP-binding cassette transporter system with an associated SQ binding protein. A sulfoquinovosidase hydrolyzes the SQ glycoside and the liberated SQ is acted on by a flavin mononucleotide-dependent sulfoquinovose monooxygenase, in concert with an NADH-dependent flavin reductase, to release sulfite and 6-oxo-glucose. An NAD(P)H-dependent oxidoreductase reduces the 6-oxo-glucose to glucose, enabling entry into primary metabolic pathways. Structural and biochemical studies provide detailed insights into the recognition of key metabolites by proteins in this pathway. Bioinformatic analyses reveal that the sulfoquinovose monooxygenase pathway is distributed across Alpha- and Betaproteobacteria and is especially prevalent within the Rhizobiales order. This strategy for SQ catabolism is distinct from previously described pathways because it enables the complete utilization of all carbons within SQ by a single organism with concomitant production of inorganic sulfite.


Assuntos
Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Redes e Vias Metabólicas , Metilglucosídeos/metabolismo , Estresse Oxidativo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Metabolismo dos Carboidratos , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Enxofre/metabolismo
2.
J Infect Dis ; 228(11): 1482-1490, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37804520

RESUMO

BACKGROUND: Two crowdsourcing open calls were created to enhance community engagement in dengue control in Sri Lanka. We analyzed the process and outcomes of these digital crowdsourcing open calls. METHODS: We used standard World Health Organization methods to organize the open calls, which used exclusively digital methods because of coronavirus disease 2019 (COVID-19). We collected and analyzed sociodemographic information and digital engagement metrics from each submission. Submissions in the form of textual data describing community-led strategies for mosquito release were coded using grounded theory. RESULTS: The open calls received 73 submissions. Most people who submitted ideas spoke English, lived in Sri Lanka, and were 18 to 34 years old. The total Facebook reach was initially limited (16 161 impressions), prompting expansion to a global campaign, which reached 346 810 impressions over 14 days. Diverse strategies for the distribution of Wolbachia-infected mosquito boxes were identified, including leveraging traditional festivals, schools, and community networks. Fifteen submissions (21%) suggested the use of digital tools for monitoring and evaluation, sharing instructions, or creating networks. Thirteen submissions (18%) focused on social and economic incentives to prompt community engagement and catalyze community-led distribution. CONCLUSIONS: Our project demonstrates that digital crowdsourcing open calls are an effective way to solicit creative and innovative ideas in a resource-limited setting.


Assuntos
Crowdsourcing , Culicidae , Dengue , Animais , Humanos , Adolescente , Adulto Jovem , Adulto , Crowdsourcing/métodos , Sri Lanka/epidemiologia , Participação da Comunidade , Dengue/epidemiologia , Dengue/prevenção & controle , Controle de Mosquitos
3.
Appl Environ Microbiol ; 89(2): e0201622, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36728421

RESUMO

Sulfoquinovose (SQ) is a major metabolite in the global sulfur cycle produced by nearly all photosynthetic organisms. One of the major pathways involved in the catabolism of SQ in bacteria such as Escherichia coli is a variant of the glycolytic Embden-Meyerhof-Parnas (EMP) pathway termed the sulfoglycolytic EMP (sulfo-EMP) pathway, which leads to the consumption of three of the six carbons of SQ and the excretion of 2,3-dihydroxypropanesulfonate (DHPS). Comparative metabolite profiling of aerobically glucose (Glc)-grown and SQ-grown E. coli cells was undertaken to identify the metabolic consequences of the switch from glycolysis to sulfoglycolysis. Sulfoglycolysis was associated with the diversion of triose phosphates (triose-P) to synthesize sugar phosphates (gluconeogenesis) and an unexpected accumulation of trehalose and glycogen storage carbohydrates. Sulfoglycolysis was also associated with global changes in central carbon metabolism, as indicated by the changes in the levels of intermediates in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), polyamine metabolism, pyrimidine metabolism, and many amino acid metabolic pathways. Upon entry into stationary phase and the depletion of SQ, E. coli cells utilize their glycogen, indicating a reversal of metabolic fluxes to allow glycolytic metabolism. IMPORTANCE The sulfosugar sulfoquinovose is estimated to be produced on a scale of 10 billion metric tons per annum, making it a major organosulfur species in the biosulfur cycle. The microbial degradation of sulfoquinovose through sulfoglycolysis allows the utilization of its carbon content and contributes to the biomineralization of its sulfur. However, the metabolic consequences of microbial growth on sulfoquinovose are unclear. We use metabolomics to identify the metabolic adaptations that Escherichia coli undergoes when grown on sulfoquinovose versus glucose. This revealed the increased flux into storage carbohydrates through gluconeogenesis and the reduced flux of carbon into the TCA cycle and downstream metabolism. These changes are relieved upon entry into stationary phase and reversion to glycolytic metabolism. This work provides new insights into the metabolic consequences of microbial growth on an abundant sulfosugar.


