RESUMO
Intermolecular interactions in several dimer aromatic systems were analyzed to determine how various energy contributions (electrostatic, exchange, repulsion, and polarization) change depending on the value of monomers separation. Different contributions to the intermolecular energy interactions between imidazole-imidazole and benzene-imidazole dimers are studied using the aug-cc-pVDZ basis set in the framework of ab initio Hartree-Fock and second-order Møller-Plesset perturbation theory methods. Special attention is paid to the exchange and dispersion energy binding contributions.
Assuntos
Benzeno/química , Imidazóis/química , TermodinâmicaRESUMO
Mutations in the presenilin genes cause the majority of early-onset familial Alzheimer's disease. Recently, presenilin mutations have been identified in patients with dilated cardiomyopathy (DCM), a common cause of heart failure and the most prevalent diagnosis in cardiac transplantation patients. However, the molecular mechanisms, by which presenilin mutations lead to either AD or DCM, are not yet understood. We have employed transgenic Drosophila models and optical coherence tomography imaging technology to analyze cardiac function in live adult Drosophila. Silencing of Drosophila ortholog of presenilins (dPsn) led to significantly reduced heart rate and remarkably age-dependent increase in end-diastolic vertical dimensions. In contrast, overexpression of dPsn increased heart rate. Either overexpression or silencing of dPsn resulted in irregular heartbeat rhythms accompanied by cardiomyofibril defects and mitochondrial impairment. The calcium channel receptor activities in cardiac cells were quantitatively determined via real-time RT-PCR. Silencing of dPsn elevated dIP3R expression, and reduced dSERCA expression; overexprerssion of dPsn led to reduced dRyR expression. Moreover, overexpression of dPsn in wing disc resulted in loss of wing phenotype and reduced expression of wingless. Our data provide novel evidence that changes in presenilin level leads to cardiac dysfunction, owing to aberrant calcium channel receptor activities and disrupted Wnt signaling transduction, indicating a pathogenic role for presenilin mutations in DCM pathogenesis.
Assuntos
Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Drosophila melanogaster/genética , Presenilinas/genética , Doença de Alzheimer/genética , Animais , Animais Geneticamente Modificados , Western Blotting , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Drosophila/genética , Reação em Cadeia da Polimerase em Tempo Real , Tomografia de Coerência Óptica , Via de Sinalização Wnt/genéticaRESUMO
A laser flash photolysis-resonance fluorescence technique has been employed to study the kinetics of the reaction of atomic chlorine with pyridine (C(5)H(5)N) as a function of temperature (215-435 K) and pressure (25-250 Torr) in nitrogen bath gas. At T> or = 299 K, measured rate coefficients are pressure independent and a significant H/D kinetic isotope effect is observed, suggesting that hydrogen abstraction is the dominant reaction pathway. The following Arrhenius expression adequately describes all kinetic data at 299-435 K for C(5)H(5)N: k(1a) = (2.08 +/- 0.47) x 10(-11) exp[-(1410 +/- 80)/T] cm(3) molecule(-1) s(-1) (uncertainties are 2sigma, precision only). At 216 K < or =T< or = 270 K, measured rate coefficients are pressure dependent and are much faster than computed from the above Arrhenius expression for the H-abstraction pathway, suggesting that the dominant reaction pathway at low temperature is formation of a stable adduct. Over the ranges of temperature, pressure, and pyridine concentration investigated, the adduct undergoes dissociation on the time scale of our experiments (10(-5)-10(-2) s) and establishes an equilibrium with Cl and pyridine. Equilibrium constants for adduct formation and dissociation are determined from the forward and reverse rate coefficients. Second- and third-law analyses of the equilibrium data lead to the following thermochemical parameters for the addition reaction: Delta(r)H = -47.2 +/- 2.8 kJ mol(-1), Delta(r)H = -46.7 +/- 3.2 kJ mol(-1), and Delta(r)S = -98.7 +/- 6.5 J mol(-1) K(-1). The enthalpy changes derived from our data are in good agreement with ab initio calculations reported in the literature (which suggest that the adduct structure is planar and involves formation of an N-Cl sigma-bond). In conjunction with the well-known heats of formation of atomic chlorine and pyridine, the above Delta(r)H values lead to the following heats of formation for C(5)H(5)N-Cl at 298 K and 0 K: Delta(f)H = 216.0 +/- 4.1 kJ mol(-1), Delta(f)H = 233.4 +/- 4.6 kJ mol(-1). Addition of Cl to pyridine could be an important atmospheric loss process for pyridine if the C(5)H(5)N-Cl product is chemically degraded by processes that do not regenerate pyridine with high yield.
Assuntos
Físico-Química/métodos , Cloro/química , Piridinas/química , Compostos Clorados/química , Temperatura Alta , Hidrogênio/química , Cinética , Modelos Químicos , Modelos Teóricos , Estrutura Molecular , Fotólise , Pressão , Temperatura , Termodinâmica , Fatores de TempoRESUMO
A laser flash photolysis-resonance fluorescence technique has been employed to study the kinetics of the reaction of chlorine atoms with dimethyl sulfoxide (CH3S(O)CH3; DMSO) as a function of temperature (270-571 K) and pressure (5-500 Torr) in nitrogen bath gas. At T = 296 K and P > or = 5 Torr, measured rate coefficients increase with increasing pressure. Combining our data with literature values for low-pressure rate coefficients (0.5-3 Torr He) leads to a rate coefficient for the pressure independent H-transfer channel of k1a = 1.45 x 10(-11) cm3 molecule(-1) s(-1) and the following falloff parameters for the pressure-dependent addition channel in N2 bath gas: k(1b,0) = 2.53 x 10(-28) cm6 molecule(-2) s(-1); k(1b,infinity) = 1.17 x 10(-10) cm3 molecule(-1) s(-1), F(c) = 0.503. At the 95% confidence level, both k1a and k1b(P) have estimated accuracies of +/-30%. At T > 430 K, where adduct decomposition is fast enough that only the H-transfer pathway is important, measured rate coefficients are independent of pressure (30-100 Torr N2) and increase with increasing temperature. The following Arrhenius expression adequately describes the temperature dependence of the rate coefficients measured at over the range 438-571 K: k1a = (4.6 +/- 0.4) x 10(-11) exp[-(472 +/- 40)/T) cm3 molecule(-1) s(-1) (uncertainties are 2sigma, precision only). When our data at T > 430 K are combined with values for k1a at temperatures of 273-335 K that are obtained by correcting reported low-pressure rate coefficients from discharge flow studies to remove the contribution from the pressure-dependent channel, the following modified Arrhenius expression best describes the derived temperature dependence: k1a = 1.34 x 10(-15)T(1.40) exp(+383/T) cm3 molecule(-1) s(-1) (273 K < or = T < or = 571 K). At temperatures around 330 K, reversible addition is observed, thus allowing equilibrium constants for Cl-DMSO formation and dissociation to be determined. A third-law analysis of the equilibrium data using structural information obtained from electronic structure calculations leads to the following thermochemical parameters for the association reaction: delta(r)H(o)298 = -72.8 +/- 2.9 kJ mol(-1), deltaH(o)0 = -71.5 +/- 3.3 kJ mol(-1), and delta(r)S(o)298 = -110.6 +/- 4.0 J K(-1) mol(-1). In conjunction with standard enthalpies of formation of Cl and DMSO taken from the literature, the above values for delta(r)H(o) lead to the following values for the standard enthalpy of formation of Cl-DMSO: delta(f)H(o)298 = -102.7 +/- 4.9 kJ mol(-1) and delta(r)H(o)0 = -84.4 +/- 5.8 kJ mol(-1). Uncertainties in the above thermochemical parameters represent estimated accuracy at the 95% confidence level. In agreement with one published theoretical study, electronic structure calculations using density functional theory and G3B3 theory reproduce the experimental adduct bond strength quite well.
Assuntos
Bacillus anthracis/metabolismo , Técnicas de Química Analítica/métodos , Cromatografia Gasosa/métodos , Ésteres/química , Ácidos Graxos/química , Ágar/química , Calibragem , Meios de Cultura/farmacologia , Metilação , Modelos Estatísticos , Controle de Qualidade , Glycine max/química , Esporos Bacterianos/metabolismo , Fatores de TempoRESUMO
Two pathways have been considered in the fluorination of CB(5)H(6)(-) and CB(9)H(10)(-) by HF. In the ionic HF fluorination pathway, the monocarborane anion cage is first protonated in a BBB face followed by H(2) elimination and fluoride anion addition. In the covalent HF fluorination pathway, HF is first coordinated through hydrogen to the BBB face. Next, the fluorine can add to either an axial or equatorial boron atom which opens the cage to a nido structure with an endo fluoride substituent. Endo to exo rearrangement occurs with a small activation barrier followed by H(2) elimination. In both pathways, fluorination at the equatorial boron position is predicted to have smaller activation barriers even though substitution at the axial position leads to the more stable products.
RESUMO
The potential energy surface for the reaction of atomic carbon with thiophene has been studied computationally. Intermediates which are energetically viable include the 2- and 3-thienylcarbenes 8 and 11, thiacyclohexa-3,5-dien-2-ylidene, 10, and thiacyclohexa-2,3,5-triene, 6. In accord with experimental data, 6 and 8 are in equilibrium. The lowest-energy pathway for rearrangement of 6 to 8, which is endothermic by 14.5 kcal/mol, involves ring opening to Z-2-penten-4-ynthial which then recloses to carbene 8. A 1,4 addition of C across the diene system in thiophene generates an ylid which rearranges with little or no barrier to cyclopentadienethione, the global minimum on this potential energy surface.
RESUMO
In the presence of Fe(+) catalyst, the retro Diels-Alder reaction of norbornadiene (NBD) is predicted to be stepwise with an activation barrier of 18.8 kcal/mol, which is 3.1 kcal/mol lower than the concerted retro reaction. For norbornene (NBN), the Fe(+)-catalyzed retro reaction is also calculated to be stepwise with an activation barrier of 24.9 kcal/mol, which is 8.5 kcal/mol lower than the uncatalyzed stepwise reaction but 3.8 kcal/mol higher than the concerted reaction. The intermediates from the NBD and NBN retro Diels-Alder reactions, C(5)H(6)FeC(2)H(2)(+) and C(5)H(6)FeC(2)H(4)(+), are predicted to have low activation barriers for ligand-to-ligand hydrogen transfers (through an iron-hydrido intermediate) to form CpFeC(2)H(3)(+) and CpFeC(2)H(5)(+) and, ultimately, vinyl- and ethyl-substituted cyclopentadiene-iron complexes, respectively. In contrast to FeC(2)H(2)(+) and FeC(2)H(4)(+), the lowest-energy pathways on the C(5)H(6)FeC(2)H(2)(+) and C(5)H(6)FeC(2)H(4)(+) potential energy surfaces involve only one multiplicity (quartet). The C(2)H(2) and C(2)H(4) complexes of CpFe(+) and C(5)H(6)Fe(+) are compared.
RESUMO
The reactions of OH with CS2, OCS, and 3SO and of 3O2 with CS2, SCSOH, and HOSO have been studied by optimizing minima and transition states with B3LYP/6-31+G(d) and carrying out higher-level ab initio calculations on fixed geometries. The combined calculations provide valuable insight into the mechanism for the atmospheric oxidation of CS2. The initial step is the formation of the SCSOH complex (1) which readily adds molecular oxygen to form the SC(OO)SOH complex (8). A key step is the oxygen atom transfer to the sulfur bearing the hydroxyl group which leads directly to OCS plus HOSO. The HOSO + 3O2 reaction has a near zero calculated activation barrier so generation of O2H + SO2 should proceed readily in the atmosphere.
Assuntos
Dissulfeto de Carbono/metabolismo , OxirreduçãoRESUMO
A thorough study of the reaction of singlet oxygen with 1,3-cyclohexadiene has been made at the B3LYP/6-31G(d) and CASPT2(12e,10o) levels. The initial addition reaction follows a stepwise diradical pathway to form cyclohexadiene endoperoxide with an activation barrier of 6.5 kcal/mol (standard level = CASPT2(12e,10o)/6-31G(d); geometries and zero-point corrections at B3LYP/6-31G(d)), which is consistent with an experimental value of 5.5 kcal/mol. However, as the enthalpy of the transition structure for the second step is lower than the diradical intermediate, the reaction might also be viewed as a nonsynchronous concerted reaction. In fact, the concertedness of the reaction is temperature dependent since entropy differences create a free energy barrier for the second step of 1.8 kcal/mol at 298 K. There are two ene reactions; one is a concerted mechanism (DeltaH(double dagger) = 8.8 kcal/mol) to 1-hydroperoxy-2,5-cyclohexadiene (5), while the other, which forms 1-hydroperoxy-2,4-cyclohexadiene (18), passes through the same diradical intermediate (9) as found on the pathway to endoperoxide. The major pathway from the endoperoxide is O-O bond cleavage (22.0 kcal/mol barrier) to form a 1,4-diradical (25), which is 13.9 kcal/mol less stable than the endoperoxide. From the diradical, two low-energy pathways exist, one to epoxyketone (29) and the other to the diepoxide (27), where both products are known to be formed experimentally with a product ratio sensitive to the nature of substitutents. A significantly higher activation barrier leads to C-C bond cleavage and direct formation of maleic aldehyde plus ethylene.
RESUMO
Pseudomonas aeruginosa exotoxin A (PE) is a 67-kDa protein expressed under the selective pressure of a low iron environment. Previous studies using non-toxic PE chimeras containing a viral surface antigen, the V3 loop of MN gp120 from human immunodeficiency virus type 1 (HIV-1), resulted in not only an effective mucosal immunization but also a striking systemic immune response following epithelial application. Presently, we have examined the possibility that such a strong dual immune response was generated by the efficient targeting of critical cells of the immune system. Mice were dosed with 10 microg of toxic PE or a non-toxic mutant of PE (ntPE) by intratracheal instillation. Examination of lung, liver and spleen tissues isolated 4, 8 and 12 h following intratracheal instillation with PE demonstrated specific cell damage in these tissues which was not observed in mice dosed with ntPE. Based upon the location and characteristics of observed responses, the cells targeted by PE appear to be involved in the antigen presentation arm of the immune response. Since ntPE chimeras with inserted peptide antigen epitopes from a wide variety of pathogens are easy to prepare and administer, these results support this approach for mucosal immunization.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas/administração & dosagem , Sistemas de Liberação de Medicamentos , Exotoxinas/administração & dosagem , Fígado/metabolismo , Linfonodos/metabolismo , Baço/metabolismo , Fatores de Virulência , Administração Tópica , Animais , Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/farmacologia , Células Epiteliais/metabolismo , Exotoxinas/farmacocinética , Exotoxinas/farmacologia , Feminino , Sistema Imunitário/metabolismo , Fígado/citologia , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Pseudomonas aeruginosa/metabolismo , Baço/citologia , Traqueia/citologia , Traqueia/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMO
A comprehensive survey of the (CH)2(BH)2 potential energy surface was carried out at the [MP4/6-311 + G(d,p)]//MP2/6-31G(d) level. Many of the classical and nonclassical isomers of the carborane surface are separated by high activation barriers, which explains why derivatives of most isomers could be prepared as stable compounds at room temperature. The transition states are grouped into two types, hydrogen migration (terminal-to-bridge and bridge-to-terminal) and group migration (BH, CH, and CH2). The rearrangement of 1,3-diamino-1,3-diboretene (1-NH2) to 1,2-diamino-1,2-diboretene (2-NH2) was computed and compared to the rearrangement in the parent (1-->2). The effect of the amino group is to substantially increase the barrier height and stabilize the product, 2-NH2.
RESUMO
Upon entering mammalian cells, Pseudomonas exotoxin A (PE) is proteolytically processed by furin to produce an N-terminal fragment of 28 kDa and a C-terminal fragment of 37 kDa. Cleavage is followed by the reduction of a key disulfide bond (cysteines 265-287). This combination of proteolysis and reduction releases the 37 kDa C-terminal fragment, which then translocates to the cytosol where it ADP-ribosylates elongation factor 2 and inhibits protein synthesis. To investigate toxin reduction, furin-nicked PE or a hypercleavable mutant, PEW281A, was subjected to various treatments and then analyzed for fragment production. Reduction was evident only when unfolding conditions and a reducing agent were applied. Thermal unfolding of PE, as evidenced by changes in alpha-helical content and increased sensitivity to trypsin, rendered nicked toxin susceptible to protein disulfide isomerase- (PDI-) mediated reduction. When subcellular fractions from toxin-sensitive cells were incubated with nicked PE, toxin unfolding and reducing activities were present in the membrane fraction but not the soluble fraction. These data indicate that PE reduction is a two-step process: unfolding that allows access to the Cys265-287 disulfide bond, followed by reduction of the sulfur-sulfur bond by PDI or a PDI-like enzyme. With regard to cellular processing, we propose that the toxin's three-dimensional structure retains a "closed" conformation that restricts solvent access to the Cys265-287 disulfide bond until after a cell-mediated unfolding event.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas/química , Exotoxinas/química , Dobramento de Proteína , Subtilisinas/química , Fatores de Virulência , Animais , Toxinas Bacterianas/metabolismo , Células CHO , Fracionamento Celular , Cricetinae , Exotoxinas/metabolismo , Furina , Temperatura Alta , Hidrólise , Células L , Camundongos , Oxirredução , Pseudomonas aeruginosa , Subtilisinas/metabolismo , Tripsina/química , Exotoxina A de Pseudomonas aeruginosaRESUMO
We examined the development of the corticostriate pathway in mice by labeling corticofugal axons with the carbocyanine dye 1, 1'-dioctadecyl-3,3,3'-3'-tetramethylindocarbocyanine perchlorate (DiI). Growth cones of corticofugal axons enter the developing striatum on embryonic day 12 (E12; conception is on E0). By E15 corticofugal axons pass through the developing striatum in the internal capsule but do not produce striatal collaterals. Corticostriate collaterals are seen for the first time on E18, 6 days after the earliest arriving axons enter the striatum. At that time, presumptive synaptic contacts form between cortical axons and striatal neurons. Corticostriate collaterals arise from corticofugal axon trunks at or near axonal varicosities. Primitive corticostriate arbors form by postnatal day 2 (P2; day of birth is P0) and develop further by P7. Thus, corticostriate connections develop in three morphologically defined stages: first cortical axons elongate through the striatum to other subcortical targets, next they produce striatal collaterals, and finally they elaborate terminal arbors. The transition from elongation to collateralization stage may be triggered, among other factors, by signals from striatal neurons relayed via the synaptic contacts.
Assuntos
Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Corpo Estriado/embriologia , Corpo Estriado/crescimento & desenvolvimento , Vias Eferentes/embriologia , Vias Eferentes/crescimento & desenvolvimento , 3,3'-Diaminobenzidina , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Axônios/fisiologia , Axônios/ultraestrutura , Carbocianinas , Dendritos/fisiologia , Dendritos/ultraestrutura , Desenvolvimento Embrionário e Fetal/fisiologia , Corantes Fluorescentes , Camundongos , Camundongos Endogâmicos , Microscopia EletrônicaRESUMO
To develop a candidate vaccine for human immunodeficiency virus, type 1 (HIV-1), chimeric proteins were constructed by inserting sequences derived from the V3 loop of gp120 into a nontoxic form of Pseudomonas exotoxin (PE). Inserts of 14 or 26 amino acids, constrained by a disulfide bond, were introduced between domains II and III of PE. V3 loop-toxin proteins expressed in Escherichia coli and corresponding to either MN (subtype B) or Thai (subtype E) strains, were recognized by strain-specific monoclonal anti-gp120 antibodies. When loop sequences were introduced into an enzymatically active form of the toxin, there was no loss of toxin-mediated cell killing, suggesting that these sequences were co-transported to the cytosol. Sera from rabbits injected with nontoxic PE-V3 loop chimeras were reactive for strain-specific gp120s in Western blots, immunocapture assays, enzyme-linked immunosorbent assays, and neutralized HIV-1 infectivity. Since toxin vectors were designed to receive oligonucleotide duplexes encoding any V3 loop sequence, this approach should allow for the production of V3 loop-toxin chimeras corresponding to multiple HIV isolates.
Assuntos
ADP Ribose Transferases , Vacinas contra a AIDS , Toxinas Bacterianas , Exotoxinas/toxicidade , Proteína gp120 do Envelope de HIV/toxicidade , HIV-1/efeitos dos fármacos , Pseudomonas aeruginosa , Vacinas Sintéticas , Fatores de Virulência , Síndrome da Imunodeficiência Adquirida/sangue , Síndrome da Imunodeficiência Adquirida/imunologia , Sequência de Aminoácidos , Animais , Carcinoma de Células Escamosas , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Exotoxinas/química , Exotoxinas/imunologia , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Conformação Proteica , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/toxicidade , Células Tumorais Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMO
Intimin, the product of the eaeA gene in enterohemorrhagic Escherichia coli O157:H7 (EHEC), is required for intimate adherence of these organisms to tissue culture cells and formation of the attaching and effacing lesion in the gnotobiotic pig. Because of the importance of intimin in the pathogenesis of EHEC O157:H7 infection in this animal model, we began a structure-function analysis of EaeA. For this purpose, we constructed amino-terminal fusions of the intimin protein with six histidine residues to form two independent fusions. The longer fusion, RIHisEae, contained 900 of the 935 predicted amino acids and included all but the extreme amino terminus. The second fusion, RVHdHisEae, consisted of the carboxyl two-thirds of the protein. Purified extracts of either construct enhanced binding of wild-type 86-24 to HEp-2 cells and conferred HEp-2 cell adherence on 86-24eaeDelta10, an eaeA deletion mutant, and B2F1, an EHEC O91:1-121 eaeA mutant strain. When 86-24eaeDelta10 was transformed with either of the plasmids encoding the intimin fusion proteins, the transformant behaved like the wild-type parent strain and displayed localized adherence to HEp-2 cells, with positive fluorescent-actin staining. In addition, polyclonal antisera raised against RIHisEae reacted with both fusion constructs and recognized an outer membrane protein of the same mass as intimin (97 kDa) in EHEC and enteropathogenic E. coli but not E. coli K-12. The intimin-specific antisera also blocked adherence of EHEC to HEp-2 cells. Thus, intimin (i) is a 97-kDa outer membrane protein in EHEC that serves as a requisite adhesin for attachment of the bacteria to epithelial cells, even when the protein is truncated by one-third at its amino terminus and (ii) can be added exogenously to specifically facilitate HEp-2 cell adherence of EHEC but not E. coli K-12.
Assuntos
Adesinas Bacterianas , Aderência Bacteriana/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/farmacologia , Proteínas de Transporte , Proteínas de Escherichia coli , Escherichia coli/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Feminino , Humanos , Soros Imunes/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Regiões Promotoras Genéticas , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insertion in the eaeA gene encoding intimin was used to establish that intimin is required for the organism to attach to and efface microvilli in the piglet intestine (M. S. Donnenberg, S. Tzipori, M. L. McKee, A. D. O'Brien, J. Alroy, and J. B. Kaper, J. Clin. Invest. 92:1418-1424, 1993). However, in the same investigation, a role for intimin in EHEC adherence to HEp-2 cells could not be definitively demonstrated. To analyze the basis for this discrepancy, we constructed an in-frame deletion of eaeA and compared the adherence capacity of this mutant with that of the wild-type strain in vitro and in vivo. We observed a direct correlation between the requisite for intimin in EHEC O157:H7 colonization of the gnotobiotic piglet intestine and adherence of the bacterium to HEp-2 cells. The in vitro-in vivo correlation lends credence to the use of the HEp-2 cell adherence model for further study of the intimin protein.
Assuntos
Adesinas Bacterianas , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Transporte , Proteínas de Escherichia coli , Escherichia coli/patogenicidade , Intestinos/microbiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Linhagem Celular , Vida Livre de Germes , Dados de Sequência Molecular , SuínosRESUMO
In this study, the interactions of enterohemorrhagic Escherichia coli (EHEC) O157 strains with human ileocecal (HCT-8) epithelial cells and HEp-2 cells were examined. EHEC adhered to, but did not invade, HCT-8 cells by the localized adherence mechanism and a heretofore unrecognized pattern which we called log jam. The log jam formation was (i) not observed on HEp-2 cells, (ii) independent of the EHEC eaeA gene required for localized adherence, and (iii) shared by pathogenic and nonpathogenic E. coli strains but not K-12 strains. The log jam phenotype may represent a basal means by which E. coli bacteria attach to the human intestine.
Assuntos
Aderência Bacteriana/imunologia , Escherichia coli/patogenicidade , Intestinos/microbiologia , Ceco/citologia , Células Cultivadas , Escherichia coli/classificação , Hemorragia , Humanos , Íleo/citologia , Laringe/citologia , Antígenos O , Polissacarídeos Bacterianos , SorotipagemRESUMO
We analyzed Escherichia coli O157:H7 isolates from stool samples of five patients who had bloody diarrhea and were infected during a large food-borne outbreak of hemorrhagic colitis in Washington state. The isolates were assessed for Shiga-like toxin profile, adherence and plasmid traits, mouse virulence, capsule, and enterohemolysin production. The profiles of the five isolates were indistinguishable from each other and similar to that of E. coli O157:H7 strain EDL933, an organism responsible for a similar hamburger-associated food poisoning episode in 1982.
Assuntos
Surtos de Doenças , Escherichia coli/patogenicidade , Microbiologia de Alimentos , Hemorragia Gastrointestinal/epidemiologia , Síndrome Hemolítico-Urêmica/epidemiologia , Carne/intoxicação , Adolescente , Adulto , Animais , Toxinas Bacterianas/análise , Criança , Feminino , Hemorragia Gastrointestinal/etiologia , Síndrome Hemolítico-Urêmica/etiologia , Humanos , Masculino , Camundongos , Toxina Shiga II , Virulência , WashingtonRESUMO
The eaeA gene of enteropathogenic Escherichia coli (EPEC) is necessary for intimate attachment to epithelial cells in vitro. Enterohemorrhagic E. coli (EHEC) strains also possess an eae gene and are capable of intimate attachment and microvillus effacement in vitro and in animal models. To assess the role of the EHEC eae gene in intimate attachment, we constructed an eae deletion/insertion mutation in wild-type EHEC O157:H7 strain 86-24 by using linear electroporation of a recombinant allele. The mutant obtained was deficient in inducing f-actin accumulation in HEp-2 cells and was incapable of attaching intimately to colonic epithelial cells in a newborn piglet model of infection. Intimate attachment in vivo was restored when the EHEC eae gene or the eaeA gene of EPEC was introduced into the mutant on a plasmid. These results indicate that the eae gene is necessary for intimate attachment of EHEC in vivo. In addition, the complementation achieved by the EPEC locus indicates that the eae gene of EHEC and the eaeA gene of EPEC are functionally homologous.