Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line.

Serosal application of carbachol to T84 cell monolayers mounted in an Ussing chamber caused an immediate increase in short circuit current (Isc) that peaked within 5 min and declined rapidly thereafter, although a small increase in Isc persisted for approximately 30 min. The increase in Isc was detectable with 1 microM carbachol; half-maximal with 10 microM carbachol; and maximal with 100 microM carbachol. Unidirectional Na+ and Cl- flux measurements indicated that the increase in Isc was due to net Cl- secretion. Carbachol did not alter cellular cAMP, but caused a transient increase in free cytosolic Ca2+ ([Ca2+]i) from 117 +/- 7 nM to 160 +/- 15 nM. The carbachol-induced increase in Isc was potentiated by either prostaglandin E1 (PGE1) or vasoactive intestinal polypeptide (VIP), agents that act by increasing cAMP. Measurements of cAMP and [Ca2+]i indicated that the potentiated response was not due to changes in these second messengers. Studies of the effects of these agents on ion transport pathways indicated that carbachol, PGE1, or VIP each increased basolateral K+ efflux by activating two different K+ transport pathways on the basolateral membrane. The pathway activated by carbachol was not sensitive to barium, while that activated by PGE1 or VIP was; furthermore, their action on K+ efflux are additive. Our study indicates that carbachol causes Cl- secretion, and that this action may result from its ability to increase [Ca2+]i and basolateral K+ efflux. Carbachol's effect on Cl- secretion is greatly augmented in the presence of VIP or PGE1, which open a cAMP-sensitive Cl- channel on the apical membrane, accounting for a potentiated response.     

Mechanism of action of quinolones against Escherichia coli DNA gyrase.

The mechanism of action of quinolones was investigated by use of various DNA gyrases reconstituted from wild-type and mutant GyrA and GyrB proteins of Escherichia coli. The quinolone sensitivities of the DNA supercoiling activity of the gyrases were generally parallel to the quinolone susceptibilities of strains having the corresponding enzymes and depended on gyrase subunits but not on substrate DNA. [3H]Enoxacin did not bind to gyrase alone or DNA alone but bound to gyrase-DNA complexes when measured by a gel filtration method. There appeared to be two enoxacin binding phases, at low and high enoxacin concentrations, for the wild-type gyrase-DNA and type 2 GyrB (Lys-447 to Glu) mutant gyrase-DNA complexes but only one enoxacin binding phase at the concentrations used for the GyrA (Ser-83 to Leu) mutant gyrase-DNA and type 1 GyrB (Asp-426 to Asn) mutant gyrase-DNA complexes. New enoxacin binding sites appeared in the presence of enoxacin, and the enoxacin binding affinities for the sites, especially at low enoxacin concentrations, near the MICs for the strains having the corresponding gyrases, correlated well with the enoxacin sensitivities of the gyrases and the MICs. From the results obtained, we propose a quinolone pocket model as the mechanism of action of quinolones, in which quinolones exert their action through binding to a gyrase-DNA complex and the quinolone binding affinities for the complex are determined by both GyrA and GyrB subunits in concert.     

Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

Chemically transformed mouse fibroblasts did not raise their cyclic AMP level in response to Escherichia coli heat-labile enterotoxin. These fibroblasts did, however, incorporate exogenous mono-, di-, and trisialogangliosides. After the uptake of monosialoganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM1), the cells responded to E. coli heat-labile enterotoxin. The di- and trisialogangliosides were considerably less effective. GM1, the putative cholera toxin (choleragen) receptor, has been implicated previously as the receptor for E. coli heat-labile enterotoxin based on the ability of the free ganglioside to inhibit the effects of toxin. This investigation establishes that the ganglioside, when incorporated into fibroblasts, serves a functional role in mediating the responsiveness to the toxin.     

Synergistic action of cyclic adenosine monophosphate- and calcium-mediated chloride secretion in a colonic epithelial cell line

Vasoactive intestinal polypeptide (VIP) and the calcium ionophore A23187 caused dose-dependent changes in the potential difference and the short circuit current (Isc) across confluent T84 cell monolayers mounted in modified Ussing chambers. Both VIP and A23187 stimulated net chloride secretion without altering sodium transport. Net chloride secretion accounted for the increase in Isc. When A23187 was tested in combination with VIP, net chloride secretion was significantly greater than predicted from the calculated sum of their individual responses indicating a synergistic effect. VIP increased cellular cyclic AMP (cAMP) production in a dose-dependent manner, whereas A23187 had no effect on cellular cAMP. We then determined whether VIP and A23187 activated different transport pathways. Earlier studies suggest that VIP activates a basolaterally localized, barium-sensitive potassium channel as well as an apically localized chloride conductance pathway. In this study, stimulation of basolateral membrane potassium efflux by A23187 was documented by preloading the monolayers with 86Rb+. Stimulation of potassium efflux by A23187 was additive to the VIP-stimulated potassium efflux. By itself, 0.3 microM A23187 did not alter transepithelial chloride permeability, and its stimulation of basolateral membrane potassium efflux caused only a relatively small amount of chloride secretion. However, in the presence of an increased transepithelial chloride permeability induced by VIP, the effectiveness of A23187 on chloride secretion was greatly augmented. Our studies suggest that cAMP and calcium each activate basolateral potassium channels, but cAMP also activates an apically localized chloride channel. Synergism results from cooperative interaction of potassium channels and the chloride channel.     

Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity.

Defensins are small, cysteine-rich antimicrobial peptides that are abundant in human, rabbit, and guinea pig neutrophils (PMN). Three defensins (human neutrophil peptide defensin [HNP]-1, HNP-2, and HNP-3) constitute between 30 and 50% of the total protein in azurophil granules of human PMN. We examined the mechanism of HNP-mediated bactericidal activity against Escherichia coli ML-35 (i-, y-, z+) and its pBR322-transformed derivative, E. coli ML-35p. Under conditions that supported bactericidal activity, HNP-1 sequentially permeabilized the outer membrane (OM) and inner membrane (IM) of E. coli. Coincident with these events, bacterial synthesis of DNA, RNA, and protein ceased and the colony count fell. Although these events were closely coupled under standard assay conditions, OM permeabilization was partially dissociated from IM permeabilization when experiments were performed with E. coli that had been plasmolyzed by mannitol. Under such conditions, the rate and extent of bacterial death more closely paralled loss of IM integrity than OM permeabilization. Electron microscopy of E. coli that had been killed by defensins revealed the presence of striking electron-dense deposits in the periplasmic space and affixed to the OM. Overall, these studies show that HNP-mediated bactericidal activity against E. coli ML-35 is associated with sequential permeabilization of the OM and IM, and that inner membrane permeabilization appears to be the lethal event.     

Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model.

Enteroaggregative Escherichia coli (EAggEC) have been associated with persistent diarrhea in young children, but little is known about its pathogenesis. We assayed for enterotoxic activity in culture filtrates (CF) of EAggEC strains in Ussing chambers mounted with rabbit ileal mucosa. CF from strain 17-2, a prototype Chilean EAggEC strain, caused a greater rise in potential difference and short circuit current (SCC) than that seen in HB101 control, and this effect was abolished by protease pretreatment and partially stable after heat treatment. Ultrafiltration of 17-2 CF preparations localized the active moiety to the 2-5 kD Mr size range. CF from HB101 transformed with the 17-2 plasmid showed Ussing chamber activity. less than 10-kD CF fractions from five of six other EAggEC strains screened in Ussing chambers gave SCC responses of similar magnitude to 17-2. The 17-2 CF activity was not neutralized after pretreatment with polyclonal anti-STa antibody. Additionally, all of the seven EAggEC strains studied were nonreactive by heat-stable enterotoxin variant STa ELISA, were negative in the suckling mouse assay, and failed to hybridize with heat-stable enterotoxin variant STh and STp DNA probes. In summary, our data indicate that 17-2 produces a low molecular weight, partially heat-stable, protease-sensitive enterotoxin which appears to be plasmid associated, and genetically and immunologically distinct from E. coli STa. Preliminary screening suggests that this tox+ phenotype may be common among EAggEC.     

Intranasal immunization with influenza vaccine and a detoxified mutant of heat labile enterotoxin from Escherichia coli (LTK63).

Groups of 10 Balb/c mice were immunized intranasally (IN) with influenza haemagglutinin (HA), and a genetically detoxified mutant of heat-labile enterotoxin from Escherichia coli (LTK63) at several different doses. IN immunization at the optimal dose combination for HA and LTK63 induced equivalent levels of serum IgG antibodies to intramuscular (IM) immunization with HA alone, and induced significantly enhanced IgA titers in nasal wash. However, haemagglutination inhibition (HI) assays showed that the IM vaccine induced approximately 10-fold higher HI titers than IN immunization with HA and LTK63. In a second study, HA and LTK63 was compared to a licensed emulsion adjuvant MF59 by the IN route. LTK63 was shown to be significantly more potent than MF59 when evaluated at the optimal dose combination with HA. Hence, the LTK63 and HA combination represents an attractive candidate for evaluation as an IN vaccine in larger animal models, or humans.     

Cl- secretion induced by bile salts. A study of the mechanism of action based on a cultured colonic epithelial cell line.

When applied to the basolateral (serosal) side of the T84 colonic epithelial monolayer, taurodeoxycholate caused net Cl- secretion in a dose-dependent manner with a threshold effect observed at 0.2 mM. In contrast, when applied to the apical (luminal) surface, concentrations of taurodeoxycholate below 1 mM had little or no effect. Only when the concentration of taurodeoxycholate present on the apical side was greater than or equal to 1 mM did apical addition results in an electrolyte transport effect. This apical effect on electrolyte transport was associated with an abrupt increase in the permeability of the monolayer. Cyclic AMP and cyclic GMP in the T84 monolayers were not increased by the bile salt, but in the presence of extracellular Ca2+, free cytosolic Ca2+ increased with a graded dose effect and time course that corresponded approximately to the changes in short circuit current (Isc). The results suggest that luminal bile salts at a relatively high concentration (greater than or equal to 1 mM) increase tight junction permeability. Once tight junction permeability increases, luminal bile salts could reach the basolateral membrane of the epithelial cells where they act to increase free cytosolic Ca2+ from extracellular sources. The resulting increases in free cytosolic Ca2+, rather than in cyclic nucleotides, appear to be involved in transcellular Cl- secretion.     

金黄色葡萄球菌肠毒素B基因在大肠杆菌中的克隆及表达

目的扩增金黄色葡萄球菌肠毒素B(SEB)基因,构建其原核表达载体,并进行诱导、表达,为其应用研究奠定基础。方法根据GenBank中SEB的基因序列,设计一对分别含BamHⅠ、XhoⅠ酶切位点的特异性引物,以金黄色葡萄球菌基因组DNA为模板进行PCR扩增后,经BamHⅠ、XhoⅠ双酶切,并与做相应酶切的pET-28α(+)连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定及测序,用IPTG诱导表达融合蛋白,SDS-PAGE和Western blot印迹鉴定表达产物。结果成功扩增出SEB基因,基因大小为801bp,重组PET-28α(+)-SEB双酶切鉴定可见目的片段,测序结果显示SEB在正确读框中,序列比对分析显示其与相关报道核苷酸序列一致性达99%。经IPTG诱导后,pET-28α(+)-SEB/BL21在相应的相对分子质量(35×103)可见融合蛋白以包涵体形式表达,免疫印迹在相应分子量检测到目的蛋白。结论克隆了SEB基因,并成功在大肠杆菌BL21中以包涵体形式表达,为肠毒素B应用研究奠定了基础。     

Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line.

Confluent T84 monolayers grown on permeable supports and mounted in a modified Ussing chamber secrete chloride (Cl-) in response to prostaglandin E1. The threshold stimulation was observed at 10(-9) M and a maximal effect at 10(-6) M. Unidirectional flux studies showed an increase in both serosal to mucosal and mucosal to serosal Cl- fluxes with 10(-6) M prostaglandin E1; the increase in serosal to mucosal Cl- flux exceeded the increase in mucosal to serosal flux, resulting in net Cl- secretion. Na+ transport was not affected in either direction and the changes in net Cl- flux correlated well with the changes in short circuit current. To identify the electrolyte transport pathways involved in the Cl- secretory process, the effect of prostaglandin E1 on ion fluxes was tested in the presence of putative inhibitors. Bumetanide was used as an inhibitor for the basolaterally localized Na+,K+,Cl- cotransport system whose existence and bumetanide sensitivity have been verified in earlier studies (Dharmsathaphorn et al. 1984. J. Clin. Invest. 75:462-471). Barium was used as an inhibitor for the K+ efflux pathway on the basolateral membrane whose existence and barium sensitivity were demonstrated in this study by preloading the monolayers with 86Rb+ (as a tracer for K+) and simultaneously measuring 86Rb+ efflux into both serosal and mucosal reservoirs. Both bumetanide and barium inhibited the net chloride secretion induced by prostaglandin E1 suggesting the involvement of the Na+,K+,Cl- cotransport and a K+ efflux pathways on the basolateral membrane in the Cl- secretory process. The activation of another Cl- transport pathway on the apical membrane by prostaglandin E1 was suggested by Cl- uptake studies. Our findings indicate that the prostaglandin E1-stimulated Cl- secretion, which is associated with an increase in cyclic AMP level, intimately involves (a) a bumetanide-sensitive Na+,K+,Cl- cotransport pathway that serves as a Cl- uptake step across the basolateral membrane, (b) the stimulation of a barium-sensitive K+ efflux mechanism on the basolateral membrane that most likely acts to recycle K+, and (c) the activation of a Cl- transport pathway on the apical membrane that serves as a Cl- exit pathway.     

Effects of Escherichia coli hemolysin on human monocytes. Cytocidal action and stimulation of interleukin 1 release.

This study reports on the potent cytocidal and interleukin-1 releasing properties of Escherichia coli hemolysin (ECH) on human monocytes. Nanomolar concentrations of purified ECH (250-2,000 ng/ml) caused rapid and irreversible depletion of cellular ATP to levels below 20% of controls within 60 min. Subcytocidal doses (10-200 ng/ml) of ECH induced rapid release within 60-120 min of large amounts of interleukin 1 beta (IL-1 beta) from cultured monocytes. IL-1 beta release occurred in the presence of actinomycin D and cycloheximide, and was thus probably due to processing and export of intracellular IL-1 beta precursor. Incubation of toxin-producing E. coli at ratios of only 0.3-3 colony-forming units per monocyte evoked approximately 50% depletion of total cellular ATP within 90 min. Toxin producers also stimulated synthesis and release of large amounts of interleukin 1, but not of tumor necrosis factor within the same time span. In contrast, non-toxin producers caused neither cell death nor rapid interleukin 1 release. Stimulation of rapid interleukin 1 release coupled with potent cytocidal effects on cells of monocytic origin may represent pathogenetically significant events incurred by bacterial strains that produce ECH and related cytolysins.     

Fluoroquinolone action in mycobacteria: similarity with effects in Escherichia coli and detection by cell lysate viscosity.

Fluoroquinolones are potent antibacterial agents that are being used as therapeutic agents for the treatment of multidrug-resistant tuberculosis. To better understand fluoroquinolone action in mycobacteria, the effects of ciprofloxacin were examined. DNA synthesis was inhibited rapidly in Mycobacterium smegmatis, DNA cleavage was readily observed by an empirical assay of cell lysate viscosity, and cell growth was blocked. These data are explained by the formation of gyrase-DNA-ciprofloxacin complexes that block replication fork movement. The bactericidal action of ciprofloxacin against M. smegmatis, Mycobacterium bovis BCG, and Escherichia coli occurred more slowly in cells with longer doubling times. The bactericidal effect against M. bovis BCG was partially blocked by pretreatment with chloramphenicol, an inhibitor of protein synthesis, and by very high concentrations of ciprofloxacin itself. Similar responses occur when E. coli is treated with ciprofloxacin. These similarities between E. coli and mycobacteria indicate that results from extensive fluoroquinolone studies with E. coli can be applied to mycobacteria. A simple viscometric assay of DNA cleavage is described. The assay is expected to be useful for screening new fluoroquinolone derivatives for increased effectiveness against clinically important bacteria.     

Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes

The contribution of Escherichia coli hemolysin (ECH) to bacterial virulence has been considered mainly in context with its hemolytic properties. We here report that this prevalent bacterial cytolysin is the most potent leukocidin known to date. Very low concentrations (approximately 1 ng/ml) of ECH evoke membrane permeability defects in PMN (2-10 x 10(6) cells/ml) leading to an efflux of cellular ATP and influx of propidium iodide. The attacked cells do not appear to repair the membrane lesions. Human serum albumin, high density and low density lipoprotein, and IgG together protect erythrocytes and platelets against attack by even high doses (5-25 micrograms/ml) of ECH. In contrast, PMN are still permeabilized by ECH at low doses (50-250 ng/ml) in the presence of these plasma inactivators. Thus, PMN become preferred targets for attack by ECH in human blood and protein-rich body fluids. Kinetic studies demonstrate that membrane permeabilization is a rapid process, ATP-release commencing within seconds after application of toxin to leukocytes. It is estimated that membrane permeabilization ensues upon binding of approximately 300 molecules ECH/PMN. This process is paralleled by granule exocytosis, and by loss of phagocytic killing capacity of the cells. The recognition that ECH directly counteracts a major immune defence mechanism of the human organism through its attack on granulocytes under physiological conditions sheds new light on its possible role and potential importance as a virulence factor of E. coli.     

Pharmacokinetic and pharmacodynamic comparisons between human granulocyte colony-stimulating factor purified from human bladder carcinoma cell line 5637 culture medium and recombinant human granulocyte colony-stimulating factor produced in Escherichia coli.

The pharmacokinetics and biological activities of recombinant human granulocyte colony-stimulating factor (hG-CSF) produced in Escherichia coli were compared with those of hG-CSF purified from human bladder carcinoma cell line 5637 culture medium (5637-hG-CSF). Recombinant hG-CSF was biologically active in a bone marrow cell proliferation assay in vitro, with a dose-response curve similar to that of 5637-hG-CSF. The effects of 5637- and recombinant hG-CSF administered via i.v. injection to rats showed similar response patterns of neutrophil counts in peripheral blood. From these results, it is concluded that the O-linked sugar chain of hG-CSF does not contribute to the in vitro and in vivo biological activities. The pharmacokinetics of both forms of hG-CSF in rats were investigated using a sandwich enzyme-linked immunosorbent assay. After i.v. administration, the serum concentration-time curves of 5637- and recombinant hG-CSF declined biexponentially. Total body clearance and steady-state volume of distribution of 5637-hG-CSF were smaller than those for the recombinant form. After s.c. administration, a lower peak serum level, smaller AUC, and lower bioavailability of 5637-hG-CSF were observed compared to recombinant hG-CSF.     

Mechanism of Escherichia coli Resistance to Pyrrhocoricin

Due to their lack of toxicity to mammalian cells and good serum stability, proline-rich antimicrobial peptides (PR-AMPs) have been proposed as promising candidates for the treatment of infections caused by antimicrobial-resistant bacterial pathogens. It has been hypothesized that these peptides act on multiple targets within bacterial cells, and therefore the likelihood of the emergence of resistance was considered to be low. Here, we show that spontaneous Escherichia coli mutants resistant to pyrrhocoricin arise at a frequency of approximately 6 × 10⁻⁷. Multiple independently derived mutants all contained a deletion in a nonessential gene that encodes the putative peptide uptake permease SbmA. Sensitivity could be restored to the mutants by complementation with an intact copy of the sbmA gene. These findings question the viability of the development of insect PR-AMPs as antimicrobials.