Assuntos
Carbono , Escherichia coli , Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicólise , Glucose/metabolismo , Glicogênio/metabolismo , Trioses/metabolismo , Enxofre/metabolismo
4.
Immunol Cell Biol ; 98(10): 832-844, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32780446

RESUMO

Leishmania are parasitic protists that cause a spectrum of diseases in humans characterized by the formation of granulomatous lesions in the skin or other tissues, such as liver and spleen. The extent to which Leishmania granulomas constrain or promote parasite growth is critically dependent on the host T-helper type 1/T-helper type 2 immune response and the localized functional polarization of infected and noninfected macrophages toward a classically (M1) or alternatively (M2) activated phenotype. Recent studies have shown that metabolic reprograming of M1 and M2 macrophages underpins the capacity of these cells to act as permissive or nonpermissive host reservoirs, respectively. In this review, we highlight the metabolic requirements of Leishmania amastigotes and the evidence that these parasites induce and/or exploit metabolic reprogramming of macrophage metabolism. We also focus on recent studies highlighting the role of key macrophage metabolic signaling pathways, such as mechanistic target of rapamycin, adenosine monophosphate-activated protein kinase and peroxisome proliferator receptor gamma in regulating the pathological progression of Leishmania granulomas. These studies highlight the intimate connectivity between Leishmania and host cell metabolism, the need to investigate these interactions in vivo and the potential to exploit host cell metabolic signaling pathways in developing new host-directed therapies.


Assuntos
Reprogramação Celular , Granuloma , Leishmania , Macrófagos , Granuloma/parasitologia , Humanos , Leishmania/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Transdução de Sinais
5.
Mol Microbiol ; 108(2): 143-158, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29411460

RESUMO

Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L. mexicana Δlmxgt1-3 mutant which lacks the major glucose transporters LmxGT1-3. We identified a novel suppressor line (Δlmxgt1-3s2 ) that has restored growth in rich culture medium and virulence in ex vivo infected macrophages, but failed to induce lesions in mice. Δlmxgt1-3s2 amastigotes had lower rates of glucose utilization than the parental line and primarily catabolized non-essential amino acids. The increased mitochondrial metabolism of this line was associated with elevated levels of intracellular reactive oxygen species, as well as increased sensitivity to inhibitors of the tricarboxylic acid (TCA) cycle, including nitric oxide. These results suggest that hardwired sugar addiction of Leishmania amastigotes contributes to the intrinsic resistance of this stage to macrophage microbicidal processes in vivo, and that these stages have limited capacity to switch to using other carbon sources.


