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1.
Methods Mol Biol ; 2194: 291-300, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32926372

RESUMO

Bile acids are important end products of cholesterol metabolism, having been shown to serve as signaling molecules and intermediates between the host and the gut microbiota. Here we describe a robust and accurate method using ultrahigh-pressure liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for the quantification of bile acids in stool/cecal and tissue samples.

2.
mBio ; 11(4)2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665268

RESUMO

Beneficial microorganisms associated with animals derive their nutritional requirements entirely from the animal host, but the impact of these microorganisms on host metabolism is largely unknown. The focus of this study was the experimentally tractable tripartite symbiosis between the pea aphid Acyrthosiphon pisum, its obligate intracellular bacterial symbiont Buchnera, and the facultative bacterium Hamiltonella which is localized primarily to the aphid hemolymph (blood). Metabolome experiments on, first, multiple aphid genotypes that naturally bear or lack Hamiltonella and, second, one aphid genotype from which Hamiltonella was experimentally eliminated revealed no significant effects of Hamiltonella on aphid metabolite profiles, indicating that Hamiltonella does not cause major reconfiguration of host metabolism. However, the titer of just one metabolite, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), displayed near-significant enrichment in Hamiltonella-positive aphids in both metabolome experiments. AICAR is a by-product of biosynthesis of the essential amino acid histidine in Buchnera and, hence, an index of histidine biosynthetic rates, suggesting that Buchnera-mediated histidine production is elevated in Hamiltonella-bearing aphids. Consistent with this prediction, aphids fed on [13C]histidine yielded a significantly elevated 12C/13C ratio of histidine in Hamiltonella-bearing aphids, indicative of increased (∼25%) histidine synthesized de novo by Buchnera However, in silico analysis predicted an increase of only 0.8% in Buchnera histidine synthesis in Hamiltonella-bearing aphids. We hypothesize that Hamiltonella imposes increased host demand for histidine, possibly for heightened immune-related functions. These results demonstrate that facultative bacteria can alter the dynamics of host metabolic interactions with co-occurring microorganisms, even when the overall metabolic homeostasis of the host is not substantially perturbed.IMPORTANCE Although microbial colonization of the internal tissues of animals generally causes septicemia and death, various animals are persistently associated with benign or beneficial microorganisms in their blood or internal organs. The metabolic consequences of these persistent associations for the animal host are largely unknown. Our research on the facultative bacterium Hamiltonella, localized primarily to the hemolymph of pea aphids, demonstrated that although Hamiltonella imposed no major reconfiguration of the aphid metabolome, it did alter the metabolic relations between the aphid and its obligate intracellular symbiont, Buchnera Specifically, Buchnera produced more histidine in Hamiltonella-positive aphids to support both Hamiltonella demand for histidine and Hamiltonella-induced increase in host demand. This study demonstrates how microorganisms associated with internal tissues of animals can influence specific aspects of metabolic interactions between the animal host and co-occurring microorganisms.

3.
Gut Microbes ; 11(4): 979-996, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32138583

RESUMO

Bile acids are potent antibacterial compounds and play an important role in shaping the microbial ecology of the gut. Here, we combined flow cytometry, growth rate measurements (OD600), and NMR- and mass spectrometry-based metabolomics to systematically profile the impact of bile acids on the microbiome using in vitro and in vivo models. This study confirmed that (1) unconjugated bile acids possess more potent antibacterial activity than conjugated bile acids; (2) Gram-positive bacteria are more sensitive to bile acids than Gram-negative bacteria; (3) some probiotic bacteria such as Lactobacillus and Bifidobacterium and 7α-dehydroxylating bacteria such as Clostridium scindens show bile acid resistance that is associated with activation of glycolysis. Moreover, we demonstrated that (4) as one of most hydrophobic bile acids, lithocholic acid (LCA) shows reduced toxicity to bacteria in the cecal microbiome in both in vivo and in vitro models; (5) bile acids directly and rapidly affect bacterial global metabolism including membrane damage, disrupted amino acid, nucleotide, and carbohydrate metabolism; and (6) in vivo, short-term exposure to bile acids significantly affected host metabolism via alterations of the bacterial community structure. This study systematically profiled interactions between bile acids and gut bacteria providing validation of previous observation and new insights into the interaction of bile acids with the microbiome and mechanisms related to bile acid tolerance.

4.
Sci Transl Med ; 11(510)2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31534021

RESUMO

Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage Plasmodium falciparum parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of P. falciparum infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl-coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl-coenzyme A synthetase and acyl-coenzyme A synthetase 11. Introduction and reversion of these mutations in P. falciparum using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.

5.
Appl Environ Microbiol ; 85(15)2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31152019

RESUMO

The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents.IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

6.
J Infect Dis ; 219(11): 1766-1776, 2019 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-30566600

RESUMO

BACKGROUND: Acidosis in severe Plasmodium falciparum malaria is associated with high mortality, yet the pathogenesis remains incompletely understood. The aim of this study was to determine the nature and source of metabolic acids contributing to acidosis in patients with severe falciparum malaria. METHODS: A prospective observational study was conducted to characterize circulating acids in adults with P. falciparum malaria (n = 107) and healthy controls (n = 45) from Bangladesh using high-resolution liquid chromatography-mass spectrometry metabolomics. Additional in vitro P. falciparum culture studies were performed to determine if parasites release the acids detected in plasma from patients with severe malaria acidosis. RESULTS: We identified previously unmeasured plasma acids strongly associated with acidosis in severe malaria. Metabolomic analysis of P. falciparum parasites in vitro showed no evidence that these acids are released by the parasite during its life cycle. Instead, 10 of the plasma acids could be mapped to a gut microbial origin. Patients with malaria had low L-citrulline levels, a plasma marker indicating reduced gut barrier integrity. Longitudinal data showed the clearance of these newly identified acids was delayed in fatal cases. CONCLUSIONS: These data suggest that a compromise in intestinal barrier function may contribute significantly to the pathogenesis of life-threatening acidosis in severe falciparum malaria. CLINICAL TRIALS REGISTRATION: NCT02451904.


Assuntos
Acidose/metabolismo , Ácidos/metabolismo , Malária Falciparum/metabolismo , Metabolômica , Plasmodium falciparum/fisiologia , Acidose/complicações , Acidose/parasitologia , Adulto , Biomarcadores/sangue , Cromatografia Líquida , Feminino , Humanos , Mucosa Intestinal , Malária Falciparum/complicações , Malária Falciparum/parasitologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
7.
Antimicrob Agents Chemother ; 60(11): 6635-6649, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27572391

RESUMO

The threat of widespread drug resistance to frontline antimalarials has renewed the urgency for identifying inexpensive chemotherapeutic compounds that are effective against Plasmodium falciparum, the parasite species responsible for the greatest number of malaria-related deaths worldwide. To aid in the fight against malaria, a recent extensive screening campaign has generated thousands of lead compounds with low micromolar activity against blood stage parasites. A subset of these leads has been compiled by the Medicines for Malaria Venture (MMV) into a collection of structurally diverse compounds known as the MMV Malaria Box. Currently, little is known regarding the activity of these Malaria Box compounds on parasite metabolism during intraerythrocytic development, and a majority of the targets for these drugs have yet to be defined. Here we interrogated the in vitro metabolic effects of 189 drugs (including 169 of the drug-like compounds from the Malaria Box) using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). The resulting metabolic fingerprints provide information on the parasite biochemical pathways affected by pharmacologic intervention and offer a critical blueprint for selecting and advancing lead compounds as next-generation antimalarial drugs. Our results reveal several major classes of metabolic disruption, which allow us to predict the mode of action (MoA) for many of the Malaria Box compounds. We anticipate that future combination therapies will be greatly informed by these results, allowing for the selection of appropriate drug combinations that simultaneously target multiple metabolic pathways, with the aim of eliminating malaria and forestalling the expansion of drug-resistant parasites in the field.


Assuntos
Antimaláricos/farmacologia , Quimioterapia Combinada/métodos , Estágios do Ciclo de Vida/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , Plasmodium falciparum/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Antimaláricos/química , Atovaquona/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Bases de Dados de Compostos Químicos , Resistência a Medicamentos/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Estágios do Ciclo de Vida/fisiologia , Metabolômica/métodos , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Bibliotecas de Moléculas Pequenas/química , Espectrometria de Massas em Tandem
8.
Am J Physiol Cell Physiol ; 310(3): C233-42, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26561640

RESUMO

Calcineurin B homologous proteins (CHP) are N-myristoylated, EF-hand Ca(2+)-binding proteins that bind to and regulate Na(+)/H(+) exchangers, which occurs through a variety of mechanisms whose relative significance is incompletely understood. Like mammals, Caenorhabditis elegans has three CHP paralogs, but unlike mammals, worms can survive CHP loss-of-function. However, mutants for the CHP ortholog PBO-1 are unfit, and PBO-1 has been shown to be required for proton signaling by the basolateral Na(+)/H(+) exchanger NHX-7 and for proton-coupled intestinal nutrient uptake by the apical Na(+)/H(+) exchanger NHX-2. Here, we have used this genetic model organism to interrogate PBO-1's mechanism of action. Using fluorescent tags to monitor Na(+)/H(+) exchanger trafficking and localization, we found that loss of either PBO-1 binding or activity caused NHX-7 to accumulate in late endosomes/lysosomes. In contrast, NHX-2 was stabilized at the apical membrane by a nonfunctional PBO-1 protein and was only internalized following its complete loss. Additionally, two pbo-1 paralogs were identified, and their expression patterns were analyzed. One of these contributed to the function of the excretory cell, which acts like a kidney in worms, establishing an alternative model for testing the role of this protein in membrane transporter trafficking and regulation. These results lead us to conclude that the role of CHP in Na(+)/H(+) exchanger regulation differs between apical and basolateral transporters. This further emphasizes the importance of proper targeting of Na(+)/H(+) exchangers and the critical role of CHP family proteins in this process.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Calcineurina/metabolismo , Membrana Celular/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Calcineurina/genética , Endossomos/metabolismo , Regulação da Expressão Gênica , Lisossomos/metabolismo , Dados de Sequência Molecular , Mutação , Estabilidade Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Trocadores de Sódio-Hidrogênio/genética
9.
PLoS Biol ; 11(7): e1001613, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23935448

RESUMO

For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters--not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals--e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death.


Assuntos
Caenorhabditis elegans/química , Fluorescência , ortoaminobenzoatos/química , Animais , Ésteres/química , Estresse Oxidativo
10.
J Biol Chem ; 288(8): 5886-95, 2013 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-23319594

RESUMO

Membrane proton transporters contribute to pH homeostasis but have also been shown to transmit information between cells in close proximity through regulated proton secretion. For example, the nematode intestinal Na(+)/H(+) exchanger NHX-7 causes adjacent muscle cells to contract by transiently acidifying the extracellular space between the intestine and muscle. NHX-7 operates during a Ca(2+)-dependent rhythmic behavior and contains several conserved motifs for regulation by Ca(2+) input, including motifs for calmodulin and phosphatidylinositol 4,5-bisphosphate binding, protein kinase C- and calmodulin-dependent protein kinase type II phosphorylation, and a binding site for calcineurin homologous protein. Here, we tested the idea that Ca(2+) input differentiates proton signaling from pH housekeeping activity. Each of these motifs was mutated, and their contribution to NHX-7 function was assessed. These functions included pH recovery from acidification in cells in culture expressing recombinant NHX-7, extracellular acidification measured during behavior in live moving worms, and muscle contraction strength as a result of this acidification. Our data suggest that multiple levels of Ca(2+) input regulate NHX-7, whose transport capacity normally exceeds the minimum necessary to cause muscle contraction. Furthermore, extracellular acidification limits NHX-7 proton transport through feedback inhibition, likely to prevent metabolic acidosis from occurring. Our findings are consistent with an integrated network whereby both Ca(2+) and pH contribute to proton signaling. Finally, our results obtained by expressing rat NHE1 in Caenorhabditis elegans suggest that a conserved mechanism of regulation may contribute to cell-cell communication or proton signaling by Na(+)/H(+) exchangers in mammals.


Assuntos
Caenorhabditis elegans/fisiologia , Sinalização do Cálcio , Cálcio/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Motivos de Aminoácidos , Animais , Técnicas Biossensoriais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans , Cálcio/química , Calmodulina/química , Concentração de Íons de Hidrogênio , Modelos Biológicos , Mutagênese , Prótons , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/genética , Relação Estrutura-Atividade
11.
Curr Biol ; 22(23): 2213-20, 2012 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-23141108

RESUMO

BACKGROUND: Rhythmic behaviors are ubiquitous phenomena in animals. In C. elegans, defecation is an ultradian rhythmic behavior: every ∼50 s a calcium wave initiating in the posterior intestinal cells triggers the defecation motor program that comprises three sequential muscle contractions. Oscillatory calcium signaling is central to the periodicity of defecation. The posteriormost intestinal cells function as the pacemaker for this rhythmic behavior, although it is unclear how the supremacy of these cells for calcium-wave initiation is controlled. RESULTS: We describe how the loss of the mir-240/786 microRNA cluster, which results in arrhythmic defecation, causes ectopic intestinal calcium-wave initiation. mir-240/786 expression in the intestine is restricted to the posterior cells that function as the defecation pacemaker. Genetic data indicate that mir-240/786 functions upstream of the inositol 1,4,5-trisphosphate (IP(3)) receptor. Through rescue analysis, it was determined that miR-786 functions to regulate defecation. Furthermore, we identified elo-2, a fatty-acid elongase with a known role in defecation cycling, as a direct target for miR-786. We propose that the regulation of palmitate levels through repression of elo-2 activity is the likely mechanistic link to defecation. CONCLUSIONS: Together, these data indicate that miR-786 confers pacemaker status on posterior intestinal cells for the control of calcium-wave initiation through the regulation of elo-2 and, subsequently, palmitate levels. We propose that a difference in fatty-acid composition in the posterior intestinal cells may alter the activities of membrane proteins, such as IP(3)-receptor or TRPM channels, that control pacemaker activity in the C. elegans intestine.


Assuntos
Acetiltransferases/metabolismo , Relógios Biológicos , Sinalização do Cálcio , Defecação , MicroRNAs/metabolismo , Animais , Caenorhabditis elegans , Cálcio/metabolismo , Elongases de Ácidos Graxos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mucosa Intestinal/metabolismo , Ácidos Palmíticos/metabolismo
12.
Am J Physiol Cell Physiol ; 302(7): C1045-54, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22237403

RESUMO

Mitochondrial morphology is determined by the balance between the opposing processes of fission and fusion, each of which is regulated by a distinct set of proteins. Abnormalities in mitochondrial dynamics have been associated with a variety of diseases, including neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and dominant optic atrophy. Although the genetic determinants of fission and fusion are well recognized, less is known about the mechanism(s) whereby altered morphology contributes to the underlying pathophysiology of these disease states. Previous work from our laboratory identified a role for mitochondrial dynamics in intracellular pH homeostasis in both mammalian cell culture and in the genetic model organism Caenorhabditis elegans. Here we show that the acidification seen in mutant animals that have lost the ability to fuse their mitochondrial inner membrane occurs through a reactive oxygen species (ROS)-dependent mechanism and can be suppressed through the use of pharmacological antioxidants targeted specifically at the mitochondrial matrix. Physiological approaches examining the activity of endogenous mammalian acid-base transport proteins in rat liver Clone 9 cells support the idea that ROS signaling to sodium-proton exchangers contributes to acidification. Because maintaining pH homeostasis is essential for cell function and viability, the results of this work provide new insight into the pathophysiology associated with the loss of inner mitochondrial membrane fusion.


Assuntos
Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Antioxidantes/farmacologia , Células CHO , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Cricetinae , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/genética , Mutação , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Ratos , Transdução de Sinais/fisiologia , Trocadores de Sódio-Hidrogênio/genética
13.
Am J Physiol Cell Physiol ; 301(6): C1389-403, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21865588

RESUMO

Caenorhabditis elegans defecation is a rhythmic behavior, composed of three sequential muscle contractions, with a 50-s periodicity. The motor program is driven by oscillatory calcium signaling in the intestine. Proton fluxes, which require sodium-proton exchangers at the apical and basolateral intestinal membranes, parallel the intestinal calcium flux. These proton shifts are critical for defecation-associated muscle contraction, nutrient uptake, and longevity. How sodium-proton exchangers are activated in time with intestinal calcium oscillation is not known. The posterior body defecation contraction mutant (pbo-1) encodes a calcium-binding protein with homology to calcineurin homologous proteins, which are putative cofactors for mammalian sodium-proton exchangers. Loss of pbo-1 function results in a weakened defecation muscle contraction and a caloric restriction phenotype. Both of these phenotypes also arise from dysfunctions in pH regulation due to mutations in intestinal sodium-proton exchangers. Dynamic, in vivo imaging of intestinal proton flux in pbo-1 mutants using genetically encoded pH biosensors demonstrates that proton movements associated with these sodium-proton exchangers are significantly reduced. The basolateral acidification that signals the first defecation motor contraction is scant in the mutant compared with a normal animal. Luminal and cytoplasmic pH shifts are much reduced in the absence of PBO-1 compared with control animals. We conclude that pbo-1 is required for normal sodium-proton exchanger activity and may couple calcium and proton signaling events.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Calcineurina/metabolismo , Defecação/fisiologia , Intestinos/fisiologia , Contração Muscular/fisiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Calcineurina/genética , Cálcio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Trocadores de Sódio-Hidrogênio/genética
14.
Am J Physiol Cell Physiol ; 297(5): C1071-81, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19741196

RESUMO

In Caenorhabditis elegans, oscillations of intestinal pH contribute to the rhythmic defecation behavior, but the acid-base transport mechanisms that facilitate proton movement are not well understood. Here, we demonstrate that VHA-6, an intestine-specific a-subunit of the H(+)-K(+)-ATPase complex (V-ATPase), resides in the apical membrane of the intestinal epithelial cells and is required for luminal acidification. Disruption of the vha-6 gene led to early developmental arrest; the arrest phenotype could be complemented by expression of a fluorescently labeled vha-6 transgene. To study the contribution of vha-6 to pH homeostasis in larval worms, we used a partial reduction of function through postembryonic single-generation RNA interference. We demonstrate that the inability to fully acidify the intestinal lumen coincides with a defect in pH recovery of the intestinal epithelial cells, suggesting that VHA-6 is essential for proton pumping following defecation. Moreover, intestinal dipeptide accumulation and fat storage are compromised by the loss of VHA-6, suggesting that luminal acidification promotes nutrient uptake in worms, as well as in mammals. Since acidified intracellular vesicles and autofluorescent storage granules are indistinguishable between the vha-6 mutant and controls, it is likely that the nutrient-restricted phenotype is due to a loss of plasma membrane V-ATPase activity specifically. These data establish a simple genetic model for proton pump-driven acidification. Since defecation occurs at 45-s intervals in worms, this model represents an opportunity to study acute regulation of V-ATPase activity on a short time scale and may be useful in the study of alternative treatments for acid-peptic disorders.


Assuntos
Comportamento Animal/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Ritmo Circadiano/fisiologia , Mucosa Intestinal/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Caenorhabditis elegans , Defecação/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Concentração de Íons de Hidrogênio , Microscopia Confocal , Subunidades Proteicas/metabolismo , Interferência de RNA , ATPases Vacuolares Próton-Translocadoras/genética
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