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1.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
2.
iScience ; 15: 39-54, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-31030181

RESUMO

Clostridium perfringens epsilon toxin (ETX) is hypothesized to mediate blood-brain barrier (BBB) permeability by binding to the myelin and lymphocyte protein (MAL) on the luminal surface of endothelial cells (ECs). However, the kinetics of this interaction and a general understanding of ETX's behavior in a live organism have yet to be appreciated. Here we investigate ETX binding and BBB breakdown in living Danio rerio (zebrafish). Wild-type zebrafish ECs do not bind ETX. When zebrafish ECs are engineered to express human MAL (hMAL), proETX binding occurs in a time-dependent manner. Injection of activated toxin in hMAL zebrafish initiates BBB leakage, hMAL downregulation, blood vessel stenosis, perivascular edema, and blood stasis. We propose a kinetic model of MAL-dependent ETX binding and neurovascular pathology. By generating a humanized zebrafish BBB model, this study contributes to our understanding of ETX-induced BBB permeability and strengthens the proposal that MAL is the ETX receptor.

3.
Mult Scler ; : 1352458518767327, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29681209

RESUMO

BACKGROUND: It was recently reported that, using Western blotting, some multiple sclerosis (MS) patients in the United States had antibodies against epsilon toxin (Etx) from Clostridium perfringens, suggesting that the toxin may play a role in the disease. OBJECTIVE: We investigated for serum antibodies against Etx in UK patients with clinically definite multiple sclerosis (CDMS) or presenting with clinically isolated syndrome (CIS) or optic neuritis (ON) and in age- and gender-matched controls. METHODS: We tested sera from CDMS, CIS or ON patients or controls by Western blotting. We also tested CDMS sera for reactivity with linear overlapping peptides spanning the amino acid sequence (Pepscan) of Etx. RESULTS: Using Western blotting, 24% of sera in the combined CDMS, CIS and ON groups ( n = 125) reacted with Etx. In the control group ( n = 125), 10% of the samples reacted. Using Pepscan, 33% of sera tested reacted with at least one peptide, whereas in the control group only 16% of sera reacted. Out of 61 samples, 21 (43%) were positive to one or other testing methodology. Three samples were positive by Western blotting and Pepscan. CONCLUSION: Our results broadly support the previous findings and the role of Etx in the aetiology of MS warrants further investigation.

4.
Nat Commun ; 7: 11293, 2016 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-27048994

RESUMO

Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small ß-pore-forming toxins (ß-PFTs), some members of which are pathogenic to humans and animals. Despite efforts, a high-resolution structure of a channel for this family of proteins has been elusive and therefore the mechanism of activation and membrane insertion remains unclear. Here we determine the pore structure of lysenin by single particle cryo-EM, to 3.1 Å resolution. The nonameric assembly reveals a long ß-barrel channel spanning the length of the complex that, unexpectedly, includes the two pre-insertion strands flanking the hypothetical membrane-insertion loop. Examination of other members of the aerolysin family reveals high structural preservation in this region, indicating that the membrane-insertion pathway in this family is conserved. For some toxins, proteolytic activation and pro-peptide removal will facilitate unfolding of the pre-insertion strands, allowing them to form the ß-barrel of the channel.


Assuntos
Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Toxinas Biológicas/química , Humanos , Lipídeos/química , Modelos Moleculares , Estrutura Secundária de Proteína , Solubilidade , Água/química
5.
J Biol Chem ; 291(19): 10210-27, 2016 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-26984406

RESUMO

This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell.


Assuntos
Diabetes Mellitus/metabolismo , Membrana Eritrocítica , Potenciais da Membrana/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Streptococcus pneumoniae/química , Estreptolisinas/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacologia , Membrana Eritrocítica/metabolismo , Feminino , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacologia , Humanos , Masculino , Estreptolisinas/química
6.
Avian Pathol ; 45(3): 381-8, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26743457

RESUMO

Necrotic enteritis toxin B (NetB) is a pore-forming toxin produced by Clostridium perfringens and has been shown to play a key role in avian necrotic enteritis, a disease causing significant costs to the poultry production industry worldwide. The aim of this work was to determine whether immunization with a non-toxic variant of NetB (NetB W262A) and the C-terminal fragment of C. perfringens alpha-toxin (CPA247-370) would provide protection against experimental necrotic enteritis. Immunized birds with either antigen or a combination of antigens developed serum antibody levels against NetB and CPA. When CPA247-370 and NetB W262A were used in combination as immunogens, an increased protection was observed after oral challenge by individual dosing, but not after in-feed-challenge.


Assuntos
Anticorpos Antibacterianos/sangue , Toxinas Bacterianas/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Infecções por Clostridium/veterinária , Clostridium perfringens/imunologia , Enterite/veterinária , Enterotoxinas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Fosfolipases Tipo C/imunologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/genética , Bélgica , Proteínas de Ligação ao Cálcio/genética , Galinhas , Infecções por Clostridium/imunologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Enterite/imunologia , Enterite/microbiologia , Enterite/prevenção & controle , Enterotoxinas/genética , Feminino , Imunização/veterinária , Masculino , Necrose/veterinária , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Fosfolipases Tipo C/genética
7.
Vaccine ; 32(23): 2682-7, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24709588

RESUMO

Epsilon toxin (Etx) is a ß-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Enterotoxemia/prevenção & controle , Animais , Cães , Feminino , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Testes de Neutralização , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/imunologia
8.
Toxins (Basel) ; 6(3): 1049-61, 2014 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-24625763

RESUMO

Necrotic enteritis toxin B (NetB) is a ß-pore-forming toxin produced by Clostridium perfringens and has been identified as a key virulence factor in the pathogenesis of avian necrotic enteritis, a disease causing significant economic damage to the poultry industry worldwide. In this study, site-directed mutagenesis was used to identify amino acids that play a role in NetB oligomerisation and pore-formation. NetB K41H showed significantly reduced toxicity towards LMH cells and human red blood cells relative to wild type toxin. NetB K41H was unable to oligomerise and form pores in liposomes. These findings suggest that NetB K41H could be developed as a genetic toxoid vaccine to protect against necrotic enteritis.


Assuntos
Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aminoácidos/química , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Galinhas , Enterotoxinas/genética , Enterotoxinas/metabolismo , Eritrócitos/metabolismo , Fluoresceínas/metabolismo , Hemólise , Humanos , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Estrutura Secundária de Proteína
9.
Vaccine ; 31(37): 4003-8, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23727000

RESUMO

NetB (necrotic enteritis toxin B) is a recently identified ß-pore-forming toxin produced by Clostridium perfringens. This toxin has been shown to play a major role in avian necrotic enteritis. In recent years, a dramatic increase in necrotic enteritis has been observed, especially in countries where the use of antimicrobial growth promoters in animal feedstuffs has been banned. The aim of this work was to determine whether immunisation with a NetB toxoid would provide protection against necrotic enteritis. The immunisation of poultry with a formaldehyde NetB toxoid or with a NetB genetic toxoid (W262A) resulted in the induction of antibody responses against NetB and provided partial protection against disease.


Assuntos
Toxinas Bacterianas/imunologia , Infecções por Clostridium/veterinária , Clostridium perfringens/genética , Enterite/veterinária , Toxoides/farmacologia , Animais , Anticorpos Antibacterianos/análise , Toxinas Bacterianas/genética , Galinhas/imunologia , Galinhas/microbiologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Eletroforese em Gel de Poliacrilamida , Enterite/imunologia , Enterite/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Formaldeído/imunologia , Imunização/métodos , Mutação , Doenças das Aves Domésticas/microbiologia , Toxoides/imunologia
10.
Protein Sci ; 22(5): 650-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23504825

RESUMO

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (ß-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.


Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Infecções por Clostridium/microbiologia , Clostridium perfringens/fisiologia , Interações Hospedeiro-Patógeno , Receptores de Superfície Celular/metabolismo , Animais , Toxinas Bacterianas/química , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular , Clostridium perfringens/química , Clostridium perfringens/genética , Cães , Células Madin Darby de Rim Canino , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Conformação Proteica
11.
Int J Antimicrob Agents ; 41(4): 330-6, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23402703

RESUMO

Mammalian models of infection are paramount to elucidating the mechanisms of bacterial pathogenesis and are also used for evaluating the efficacy of novel antimicrobials before the commencement of human trials. In this study, Galleria mellonella was used to determine the efficacy of antibiotics towards a Burkholderia thailandensis infection in G. mellonella larvae. Kanamycin, imipenem, ceftazidime, doxycycline and ciprofloxacin could all provide some protection when given 1 h before challenge with B. thailandensis; however, at 2 h or 6 h post challenge, imipenem and kanamycin were unable to rescue larvae. The most effective antibiotic for the prevention or treatment of disease was ceftazidime. Pharmacokinetic properties of a single dose of these antibiotics in G. mellonella larvae were also determined, and it was demonstrated that this model is useful for approximating the antibiotic response in humans. The G. mellonella model was used to screen a panel of novel antimicrobials for activity towards B. thailandensis and Burkholderia pseudomallei, and three novel compounds with antibiotic activity were identified. These results support the hypothesis that G. mellonella can be used to screen antimicrobial efficacy. This is the first study to determine the pharmacokinetic parameters of clinically relevant antibiotics in this model system.


Assuntos
Antibacterianos/farmacocinética , Antibacterianos/uso terapêutico , Burkholderia pseudomallei/efeitos dos fármacos , Modelos Animais de Doenças , Lepidópteros/efeitos dos fármacos , Melioidose/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Burkholderia pseudomallei/patogenicidade , Humanos , Larva/efeitos dos fármacos , Larva/microbiologia , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/microbiologia , Melioidose/microbiologia
12.
J Biol Chem ; 288(5): 3512-22, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23239883

RESUMO

NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Clostridium perfringens/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Galinhas , Colesterol/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Fosfolipídeos/metabolismo , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Solubilidade , Eletricidade Estática
13.
Science ; 334(6057): 821-4, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-22076380

RESUMO

The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei, reveals a similarity to Escherichia coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of glutamine-339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia lethal factor 1.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Burkholderia pseudomallei/química , Burkholderia pseudomallei/patogenicidade , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Citotoxinas/química , Citotoxinas/genética , Citotoxinas/metabolismo , Citotoxinas/toxicidade , Proteínas de Escherichia coli/química , Fator de Iniciação 4A em Eucariotos/metabolismo , Glutamina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Proteínas Mutantes/toxicidade , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
FEBS J ; 278(23): 4589-601, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21518257

RESUMO

Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.


Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Animais , Toxinas Bacterianas/toxicidade , Clostridium perfringens/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Ratos , Sinaptossomos/metabolismo
15.
Hum Mol Genet ; 18(1): 65-74, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18826960

RESUMO

Maintenance of an intact mitochondrial genome is essential for oxidative phosphorylation in all eukaryotes. Depletion of mitochondrial genome copy number can have severe pathological consequences due to loss of respiratory capacity. In Saccharomyces cerevisiae, several bifunctional metabolic enzymes have been shown to be required for mitochondrial DNA (mtDNA) maintenance. For example, Ilv5 is required for branched chain amino acid biosynthesis and mtDNA stability. We have identified OXA1 and TIM17 as novel multicopy suppressors of mtDNA instability in ilv5 cells. In addition, overexpression of TIM17, but not OXA1, prevents the complete loss of mtDNA in cells lacking the TFAM homologue Abf2. Introduction of the disease-associated A3243G mutant mtDNA into human NT2 teratocarcinoma cells frequently causes mtDNA loss. Yet when human TIM17A is overexpressed in NT2 cybrids carrying A3243G mtDNA, the proportion of cybrid clones maintaining mtDNA increases significantly. TIM17A overexpression results in long-term mtDNA stabilization, since NT2 cybrids overexpressing TIM17A maintain mtDNA at levels similar to controls for several months. Tim17 is a conserved suppressor of mtDNA instability and is the first factor to be identified that can prevent mtDNA loss in a human cellular model of mitochondrial disease.


Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/genética , Doenças Mitocondriais/genética , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Mutação Puntual , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Células Tumorais Cultivadas
16.
Hum Mol Genet ; 16(19): 2306-14, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17666405

RESUMO

Rearrangements of mitochondrial DNA (mtDNA) are a well-recognized cause of human disease; deletions are more frequent, but duplications are more readily transmitted to offspring. In theory, partial duplications of mtDNA can be resolved to partially deleted and wild-type (WT) molecules, via homologous recombination. Therefore, the yeast CCE1 gene, encoding a Holliday junction resolvase, was introduced into cells carrying partially duplicated or partially triplicated mtDNA. Some cell lines carrying the CCE1 gene had substantial amounts of WT mtDNA suggesting that the enzyme can mediate intramolecular recombination in human mitochondria. However, high levels of expression of CCE1 frequently led to mtDNA loss, and so it is necessary to strictly regulate the expression of CCE1 in human cells to ensure the selection and maintenance of WT mtDNA.


Assuntos
DNA Mitocondrial/genética , Resolvases de Junção Holliday/metabolismo , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Western Blotting , Linhagem Celular Tumoral , Dosagem de Genes , Resolvases de Junção Holliday/genética , Humanos , Microscopia Confocal , Reação em Cadeia da Polimerase , Proteínas de Saccharomyces cerevisiae/genética , Transfecção
17.
Rejuvenation Res ; 9(4): 455-69, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17105386

RESUMO

Artificial transfer of mitochondrial genes to the nucleus has implications for the understanding of mitochondrial function, evolution, and human health. Therefore, we created nuclear compatible versions of human subunit a (A6) of ATP synthase, linked to a mitochondrial targeting signal. Expression and targeting of human nuclear subunit a were compared to subunit a of Chlamydomonas reinhardtii, which naturally occurs in the nucleus. Algal subunit a was targeted to mitochondria more efficiently than human nuclear subunit a variants. However, there was no evidence of improved mitochondrial function in cultured cells; on the contrary, long-term expression of algal subunit a was associated with poor survival and intolerance of growth conditions that demand heavy reliance on oxidative phosphorylation. Analysis of enriched mitochondrial membrane fractions on native gels revealed a high-molecular- weight complex containing FLAG-tagged subunit a; however, this complex did not colocalize with ATP synthase. Thus, there was no evidence of assembly of algal subunit a into holoenzyme, nor did human nuclear subunit a colocalize with ATP synthase holoenzyme. In conclusion, obstacles remain to functional expression of mitochondrial genes transferred to the nucleus.


Assuntos
Proteínas de Algas/fisiologia , Chlamydomonas reinhardtii , DNA Mitocondrial/fisiologia , Células Eucarióticas/enzimologia , Técnicas de Transferência de Genes , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Transporte Proteico/fisiologia
18.
Hum Mol Genet ; 13(24): 3219-27, 2004 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-15509589

RESUMO

Mechanisms of mitochondrial DNA (mtDNA) maintenance have recently gained wide interest owing to their role in inherited diseases as well as in aging. Twinkle is a new mitochondrial 5'-3' DNA helicase, defects of which we have previously shown to underlie a mitochondrial disease, progressive external ophthalmoplegia with multiple mtDNA deletions. Mouse Twinkle is highly similar to the human counterpart, suggesting conserved function. Here, we have characterized the mouse Twinkle gene and expression profile and report that the expression patterns are not conserved between human and mouse, but are synchronized with the adjacent gene MrpL43, suggesting a shared promoter. To elucidate the in vivo role of Twinkle in mtDNA maintenance, we generated two transgenic mouse lines overexpressing wild-type Twinkle. We could demonstrate for the first time that increased expression of Twinkle in muscle and heart increases mtDNA copy number up to 3-fold higher than controls, more than any other factor reported to date. Additionally, we utilized cultured human cells and observed that reduced expression of Twinkle by RNA interference mediated a rapid drop in mtDNA copy number, further supporting the in vivo results. These data demonstrate that Twinkle helicase is essential for mtDNA maintenance, and that it may be a key regulator of mtDNA copy number in mammals.


Assuntos
DNA Primase/metabolismo , DNA Mitocondrial , Dosagem de Genes , Mitocôndrias/genética , Animais , DNA Helicases , DNA Primase/genética , Perfilação da Expressão Gênica , Humanos , Camundongos , Proteínas Mitocondriais , Interferência de RNA
19.
Mol Microbiol ; 46(2): 439-52, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12406220

RESUMO

Progress towards understanding the molecular basis of virulence in Clostridium difficile has been hindered by the lack of effective gene transfer systems. We have now, for the first time, developed procedures that may be used to introduce autonomously replicating vectors into this organism through their conjugative, oriT-based mobilization from Escherichia coli donors. Successful transfer was achieved through the use of a plasmid replicon isolated from an indigenous C. difficile plasmid, pCD6, and through the characterization and subsequent circumvention of host restriction/modification (RM) systems. The characterized replicon is the first C. difficile plasmid replicon to be sequenced and encodes a large replication protein (RepA) and a repetitive region composed of a 35 bp iteron sequence repeated seven times. Strain CD6 has two RM systems, CdiCD6I/M.CdiCD6I and CdiCD6II/M. CdiCD6II, with equivalent specificities to Sau96I/M. Sau96I (5'-GGNMCC-3') and MboI/M. MboI (5'-GMATC-3') respectively. A second strain (CD3) possesses a type IIs restriction enzyme, Cdi I, which cleaves the sequence 5'-CATCG-3' between the fourth and fifth nucleotide to give a blunt-ended fragment. This is the first time that an enzyme with this specificity has been reported. The sequential addition of this site to vectors showed that each site caused between a five- and 16-fold reduction in transfer efficiency. The transfer efficiencies achieved with both strains equated to between 1.0 x 10-6 and 5.5 x 10-5 transconjugants per donor.


Assuntos
Clostridium difficile/genética , Conjugação Genética , Enzimas de Restrição-Modificação do DNA/metabolismo , Escherichia coli/genética , Vetores Genéticos , Sequência de Aminoácidos , Sequência de Bases , Replicação do DNA , Dados de Sequência Molecular , Plasmídeos , Origem de Replicação , Replicon
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