Assuntos
Aminoácidos/metabolismo , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/parasitologia , Macrófagos/parasitologia , Animais , Carbono/metabolismo , Ciclo do Ácido Cítrico , Modelos Animais de Doenças , Feminino , Glucose/metabolismo , Humanos , Leishmania mexicana/genética , Leishmania mexicana/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Virulência
6.
PLoS Pathog ; 11(2): e1004683, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25714830

RESUMO

Information on the growth rate and metabolism of microbial pathogens that cause long-term chronic infections is limited, reflecting the absence of suitable tools for measuring these parameters in vivo. Here, we have measured the replication and physiological state of Leishmania mexicana parasites in murine inflammatory lesions using 2H2O labeling. Infected BALB/c mice were labeled with 2H2O for up to 4 months, and the turnover of parasite DNA, RNA, protein and membrane lipids estimated from the rate of deuterium enrichment in constituent pentose sugars, amino acids, and fatty acids, respectively. We show that the replication rate of parasite stages in these tissues is very slow (doubling time of ~12 days), but remarkably constant throughout lesion development. Lesion parasites also exhibit markedly lower rates of RNA synthesis, protein turnover and membrane lipid synthesis than parasite stages isolated from ex vivo infected macrophages or cultured in vitro, suggesting that formation of lesions induces parasites to enter a semi-quiescent physiological state. Significantly, the determined parasite growth rate accounts for the overall increase in parasite burden indicating that parasite death and turnover of infected host cells in these lesions is minimal. We propose that the Leishmania response to lesion formation is an important adaptive strategy that minimizes macrophage activation, providing a permissive environment that supports progressive expansion of parasite burden. This labeling approach can be used to measure the dynamics of other host-microbe interactions in situ.


Assuntos
Óxido de Deutério , Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/patologia , Coloração e Rotulagem/métodos , Animais , Células Cultivadas , Modelos Animais de Doenças , Interações Hospedeiro-Parasita , Leishmania mexicana/metabolismo , Leishmania mexicana/fisiologia , Leishmaniose Cutânea/parasitologia , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C
7.
PLoS Pathog ; 11(9): e1005136, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26334531

RESUMO

Leishmania parasites replicate within the phagolysosome compartment of mammalian macrophages. Although Leishmania depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient Leishmania major mutant (∆gnat) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in ex vivo infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in ex vivo infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites.


Assuntos
Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Interações Hospedeiro-Parasita , Leishmania major/fisiologia , Lisossomos/parasitologia , Macrófagos/parasitologia , Fagocitose , Acetilglucosamina/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Matriz Extracelular/imunologia , Matriz Extracelular/patologia , Deleção de Genes , Hidrólise , Cinética , Leishmania major/genética , Leishmania major/crescimento & desenvolvimento , Leishmania major/imunologia , Leishmania mexicana/genética , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/imunologia , Leishmania mexicana/fisiologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Lisossomos/imunologia , Lisossomos/metabolismo , Lisossomos/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Especificidade da Espécie , Organismos Livres de Patógenos Específicos
9.
PLoS Pathog ; 10(1): e1003888, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24465208

RESUMO

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using (13)C-stable isotope resolved metabolomics and (2)H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid ß-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.


Assuntos
Aminoácidos/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Glucose/metabolismo , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/metabolismo , Mitocôndrias/metabolismo , Aminoácidos/genética , Animais , Feminino , Glucose/genética , Leishmania mexicana/genética , Leishmaniose Cutânea/genética , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/genética
10.
Eur J Med Chem ; 269: 116256, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38461679

RESUMO

Visceral leishmaniasis is a potentially fatal disease caused by infection by the intracellular protist pathogens Leishmania donovani or Leishmania infantum. Present therapies are ineffective because of high costs, variable efficacy against different species, the requirement for hospitalization, toxicity and drug resistance. Detailed analysis of previously published hit molecules suggested a crucial role of 'guanidine' linkage for their efficacy against L. donovani. Here we report the design of 2-aminoquinazoline heterocycle as a basic pharmacophore-bearing guanidine linkage. The introduction of various groups and functionality at different positions of the quinazoline scaffold results in enhanced antiparasitic potency with modest host cell cytotoxicity using a physiologically relevant THP-1 transformed macrophage infection model. In terms of the ADME profile, the C7 position of quinazoline was identified as a guiding tool for designing better molecules. The good ADME profile of the compounds suggests that they merit further consideration as lead compounds for treating visceral leishmaniasis.


Assuntos
Leishmania donovani , Leishmania infantum , Leishmaniose Visceral , Humanos , Leishmaniose Visceral/tratamento farmacológico , Antiparasitários/farmacologia , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico
11.
J Biol Chem ; 286(31): 27706-17, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21636575

RESUMO

Leishmania parasites proliferate within nutritionally complex niches in their sandfly vector and mammalian hosts. However, the extent to which these parasites utilize different carbon sources remains poorly defined. In this study, we have followed the incorporation of various (13)C-labeled carbon sources into the intracellular and secreted metabolites of Leishmania mexicana promastigotes using gas chromatography-mass spectrometry and (13)C NMR. [U-(13)C]Glucose was rapidly incorporated into intermediates in glycolysis, the pentose phosphate pathway, and the cytoplasmic carbohydrate reserve material, mannogen. Enzymes involved in the upper glycolytic pathway are sequestered within glycosomes, and the ATP and NAD(+) consumed by these reactions were primarily regenerated by the fermentation of phosphoenolpyruvate to succinate (glycosomal succinate fermentation). The initiating enzyme in this pathway, phosphoenolpyruvate carboxykinase, was exclusively localized to the glycosome. Although some of the glycosomal succinate was secreted, most of the C4 dicarboxylic acids generated during succinate fermentation were further catabolized in the TCA cycle. A high rate of TCA cycle anaplerosis was further suggested by measurement of [U-(13)C]aspartate and [U-(13)C]alanine uptake and catabolism. TCA cycle anaplerosis is apparently needed to sustain glutamate production under standard culture conditions. Specifically, inhibition of mitochondrial aconitase with sodium fluoroacetate resulted in the rapid depletion of intracellular glutamate pools and growth arrest. Addition of high concentrations of exogenous glutamate alleviated this growth arrest. These findings suggest that glycosomal and mitochondrial metabolism in Leishmania promastigotes is tightly coupled and that, in contrast to the situation in some other trypanosomatid parasites, the TCA cycle has crucial anabolic functions.


Assuntos
Ácido Aspártico/metabolismo , Ciclo do Ácido Cítrico , Ácido Glutâmico/biossíntese , Leishmania mexicana/metabolismo , Ácido Succínico/metabolismo , Animais , Sequência de Bases , Carbono/metabolismo , Primers do DNA , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Leishmania mexicana/genética , Leishmania mexicana/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética
12.
Eur J Med Chem ; 240: 114577, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-35810535

RESUMO

Visceral leishmaniasis is a potentially fatal disease caused by the parasitic protists, Leishmania donovani and L. infantum. Current treatments remain unsuitable due to cost, the need for hospitalization, variable efficacy against different species, toxicity and emerging resistance. Herein, we report the SAR exploration of the novel hit 4-Fluoro-N-(5-(4-methoxyphenyl)-1-methyl-1H-imidazole-2-yl)benzamide [1] previously identified from a high throughput screen against Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani. An extensive and informative set of analogues were synthesized incorporating key modifications around the scaffold resulting in improved potency, whilst the majority of compounds maintained low cytotoxicity against human THP-1 macrophages that are target cells for these pathogens. New lead compounds identified within this study also maintained desirable physicochemical properties, improved metabolic stability in vitro and displayed no significant mitotoxicity against HepG2 cell lines. This compound class warrants continued investigation towards development as a novel treatment for Visceral Leishmaniasis.


Assuntos
Antiprotozoários , Leishmania donovani , Leishmaniose Visceral , Trypanosoma cruzi , Antiprotozoários/química , Humanos , Imidazóis/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico
13.
Front Immunol ; 13: 926446, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36189274

RESUMO

Mucosal-associated invariant T (MAIT) cells are restricted by MR1 and are known to protect against bacterial and viral infections. Our understanding of the role of MAIT cells in parasitic infections, such as visceral leishmaniasis (VL) caused by protozoan parasites of Leishmania donovani, is limited. This study showed that in response to L. infantum, human peripheral blood MAIT cells from children with leishmaniasis produced TNF and IFN-γ in an MR1-dependent manner. The overall frequency of MAIT cells was inversely correlated with alanine aminotransferase levels, a specific marker of liver damage strongly associated with severe hepatic involvement in VL. In addition, there was a positive correlation between total protein levels and the frequency of IL-17A+ CD8+ MAIT cells, whereby reduced total protein levels are a marker of liver and kidney damage. Furthermore, the frequencies of IFN-γ+ and IL-10+ MAIT cells were inversely correlated with hemoglobin levels, a marker of severe anemia. In asymptomatic individuals and VL patients after treatment, MAIT cells also produced IL-17A, a cytokine signature associated with resistance to visceral leishmaniasis, suggesting that MAIT cells play important role in protecting against VL. In summary, these results broaden our understanding of MAIT-cell immunity to include protection against parasitic infections, with implications for MAIT-cell-based therapeutics and vaccines. At last, this study paves the way for the investigation of putative MAIT cell antigens that could exist in the context of Leishmania infection.


Assuntos
Leishmaniose Visceral , Células T Invariantes Associadas à Mucosa , Alanina Transaminase , Criança , Citocinas , Hemoglobinas , Humanos , Interleucina-10 , Interleucina-17
14.
Curr Opin Microbiol ; 63: 126-132, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34340099

RESUMO

Leishmania are unusual in being able to survive long-term in the mature phagolysosome compartment of macrophages and other phagocytic cells in their mammalian hosts. Key to their survival in this niche, Leishmania amastigotes switch to a slow growth state and activate a stringent metabolic response. The stringent metabolic response may be triggered by multiple stresses and is associated with decreased metabolic fluxes, restricted use of sugars and fatty acids as carbon sources and increased dependence on metabolic homeostasis pathways. Heterogeneity in expression of the Leishmania stringent response occurs in vivo reflects temporal and spatial heterogeneity in lesion tissues and includes non-dividing dormant stages. This response underpins the capacity of these parasites to maintain long-term chronic infections and survive drug treatments.


Assuntos
Leishmania , Parasitos , Animais , Ácidos Graxos , Leishmania/genética , Macrófagos , Fagossomos
15.
mBio ; 12(2)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824211

RESUMO

Leishmania are sandfly-transmitted protists that induce granulomatous lesions in their mammalian host. Although infected host cells in these tissues can exist in different activation states, the extent to which intracellular parasites stages also exist in different growth or physiological states remains poorly defined. Here, we have mapped the spatial distribution of metabolically quiescent and active subpopulations of Leishmania mexicana in dermal granulomas in susceptible BALB/c mice, using in vivo heavy water labeling and ultra high-resolution imaging mass spectrometry. Quantitation of the rate of turnover of parasite and host-specific lipids at high spatial resolution, suggested that the granuloma core comprised mixed populations of metabolically active and quiescent parasites. Unexpectedly, a significant population of metabolically quiescent parasites was also identified in the surrounding collagen-rich, dermal mesothelium. Mesothelium-like tissues harboring quiescent parasites progressively replaced macrophage-rich granuloma tissues following treatment with the first-line drug, miltefosine. In contrast to the granulomatous tissue, neither the mesothelium nor newly deposited tissue sequestered miltefosine. These studies suggest that the presence of quiescent parasites in acute granulomatous tissues, together with the lack of miltefosine accumulation in cured lesion tissue, may contribute to drug failure and nonsterile cure.IMPORTANCE Many microbial pathogens switch between different growth and physiological states in vivo in order to adapt to local nutrient levels and host microbicidal responses. Heterogeneity in microbial growth and metabolism may also contribute to nongenetic mechanisms of drug resistance and drug failure. In this study, we have developed a new approach for measuring spatial heterogeneity in microbial metabolism in vivo using a combination of heavy water (2H2O) labeling and imaging mass spectrometry. Using this approach, we show that lesions contain a patchwork of metabolically distinct parasite populations, while the underlying dermal tissues contain a large population of metabolically quiescent parasites. Quiescent parasites also dominate drug-depleted tissues in healed animals, providing an explanation for failure of some first line drugs to completely eradicate parasites. This approach is broadly applicable to study the metabolic and growth dynamics in other host-pathogen interactions.


Assuntos
Óxido de Deutério , Granuloma/parasitologia , Interações Hospedeiro-Parasita , Processamento de Imagem Assistida por Computador/métodos , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/parasitologia , Espectrometria de Massas/métodos , Pele/patologia , Animais , Modelos Animais de Doenças , Feminino , Marcação por Isótopo , Leishmaniose Cutânea/patologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Músculos/parasitologia , Músculos/patologia , Pele/parasitologia
16.
Parasitology ; 137(9): 1303-13, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20158936

RESUMO

Leishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in humans. Many enzymes involved in Leishmania central carbon metabolism differ from their equivalents in the mammalian host and are potential drug targets. In this review we summarize recent advances in our understanding of Leishmania central carbon metabolism, focusing on pathways of carbon utilization that are required for growth and pathogenesis in the mammalian host. While Leishmania central carbon metabolism shares many features in common with other pathogenic trypanosomatids, significant differences are also apparent. Leishmania parasites are also unusual in constitutively expressing most core metabolic pathways throughout their life cycle, a feature that may allow these parasites to exploit a range of different carbon sources (primarily sugars and amino acids) rapidly in both the insect vector and vertebrate host. Indeed, recent gene deletion studies suggest that mammal-infective stages are dependent on multiple carbon sources in vivo. The application of metabolomic approaches, outlined here, are likely to be important in defining aspects of central carbon metabolism that are essential at different stages of mammalian host infection.


Assuntos
Carbono/metabolismo , Leishmania/metabolismo , Leishmaniose/parasitologia , Animais , Metabolismo dos Carboidratos , Interações Hospedeiro-Parasita , Humanos , Espaço Intracelular/metabolismo , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Mitocôndrias/metabolismo , Parasitos/metabolismo
17.
mBio ; 11(3)2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32487758

RESUMO

Leishmania spp. are protozoan parasites that cause a spectrum of important diseases in humans. These parasites develop as extracellular promastigotes in the digestive tract of their insect vectors and as obligate intracellular amastigotes that infect macrophages and other phagocytic cells in their vertebrate hosts. Promastigote-to-amastigote differentiation is associated with marked changes in metabolism, including the upregulation of enzymes involved in fatty acid ß-oxidation, which may reflect adaptation to the intracellular niche. Here, we have investigated the function of one of these enzymes, a putative 2,4-dienoyl-coenzyme A (CoA) reductase (DECR), which is specifically required for the ß-oxidation of polyunsaturated fatty acids. The Leishmania DECR shows close homology to bacterial DECR proteins, suggesting that it was acquired by lateral gene transfer. It is present in other trypanosomatids that have obligate intracellular stages (i.e., Trypanosoma cruzi and Angomonas) but is absent from dixenous parasites with an exclusively extracellular lifestyle (i.e., Trypanosoma brucei). A DECR-green fluorescent protein (GFP) fusion protein was localized to the mitochondrion in both promastigote and amastigote stages, and the levels of expression increased in the latter stages. A Leishmania major Δdecr null mutant was unable to catabolize unsaturated fatty acids and accumulated the intermediate 2,4-decadienoyl-CoA, confirming DECR's role in ß-oxidation. Strikingly, the L. major Δdecr mutant was unable to survive in macrophages and was avirulent in BALB/c mice. These findings suggest that ß-oxidation of polyunsaturated fatty acids is essential for intracellular parasite survival and that the bacterial origin of key enzymes in this pathway could be exploited in developing new therapies.IMPORTANCE The Trypanosomatidae are protozoan parasites that infect insects, plants, and animals and have evolved complex monoxenous (single host) and dixenous (two hosts) lifestyles. A number of species of Trypanosomatidae, including Leishmania spp., have evolved the capacity to survive within intracellular niches in vertebrate hosts. The adaptations, metabolic and other, that are associated with development of intracellular lifestyles remain poorly defined. We show that genomes of Leishmania and Trypanosomatidae that can survive intracellularly encode a 2,4-dienoyl-CoA reductase that is involved in catabolism of a subclass of fatty acids. The trypanosomatid enzyme shows closest similarity to the corresponding bacterial enzymes and is located in the mitochondrion and essential for intracellular growth of Leishmania The findings suggest that acquisition of this gene by lateral gene transfer from bacteria by ancestral monoxenous Trypanosomatidae likely contributed to the development of a dixenous lifestyle of these parasites.


Assuntos
Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Leishmania major/enzimologia , Leishmania major/genética , Sequência de Aminoácidos , Animais , Ácidos Graxos Dessaturases/genética , Feminino , Leishmania major/crescimento & desenvolvimento , Leishmania mexicana/genética , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oxirredução , Filogenia
19.
Nat Commun ; 11(1): 2857, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504069

RESUMO

Virtual memory T (TVM) cells are antigen-naïve CD8+ T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (TMEM) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of TVM cells and their altered functionality with age, here we investigate TVM cell metabolism and its association with longevity and functionality. Elevated SRC is a feature of TVM, but not TMEM, cells and it increases with age in both subsets. The elevated SRC observed in aged mouse TVM cells and human CD8+ T cells from older individuals is associated with a heightened sensitivity to IL-15. We conclude that elevated SRC is a feature of TVM, but not TMEM, cells, is driven by physiological levels of IL-15, and is not indicative of enhanced functionality in CD8+ T cells.


Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/ultraestrutura , Diferenciação Celular/imunologia , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Vírus da Influenza A/imunologia , Influenza Humana/sangue , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/ultraestrutura , Adulto Jovem
20.
BMC Bioinformatics ; 8: 419, 2007 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-17963529

RESUMO

BACKGROUND: Gas chromatography-mass spectrometry (GC-MS) is a robust platform for the profiling of certain classes of small molecules in biological samples. When multiple samples are profiled, including replicates of the same sample and/or different sample states, one needs to account for retention time drifts between experiments. This can be achieved either by the alignment of chromatographic profiles prior to peak detection, or by matching signal peaks after they have been extracted from chromatogram data matrices. Automated retention time correction is particularly important in non-targeted profiling studies. RESULTS: A new approach for matching signal peaks based on dynamic programming is presented. The proposed approach relies on both peak retention times and mass spectra. The alignment of more than two peak lists involves three steps: (1) all possible pairs of peak lists are aligned, and similarity of each pair of peak lists is estimated; (2) the guide tree is built based on the similarity between the peak lists; (3) peak lists are progressively aligned starting with the two most similar peak lists, following the guide tree until all peak lists are exhausted. When two or more experiments are performed on different sample states and each consisting of multiple replicates, peak lists within each set of replicate experiments are aligned first (within-state alignment), and subsequently the resulting alignments are aligned themselves (between-state alignment). When more than two sets of replicate experiments are present, the between-state alignment also employs the guide tree. We demonstrate the usefulness of this approach on GC-MS metabolic profiling experiments acquired on wild-type and mutant Leishmania mexicana parasites. CONCLUSION: We propose a progressive method to match signal peaks across multiple GC-MS experiments based on dynamic programming. A sensitive peak similarity function is proposed to balance peak retention time and peak mass spectra similarities. This approach can produce the optimal alignment between an arbitrary number of peak lists, and models explicitly within-state and between-state peak alignment. The accuracy of the proposed method was close to the accuracy of manually-curated peak matching, which required tens of man-hours for the analyzed data sets. The proposed approach may offer significant advantages for processing of high-throughput metabolomics data, especially when large numbers of experimental replicates and multiple sample states are analyzed.


Assuntos
Algoritmos , Inteligência Artificial , Cromatografia Gasosa-Espectrometria de Massas/métodos , Perfilação da Expressão Gênica/métodos , Reconhecimento Automatizado de Padrão/métodos , Mapeamento de Peptídeos/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA