RESUMO
Two recently discovered DRD2 mutations, c.634A > T, p.Ile212Phe and c.1121T > G, p.Met374Arg, cause hyperkinetic movement disorders that have overlapping features but apparently differ in severity. The two known carriers of the Met374Arg variant had early childhood disease onset and more severe motor, cognitive, and neuropsychiatric deficits than any known carriers of the Ile212Phe variant, whose symptoms were first apparent in adolescence. Here, we evaluated if differences in the function of the two variants in cultured cells could explain differing pathogenicity. Both variants were expressed less abundantly than the wild type receptor and exhibited loss of agonist-induced arrestin binding, but differences in expression and arrestin binding between the variants were minor. Basal and agonist-induced activation of heterotrimeric Gi/o/z proteins, however, showed clear differences; agonists were generally more potent at Met374Arg than at the Ile212Phe or wild type variants. Furthermore, all Gα subtypes tested were constitutively activated more by Met374Arg than by Ile212Phe. Met374Arg produced greater constitutive inhibition of cyclic AMP accumulation than Ile212Phe or the wild type D2 receptor. Met374Arg and Ile212Phe were more sensitive to thermal inactivation than the wild type D2 receptor, as reported for other constitutively active receptors, but Ile212Phe was affected more than Met374Arg. Additional pharmacological characterization suggested that the mutations differentially affect the shape of the agonist binding pocket and the potency of dopamine, norepinephrine, and tyramine. Molecular dynamics simulations provided a structural rationale for enhanced constitutive activation and agonist potency. Enhanced constitutive and agonist-induced G protein-mediated signaling likely contributes to the pathogenicity of these novel variants.
RESUMO
A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. That allelic variant D2-I212F is a constitutively active and G protein-biased receptor. We now describe mice engineered using CRISPR-Cas9-mediated gene editing technology to carry the D2-I212F variant. Drd2I212F mice exhibited gait abnormalities resembling those in other mouse models of chorea and/or dystonia and had striatal D2 receptor expression that was decreased approximately 30% per Drd2I212F allele. Electrically evoked inhibitory postsynaptic conductances in midbrain dopamine neurons and striatum from Drd2I212F mice, caused by G protein activation of potassium channels, exhibited slow kinetics (e.g., approximately four- to sixfold slower decay) compared with Drd2 +/+ mice. Current decay initiated by photolytic release of the D2 antagonist sulpiride from CyHQ-sulpiride was also â¼fourfold slower in midbrain slices from Drd2I212F mice than Drd2 +/+ mice. Furthermore, in contrast to Drd2 +/+ mice, in which dopamine is several-fold more potent at neurons in the nucleus accumbens than in the dorsal striatum, reflecting activation of Gα o versus Gα i, dopamine had similar potencies in those two brain regions of Drd2I212F mice. Repeated cocaine treatment, which decreases dopamine potency in the nucleus accumbens of Drd2 +/+ mice, had no effect on dopamine potency in Drd2 I212F mice. The results demonstrate the pathogenicity of the D2-I212F mutation and the utility of this mouse model for investigating the role of pathogenic DRD2 variants in early-onset hyperkinetic movement disorders. SIGNIFICANCE STATEMENT: The first dopamine receptor mutation to cause a movement disorder, D2-I212F, was recently identified. The mutation makes receptor activation of G protein-mediated signaling more efficient. To confirm the pathogenesis of D2-I212F, this study reports that mice carrying this mutation have gait abnormalities consistent with the clinical phenotype. The mutation also profoundly alters D2 receptor expression and function in vivo. This mouse model will be useful for further characterization of the mutant receptor and for evaluation of potential therapeutic drugs.
Assuntos
Dopamina , Transtornos dos Movimentos , Receptores de Dopamina D2 , Animais , Humanos , Camundongos , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Marcha/genética , Hipercinese , Mutação , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , SulpiridaAssuntos
Coreia , Distonia , Criança , Coreia/genética , Mutação com Ganho de Função , Humanos , Fenótipo , Receptores de Dopamina D2/genéticaRESUMO
We previously proposed that the dopamine D2 receptor-interacting protein S100B binds to a putative S100B-binding motif at residues R233-L240 toward the N terminus of the third intracellular loop. We used in vitro pull-down assays with FLAG-tagged fragments of the rat dopamine D2 receptor third intracellular loop (D2-IC3) and in vitro-synthesized S100B to evaluate this hypothesis. Our results indicate that the putative S100B-binding motif is neither necessary nor sufficient for strong binding of S100B to D2-IC3. Instead, two residues at the junction of the fifth membrane-spanning domain and the cytoplasmic extension of that α-helical domain, K211-I212, are required for robust, calcium-sensitive binding of S100B. This is also the approximate location of previously identified determinants for the binding of arrestin and calmodulin. A D2 receptor mutation converting I212 to phenylalanine has been described in patients with a hyperkinetic movement disorder. SIGNIFICANCE STATEMENT: S100B is a small calcium-binding protein that modulates signaling by the dopamine D2 receptor. New data suggest that the previous hypothesis about the involvement of an S100B-binding motif is incorrect, and that an important determinant of S100B binding includes a residue that is mutated in patients with a hyperkinetic movement disorder.
Assuntos
Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Modelos Moleculares , Mutação , Domínios Proteicos , Ratos , Subunidade beta da Proteína Ligante de Cálcio S100/genéticaRESUMO
A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. Compared to the wild type D2 receptor, the novel allelic variant D2-I212F activates a Gαi1ß1γ2 heterotrimer with higher potency and modestly enhanced basal activity in human embryonic kidney (HEK) 293 cells and has decreased capacity to recruit arrestin3. We now report that omitting overexpressed G protein-coupled receptor kinase-2 (GRK2) decreased the potency and efficacy of quinpirole for arrestin recruitment. The relative efficacy of quinpirole for arrestin recruitment to D2-I212F compared to D2-WT was considerably lower without overexpressed GRK2 than with added GRK2. D2-I212F exhibited higher basal activation of GαoA than Gαi1 but little or no increase in the potency of quinpirole relative to D2-WT. Other signs of D2-I212F constitutive activity for G protein-mediated signaling, in addition to basal activation of Gαi/o, were enhanced basal inhibition of forskolin-stimulated cyclic AMP accumulation that was reversed by the inverse agonists sulpiride and spiperone and a â¼4-fold increase in the apparent affinity of D2-I212F for quinpirole, determined from competition binding assays. In mouse midbrain slices, inhibition of tonic current by the inverse agonist sulpiride in dopamine neurons expressing D2-I212F was consistent with our hypothesis of enhanced constitutive activity and sensitivity to dopamine relative to D2-WT. Molecular dynamics simulations with D2 receptor models suggested that an ionic lock between the cytoplasmic ends of the third and sixth α-helices that constrains many G protein-coupled receptors in an inactive conformation spontaneously breaks in D2-I212F. Overall, these results confirm that D2-I212F is a constitutively active and signaling-biased D2 receptor mutant and also suggest that the effect of the likely pathogenic variant in a given brain region will depend on the nature of G protein and GRK expression.
Assuntos
Receptores de Dopamina D2 , Transdução de Sinais , Animais , AMP Cíclico , Agonistas de Dopamina/farmacologia , Células HEK293 , Humanos , Camundongos , Quimpirol/farmacologia , Receptores de Dopamina D2/genéticaRESUMO
BACKGROUND: We describe a 4-generation Dutch pedigree with a unique dominantly inherited clinical phenotype of a combined progressive chorea and cervical dystonia carrying a novel heterozygous dopamine D2 receptor (DRD2) variant. OBJECTIVES: The objective of this study was to identify the genetic cause of the disease and to further investigate the functional consequences of the genetic variant. METHODS: After detailed clinical and neurological examination, whole-exome sequencing was performed. Because a novel variant in the DRD2 gene was found as the likely causative gene defect in our pedigree, we sequenced the DRD2 gene in a cohort of 121 Huntington-like cases with unknown genetic cause (Germany). Moreover, functional characterization of the DRD2 variant included arrestin recruitment, G protein activation, and G protein-mediated inhibition of adenylyl cyclase determined in a cell model, and G protein-regulated inward-rectifying potassium channels measured in midbrain slices of mice. RESULT: We identified a novel heterozygous variant c.634A > T, p.Ile212Phe in exon 5 of DRD2 that cosegregated with the clinical phenotype. Screening of the German cohort did not reveal additional putative disease-causing variants. We demonstrated that the D2S/L -I212 F receptor exhibited increased agonist potency and constitutive activation of G proteins in human embryonic kidney 239 cells as well as significantly reduced arrestin3 recruitment. We further showed that the D2S -I212 F receptor exhibited aberrant receptor function in mouse midbrain slices. CONCLUSIONS: Our results support an association between the novel p.Ile212Phe variant in DRD2, its modified D2 receptor activity, and the hyperkinetic movement disorder reported in the 4-generation pedigree. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Coreia , Distonia , Animais , Coreia/genética , Mutação com Ganho de Função , Alemanha , Camundongos , Fenótipo , Receptores de Dopamina D2/genéticaRESUMO
In Leishmania mexicana parasites, a unique glucose transporter, LmxGT1, is selectively targeted to the flagellar membrane, suggesting a possible sensory role that is often associated with ciliary membrane proteins. Expression of LmxGT1 is down-regulated â¼20-fold by increasing cell density but is up-regulated â¼50-fold by depleting glucose from the medium, and the permease is strongly down-regulated when flagellated insect-stage promastigotes invade mammalian macrophages and transform into intracellular amastigotes. Regulation of LmxGT1 expression by glucose and during the lifecycle operates at the level of protein stability. Significantly, a ∆lmxgt1 null mutant, grown in abundant glucose, undergoes catastrophic loss of viability when parasites deplete glucose from the medium, a property not exhibited by wild-type or add-back lines. These results suggest that LmxGT1 may function as a glucose sensor that allows parasites to enter the stationary phase when they deplete glucose and that in the absence of this sensor, parasites do not maintain viability when they run out of glucose. However, alternate roles for LmxGT1 in monitoring glucose availability are considered. The absence of known sensory receptors with defined ligands and biologic functions in Leishmania and related kinetoplastid parasites underscores the potential significance of these observations.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Leishmania mexicana/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Linhagem Celular , Feminino , Flagelos/metabolismo , Regulação da Expressão Gênica , Genes de Protozoários , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Leishmania mexicana/genética , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Proteínas de Protozoários/genética , Psychodidae/parasitologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans.
Assuntos
Gluconeogênese , Glicerol Quinase/metabolismo , Leishmania mexicana/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Protozoários/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Southern Blotting , Western Blotting , DNA de Protozoário/genética , Glucose/metabolismo , Glucose/farmacologia , Glicerol Quinase/genética , Humanos , Leishmania mexicana/genética , Leishmania mexicana/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Mutação , Fosfoenolpiruvato Carboxilase/genética , Proteínas de Protozoários/genética , Piruvato Ortofosfato Diquinase/genéticaRESUMO
The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.
Assuntos
Flagelos/metabolismo , Glucose/metabolismo , Leishmania mexicana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Cromatografia de Afinidade , Primers do DNA , Dados de Sequência Molecular , Transporte Proteico , Proteínas de Protozoários/química , Homologia de Sequência de AminoácidosRESUMO
The genome of Leishmania mexicana encompasses a cluster of three glucose transporter genes designated LmxGT1, LmxGT2 and LmxGT3. Functional and genetic studies of a cluster null mutant (Δlmxgt1-3) have dissected the roles of these proteins in Leishmania metabolism and virulence. However, null mutants were recovered at very low frequency, and comparative genome hybridizations revealed that Δlmxgt1-3 mutants contained a linear extrachromosomal 40 kb amplification of a region on chromosome 29 not amplified in wild type parasites. These data suggested a model where this 29-40k amplicon encoded a second site suppressor contributing to parasite survival in the absence of GT1-3 function. To test this, we quantified the frequency of recovery of knockouts in the presence of individual overexpressed open reading frames covering the 29-40k amplicon. The data mapped the suppressor activity to PIFTC3, encoding a component of the intraflagellar transport pathway. We discuss possible models by which PIFTC3 might act to facilitate loss of GTs specifically. Surprisingly, by plasmid segregation we showed that continued PIFTC3 overexpression was not required for Δlmxgt1-3 viability. These studies provide the first evidence that genetic suppression can occur by providing critical biological functions transiently. This novel form of genetic suppression may extend to other genes, pathways and organisms.
Assuntos
Técnicas de Inativação de Genes , Leishmania mexicana/genética , Proteínas de Transporte de Monossacarídeos/genética , Supressão Genética , Leishmania mexicana/metabolismo , Viabilidade Microbiana , Modelos BiológicosRESUMO
Many of the cilia- and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins.
Assuntos
Flagelos/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Fluorescência Verde/metabolismo , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Estrutura Terciária de ProteínaRESUMO
Glucose is a major nutrient in the insect vector stage of Leishmania parasites. Glucose transporter null mutants of Leishmania mexicana exhibit profound phenotypic changes in both insect stage promastigotes and mammalian host stage amastigotes that reside within phagolysosomes of host macrophages. Some of these phenotypic changes could be either mediated or attenuated by changes in gene expression that accompany deletion of the glucose transporter genes. To search for changes in protein expression, the profile of proteins detected on two-dimensional gels was compared for wild type and glucose transporter null mutant promastigotes. A total of 50 spots whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins is altered in expression levels in the null mutant parasites. Following identification of proteins by mass spectrometry, 3 such regulated proteins were chosen for more detailed analysis: mitochondrial aldehyde dehydrogenase, ribokinase, and hexokinase. Immunoblots employing antisera against these enzymes confirmed that their levels were upregulated, both in glucose transporter null mutants and in wild type parasites starved for glucose. Quantitative reverse transcriptase PCR (qRT-PCR) revealed that the levels of mRNAs encoding these enzymes were also enhanced. Global expression profiling using microarrays revealed a limited number of additional changes, although the sensitivity of the microarrays to detect modest changes in amplitude was less than that of two-dimensional gels. Hence, there is likely to be a network of proteins whose expression levels are altered by genetic ablation of glucose transporters, and much of this regulation may be reflected by changes in the levels of the cognate mRNAs. Some of these changes in protein expression may reflect an adaptive response of the parasites to limitation of glucose.
Assuntos
Deleção de Genes , Perfilação da Expressão Gênica , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Proteínas de Transporte de Monossacarídeos/deficiência , Proteoma/análise , Proteínas de Protozoários/análise , Eletroforese em Gel Bidimensional , Immunoblotting , Espectrometria de Massas , Análise em Microsséries , RNA Mensageiro/biossíntese , RNA de Protozoário/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Glucose is considered essential for erythrocytic stages of the malaria parasite, Plasmodium falciparum. Importance of sugar and its permease for hepatic and sexual stages of Plasmodium, however, remains elusive. Moreover, increasing global resistance to current antimalarials necessitates the search for novel drugs. Here, we reveal that hexose transporter 1 (HT1) of Plasmodium berghei can transport glucose (K(m)~87 µM), mannose (K(i)~93 µM), fructose (K(i)~0.54 mM), and galactose (K(i)~5 mM) in Leishmania mexicana mutant and Xenopus laevis; and, therefore, is functionally equivalent to HT1 of P. falciparum (Glc, K(m)~175 µM; Man, K(i)~276 µM; Fru, K(i)~1.25 mM; Gal, K(i)~5.86 mM). Notably, a glucose analog, C3361, attenuated hepatic (IC(50)~15 µM) and ookinete development of P. berghei. The PbHT1 could be ablated during intraerythrocytic stages only by concurrent complementation with PbHT1-HA or PfHT1. Together; these results signify that PbHT1 and glucose are required for the entire life cycle of P. berghei. Accordingly, PbHT1 is expressed in the plasma membrane during all parasite stages. To permit a high-throughput screening of PfHT1 inhibitors and their subsequent in vivo assessment, we have generated Saccharomyces cerevisiae mutant expressing codon-optimized PfHT1, and a PfHT1-dependent Δpbht1 parasite strain. This work provides a platform to facilitate the development of drugs against malaria, and it suggests a disease-control aspect by reducing parasite transmission.
Assuntos
Proteínas de Transporte de Monossacarídeos/metabolismo , Plasmodium berghei/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Antimaláricos/farmacologia , Sequência de Bases , Frutose/metabolismo , Galactose/metabolismo , Glucose/metabolismo , Humanos , Leishmania mexicana , Estágios do Ciclo de Vida , Manose/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Toxoplasma/efeitos dos fármacos , Xenopus laevisRESUMO
Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1-3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike Deltatgst2, the modest impairment in Deltatggt1 and Deltatggt1/Deltatgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Deltatggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in Deltatggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Deltatggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/fisiologia , Transportador de Glucose Tipo 1/fisiologia , Glutamina/metabolismo , Toxoplasma/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Glucose/metabolismo , Manose/metabolismo , Dados de Sequência Molecular , Toxoplasma/crescimento & desenvolvimentoRESUMO
A glucose transporter null mutant of the parasitic protozoan Leishmania mexicana, in which three linked glucose transporter genes have been deleted by targeted gene replacement, is unable to replicate as amastigote forms within phagolysomes of mammalian host macrophages and is avirulent. Spontaneous suppressors of the null mutant have been isolated that partially restore replication of parasites within macrophages. These suppressor mutants have amplified the gene for an alternative hexose transporter, the LmGT4 permease (previously called the D2 permease), on a circular extrachromosomal element, and they overexpress LmGT4 mRNA and protein. The suppressors have also regained the ability to transport hexoses, and they have reverted other phenotypes of the null mutant exhibiting enhanced resistance to oxidative killing, heat shock and starvation for nutrients, as well as augmented levels of the storage carbohydrate beta-mannan, increased cell size and increased growth as insect stage promastigotes compared with the unsuppressed mutant. Complementation of the null mutant with the LmGT4 gene on a multicopy episomal expression vector also reverted these phenotypes, confirming that suppression results from amplification of the LmGT4 gene. These results underscore the importance of hexose transporters for the infectious stage of the parasite life cycle.
Assuntos
Amplificação de Genes , Leishmania mexicana/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Hibridização Genômica Comparativa , Genes de Protozoários , Teste de Complementação Genética , Hexoses/metabolismo , Leishmania mexicana/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas de Protozoários/genética , RNA de Protozoário/genéticaRESUMO
Glucose is a major source of energy and carbon in promastigotes of Leishmania mexicana, and its uptake is mediated by three glucose transporters whose genes are encoded within a single cluster. A null mutant in which the glucose transporter gene cluster was deleted by homologous gene replacement was generated previously and shown to grow more slowly than wild type promastigotes but not to be viable as amastigotes in primary tissue culture macrophages or in axenic culture. Further phenotypic characterization demonstrates that the null mutant is unable to import glucose, mannose, fructose, or galactose and that each of the three glucose transporter isoforms, LmGT1, LmGT2, and LmGT3, is capable of transporting each of these hexoses. Complementation of the null mutant with each isoform is able to restore growth in each of the four hexoses to wild type levels. Null mutant promastigotes are reduced in size to about 2/3 the volume of wild type parasites. In addition, the null mutants are significantly more sensitive to oxidative stress than their wild type counterparts. These results underscore the importance of glucose transporters in the parasite life cycle and suggest reasons for their non-viability in the disease-causing amastigote stage.
Assuntos
Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Animais , Genes de Protozoários , Teste de Complementação Genética , Hexoses/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Leishmania mexicana/crescimento & desenvolvimento , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Estresse Oxidativo , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Leishmania mexicana are parasitic protozoa that express a variety of glycoconjugates that play important roles in their biology as well as the storage carbohydrate beta-mannan, which is an essential virulence factor for survival of intracellular amastigote forms in the mammalian host. Glucose transporter null mutants, which are viable as insect form promastigotes but not as amastigotes, do not take up glucose and other hexoses but are still able to synthesize these glycoconjugates and beta-mannan, although at reduced levels. Synthesis of these carbohydrate-containing macromolecules could be accounted for by incorporation of non-carbohydrate precursors into carbohydrates by gluconeogenesis. However, the significantly reduced level of the virulence factor beta-mannan in the glucose transporter null mutants compared with wild-type parasites may contribute to the non-viability of these null mutants in the disease-causing amastigote stage of the life cycle.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Leishmania mexicana/metabolismo , Leishmania mexicana/patogenicidade , Acetatos/metabolismo , Alanina/metabolismo , Animais , Ácido Aspártico/metabolismo , Linhagem Celular , Gluconeogênese , Proteínas Facilitadoras de Transporte de Glucose/deficiência , Glicerol/metabolismo , Leishmania mexicana/genética , Mananas/metabolismo , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , VirulênciaRESUMO
BACKGROUND: A protein fraction was isolated from calcareous corpuscles of Taenia solium cysticerci. The antigens in this fraction were recognized in ELISA and Western blot assays by all sera from a group of patients with active neurocysticercosis (NC) and were not recognized by the sera from patients with other neurological disorders. Western blot analysis also showed that several high molecular weight proteins were strongly recognized by antibodies in all the neurocysticercotic patient sera, suggesting a potential for serological diagnosis of neurocysticercosis. METHODS: In order to characterize these antigenic proteins, we used a monoclonal antibody raised against a high MW calcium-binding protein associated with calcareous corpuscles of Echinococcus granulosus (EgCaBP1). RESULTS: Western blot assays revealed the recognition of a protein band of about 260 kDa, appearing within the range of the high MW antigens recognized by the NC sera. Several cDNA clones were isolated through screening of a T. solium metacestode library with a DNA probe for EgCaBP1, containing partial coding sequences showing about 88% identity with the protein of E. granulosus. Moreover, a recombinant product expressed in bacteria from the partial coding sequence of T. solium showed the ability to bind Ca2+ and was recognized by the monoclonal antibody. This recombinant calcium-binding protein of T. solium was not recognized by the NC patient sera by ELISA and Western blot. CONCLUSIONS: Antigenic proteins in the calcareous corpuscles of T. solium metacestodes deserve further analysis as candidates in the development of diagnostic tools for neurocysticercosis.
Assuntos
Antígenos de Helmintos/metabolismo , Proteínas de Ligação ao Cálcio/imunologia , Corpos de Inclusão/imunologia , Taenia solium , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/isolamento & purificação , Humanos , Dados de Sequência Molecular , Neurocisticercose/imunologia , Neurocisticercose/parasitologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Suínos , Taenia solium/química , Taenia solium/citologia , Taenia solium/imunologiaRESUMO
Both insect and mammalian life cycle stages of Leishmania mexicana take up glucose and express all three isoforms encoded by the LmGT glucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of the LmGT locus that encompasses the LmGT1, LmGT2, and LmGT3 genes. This deltalmgt null mutant exhibited no detectable glucose transport activity. The growth rate of the deltalmgt knockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. deltalmgt cells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WT L. mexicana were not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of either LmGT2 or LmGT3, but not of LmGT1, in deltalmgt null mutants significantly restored growth as promastigotes, but only LmGT3 expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in deltalmgt cells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite.