The Vibrio cholerae porins OmpU and OmpT have distinct channel properties

Numerous environmental signals regulate the production of virulence factors and the composition of the outer membrane of Vibrio cholerae. In particular, bile promotes the ToxR-dependent expression of the porin OmpU. Strains expressing solely OmpU are more resistant to bile, are better able to colonize the intestine, and produce more cholera toxin than strains expressing solely the OmpT porin. To gain some understanding in the physiological relevance and the molecular mechanism underlying these porin-dependent phenotypes, we have undertaken a thorough electrophysiological characterization of the channel properties of the two porins. Purified OmpU or OmpT was reconstituted in liposomes suitable for patch clamp and in planar lipid bilayers. The high resolution of the patch clamp technique allowed us to analyze in detail the behavior of single OmpU and OmpT channels. Both channels exhibit closing transitions to various conductance states. OmpT is a much more dynamic channel than OmpU, displaying frequent and prolonged closures, even at low transmembrane potentials. With a critical voltage for closure V(c) of approximately +/-90 mV, OmpT is much more voltage-sensitive than OmpU (with a V(c) of approximately +/-160 mV), a feature that is also readily apparent in the voltage dependence of closing probability observed in patch clamp in the +/-100 mV range. OmpT has low ionic selectivity (P(K)/P(Cl) = approximately 4), whereas OmpU is more cation-selective (P(K)/P(Cl) = approximately 14). The distinct functional properties of the two porins are likely to play an integrated role with environmental regulation of their expression. For example, the higher selectivity of OmpU for cations provides a possible explanation for the protective role played by this porin in a bile-containing environment, because this type of selectivity would restrict the flux of anionic bile salts through the outer membrane and thus would reduce the exposure of the cytoplasmic membrane to this natural detergent.     

Phenotypic characterization of pore mutants of the Vibrio cholerae porin OmpU

General-diffusion porins form large β-barrel channels that control the permeability of the outer membrane of gram-negative bacteria to nutrients, some antibiotics, and external signals. Here, we have analyzed the effects of mutations in the OmpU porin of Vibrio cholerae at conserved residues that are known to affect pore properties in the Escherichia coli porins OmpF and OmpC. Various phenotypes were investigated, including sensitivity to β-lactam antibiotics, growth on large sugars, and sensitivity to and biofilm induction by sodium deoxycholate, a major bile component that acts as an external signal for multiple cellular responses of this intestinal pathogen. Overall, our results indicate that specific residues play different roles in controlling the passage of various compounds. Mutations of barrel wall arginine residues that protrude in the pore affect pore size and growth in the presence of large sugars or sodium deoxycholate. Sensitivity to large cephalosporins is mostly affected by D116, located on the L3 loop, whose homolog in E. coli, OmpF, is a known binding determinant for these drugs. L3 loop residues also affect biofilm induction. The results are interpreted in terms of a homology model based on the structures of E. coli porins.     

Unusual Constriction Zones in the Major Porins OmpU and OmpT from Vibrio cholerae

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Altered pore properties and kinetic changes in mutants of the Vibrio cholerae porin OmpU

The electrophysiological technique of patch-clamp was used to characterize the pore properties of site-directed mutants in the Vibrio cholerae general diffusion porin OmpU. Changes in conductance and selectivity were observed, thus confirming the predicted pore location of these residues, based on homology with the Escherichia coli porins OmpF and OmpC. Some mutants acquire a weak selectivity for cations, which mirrors the properties of the homologous, deoxycholic acid sensitive, OmpT porin of V. cholerae. However, the mutants remain insensitive to deoxycholic acid, like wildtype OmpU. This result suggests that channel selectivity is not an important determinant in the sensitivity to this drug, and is in agreement with our finding that the neutral deoxycholic acid, and not deoxycholate, is the actual active form in channel block. Modifications in the kinetics of spontaneous closures were also noted, and are similar to those found for the E. coli channels. In addition, mutants at the D116 residue on the L3 loop display marked transitions to sub-conductance states. The results reported here are compared to a phenotypical characterization of the mutants in terms of permeability to maltodextrins and beta-lactam antibiotic sensitivity. No strict correlations are observed, suggesting that distinct, but somewhat overlapping, molecular determinants control electrophysiological properties and substrate permeability.     

Effects of conjugated and unconjugated bile acids on the activity of the Vibrio cholerae porin OmpT

Abstract

During infection, the enteric pathogen Vibrio cholerae encounters a bile-containing environment. Previous studies have shown that bile and/or bile acids exert several effects on the virulence and physiology of the bacterial cells. These observations have led to the suggestion that bile acids may play a signaling role in infection. We have previously reported that the bile component deoxycholic acid blocks the general diffusion porin OmpT in a dose-dependent manner, presumably as it transits through the pore. V. cholerae colonizes the distal jejunum and ileum, where a mixture of various conjugated and unconjugated bile acids are found. In this work, we have used patch clamp electrophysiology to investigate the effects of six bile acids on OmpT. Two bile acids (deoxycholic and chenodeoxycholic acids) were found to block OmpT at physiological concentrations below 1 mM, while glycodeoxycholic acid was mildly effective and cholic, lithocholic and taurodeoxycholic acids were ineffective in this range. The block was also voltage-dependent. These observations suggest the presence of a specific binding site inside the OmpT pore. Since deconjugation is due to the activity of the endogenous flora, the preferential uptake of some unconjugated bile acids by OmpT may signal the presence of a hospitable environment. The results are also discussed in terms of the possible molecular interactions between the penetrating bile acid molecule and the channel wall.     

Effects of conjugated and unconjugated bile acids on the activity of the Vibrio cholerae porin OmpT

During infection, the enteric pathogen Vibrio cholerae encounters a bile-containing environment. Previous studies have shown that bile and/or bile acids exert several effects on the virulence and physiology of the bacterial cells. These observations have led to the suggestion that bile acids may play a signaling role in infection. We have previously reported that the bile component deoxycholic acid blocks the general diffusion porin OmpT in a dose-dependent manner, presumably as it transits through the pore. V. cholerae colonizes the distal jejunum and ileum, where a mixture of various conjugated and unconjugated bile acids are found. In this work, we have used patch clamp electrophysiology to investigate the effects of six bile acids on OmpT. Two bile acids (deoxycholic and chenodeoxycholic acids) were found to block OmpT at physiological concentrations below 1 mM, while glycodeoxycholic acid was mildly effective and cholic, lithocholic and taurodeoxycholic acids were ineffective in this range. The block was also voltage-dependent. These observations suggest the presence of a specific binding site inside the OmpT pore. Since deconjugation is due to the activity of the endogenous flora, the preferential uptake of some unconjugated bile acids by OmpT may signal the presence of a hospitable environment. The results are also discussed in terms of the possible molecular interactions between the penetrating bile acid molecule and the channel wall.     

Refolding and functional assembly of the Vibrio cholerae porin OmpU recombinantly expressed in the cytoplasm of Escherichia coli

OmpU is one of the major outer membrane porins of Vibrio cholerae. OmpU has been biochemically characterized previously for its 'porin'-property. However, previous studies have used the OmpU protein extracted from the bacterial outer membrane envelope fractions. Such method of isolation imposes limitations on the availability of the protein reagent, and also enhances the possibility of the OmpU preparation being contaminated with lipid molecules of bacterial outer membrane origin, especially lipopolysaccharides (LPS). Here we report a strategy of purifying the V. cholerae OmpU protein recombinantly overexpressed in heterologous protein expression system in Escherichia coli, without its being incorporated into the bacterial membrane fraction. In our strategy, the majority of the protein was expressed as insoluble inclusion body in the E. coli cytoplasm, the protein was dissolved by denaturation in 8M urea, refolded, and purified to homogeneity in presence of detergent. Our strategy allowed isolation of the recombinant OmpU protein with significantly enhanced yield as compared to that of the wild type protein extracted from the V. cholerae membrane fraction. The recombinant V. cholerae OmpU protein generated in our study displayed functional channel-forming property in the synthetic liposome membrane, thus confirming its 'porin'-property. To the best of our knowledge, this is the first report showing an efficient refolding and functional assembly of the V. cholerae OmpU porin recombinantly expressed as inclusion body in the cytoplasm of a heterologous host E. coli.     

Tamoxifen blocks estrogen-induced B cell maturation but not survival

Estrogen treatment has been shown not only to exacerbate disease activity and accelerate death in spontaneous murine models of lupus but also to induce a lupus-like phenotype in non-spontaneously autoimmune mice. In mice transgenic for the H chain of an anti-DNA Ab, estrogen rescues naive autoreactive B cells that normally are deleted and causes them to mature to a marginal zone phenotype. Estrogen further leads to the activation of this population causing an elevation of serum anti-DNA Ab titers and renal disease. This study was designed to evaluate the therapeutic potential of tamoxifen, a selective estrogen receptor modulator, on estrogen-induced lupus. Mice treated with both estradiol and tamoxifen showed no elevation in anti-DNA Ab titers and consequently no glomerular IgG. The DNA-reactive B cell population that is rescued by estrogen was present in an anergic state in mice treated with both estradiol and tamoxifen. Estradiol enhances transitional B cell resistance to apoptosis and expands the population of marginal zone B cells; tamoxifen did not impede the enhanced resistance to apoptosis, but prevented the development of autoreactive cells as marginal zone B cells. Thus, estrogen-induced autoimmunity proceeds through two distinct molecular pathways, one affecting survival and the other maturation. Activation, but not survival, of autoreactive B cells can be abrogated by tamoxifen. Drugs that modulate even some of the effects of estrogen may be beneficial in patients with lupus. Eventually, understanding the pathways involved in survival and activation of autoreactive B cells will permit the development of therapeutics that target all relevant pathways.     

Pimozide blocks establishment but not expression of cocaine-produced environment-specific conditioning

Two experiments were conducted to examine the effects of pimozide on cocaine-produced conditioning to a specific environmental context. On 8 treatment days, 12 rats were injected with cocaine (10 mg/kg i.p.) and 12 with saline prior to placement for 60 min into a test chamber outfitted with infrared emitters and detectors. Following each treatment session the saline group received cocaine in their home-cages and the cocaine group received saline. Cocaine produced a significant increase in vertical activity on treatment days. On test days all rats received saline. Significantly greater vertical activity was observed in the group previously receiving cocaine in the test environment. All rats then received 8 more treatment sessions. On saline test days, pimozide (0.4 mg/kg i.p.) pretreatment failed to antagonize expression of the conditioned effect. In experiment 2, pimozide was given prior to treatment and no evidence of conditioning was seen on saline test days. Thus, pimozide blocked the establishment but not the expression of cocaine-produced environment-specific conditioning. These results suggest that during conditioning, the effects of cocaine on dopaminergic neurons may have produced a change that subsequently influenced behaviour even when dopaminergic systems were blocked.     

Lindane but not deltamethrin blocks a component of GABA-activated chloride channels

Effects of deltamethrin, a pyrethroid insecticide, and lindane, the gamma isomer of hexachlorocyclohexane, on the gamma-aminobutyric acid (GABA)-activated ion channels were studied using cultured neurons isolated from the newborn rat dorsal root ganglia. The neuron was voltage clamped using the whole cell patch clamp technique. Two components of inward chloride currents were generated in response to bath application of GABA. One of the components was selectively blocked by 1 x 10(-5)M lindane in a stereospecific manner, but neither component was affected by 1 x 10(-5)M deltamethrin, which drastically prolonged the voltage-activated sodium channel current. Thus, the action of lindane to stimulate the nervous system is partly because of an interaction with the GABA receptor-channel complex. The target site of deltamethrin is not the GABA receptor-channel complex but the sodium channel. The results suggest a multiplicity of the GABAA receptor-chloride channel complex.     

v-myb blocks granulocyte colony-stimulating factor-induced myeloid cell differentiation but not proliferation.

To understand the effects of v-myb expression on mammalian hematopoietic cell differentiation, we have constructed a retroviral vector which can efficiently express v-myb gene product in mammalian cells. Infection of interleukin-3-dependent murine progenitor cell line 32D Cl3, which undergoes terminal differentiation to mature granulocytes in the presence of granulocyte colony-stimulating factor (GCSF), with this recombinant retrovirus does not abrogate its requirement of interleukin-3 for growth. However, expression of v-myb in these cells blocks their ability to differentiate in response to GCSF. Instead, the v-myb-infected cells proliferate indefinitely in the presence of GCSF. 32D Cl3 cells infected with empty vector carrying only the neomycin resistance gene responded to the addition of GCSF in a manner identical to that of the uninfected cells and underwent terminal differentiation into granulocytes. These results suggest that oncogenic forms of myb gene bring about transformation by blocking the differentiation signal derived by cytokines while promoting the proliferative signal transduction pathways.     

Nitrendipine blocks high potassium contractures but not twitches in rat skeletal muscle

The effects of the organic calcium channel blocker nitrendipine was tested on electrically evoked twitches and on potassium depolarization-induced contractures of rat lumbricalis muscles. Nitrendipine (10(-7) to 5 X 10(-5) M) blocked only the potassium contractures. It was concluded that blocking calcium uptake through the slow voltage-sensitive calcium channels during potassium depolarization blocks the mechanical response of the muscle. Thus extracellular calcium ions are required for the excitation-contraction (E-C) coupling during depolarization contractures. On the other hand, electrically evoked twitches were not affected by nitrendipine; therefore, extracellular calcium ions entering via the slow voltage-sensitive channels are not required for E-C coupling during the twitch.     

Maternal antibody blocks humoral but not T cell responses to BVDV.

Bovine viral diarrhoea virus (BVDV) contributes significantly to health-related economic losses in the beef and dairy industry. Antibodies of maternal origin can be protective against BVDV infection, however, calves with low titres of maternal antibody or that do not receive colostrum may be at risk for acute BVDV infection. Interference by high titres of maternal antibodies prevents the development of an antibody response following vaccination with either a killed or attenuated BVDV vaccine. However, the T cell mediated immune response to BVDV may be generated in the absence of a detectable serum neutralizing antibody response. Two trials were conducted to evaluate the potential to elicit T cell mediated immune responses to BVDV in calves with circulating maternal antibody to BVDV. In the first trial, calves with high levels of circulating maternal antibody to BVDV 1 and BVDV 2 were experimentally infected with BVDV 2 (strain 1373) at two to five weeks of age. The T-cell mediated immune responses of the experimentally infected calves and non-infected calves were monitored monthly until circulating maternal antibody was no longer detectable in either treatment group. Calves experimentally infected with BVDV developed BVDV specific CD4(+), CD8(+), and delta T cell responses while high levels of maternal antibody were circulating. A second challenge with BVDV 2 (strain 1373) was performed in the experimentally infected and control calves once maternal antibody could no longer be detected. Previous exposure to BVDV in the presence of maternal antibody protected calves from clinical signs of acute BVDV infection compared to the control calves. In the second trial, three groups of calves with circulating maternal antibody to BVDV were given either a modified live vaccine (MLV) containing BVDV 1 and BVDV 2, a killed vaccine containing BVDV 1 and BVDV 2, or no vaccine, at seven weeks of age. Serum neutralizing antibody levels and antigen specific T cell responses were monitored for 14 weeks following vaccination. Calves vaccinated with MLV BVDV developed BVDV 1 and BVDV 2 specific CD4(+)T cell responses, and BVDV 2 specific gammadelta T cell responses, in the presence of maternal antibody. Vaccination with killed BVDV did not result in the generation of measurable antigen specific T cell immune responses. In this trial, a second vaccination was performed at 14 weeks to determine whether an anamnestic antibody response could be generated when calves were vaccinated in the presence of maternal antibody. Calves vaccinated with either a MLV or killed BVDV vaccine while they had maternal antibody developed an anamnestic antibody response to BVDV 2 upon subsequent vaccination. The results of these trials indicate that vaccinating young calves against BVD while maternal antibody is present may generate BVDV specific memory T and B cells. The data also demonstrated that seronegative calves with memory T and B cells specific for BVDV may be immune to challenge with virulent BVDV.     

Transforming growth factor-beta blocks proliferation but not early mitogenic signaling events in T-lymphocytes

Transforming growth factor-beta (TGF-beta) inhibits the proliferation of T-lymphocytes in response to activation with mitogenic lectin. The influence of TGF-beta on elevation of cytosolic Ca2+, induction of proliferation-associated mRNA species, and total cellular RNA content has been studied. The cells seem to exit G0 when activated in the presence of TGF-beta, but they arrest in mid-G1 phase.     

Bombesin, but not amylin, blocks the orexigenic effect of peripheral ghrelin

The interaction between ghrelin and bombesin or amylin administered intraperitoneally on food intake and brain neuronal activity was assessed by Fos-like immunoreactivity (FLI) in nonfasted rats. Ghrelin (13 microg/kg ip) increased food intake compared with the vehicle group when measured at 30 min (g/kg: 3.66 +/- 0.80 vs. 1.68 +/- 0.42, P < 0.0087). Bombesin (8 microg/kg) injected intraperitoneally with ghrelin (13 microg/kg) blocked the orexigenic effect of ghrelin (1.18 +/- 0.41 g/kg, P < 0.0002). Bombesin alone (4 and 8 microg/kg ip) exerted a dose-related nonsignificant reduction of food intake (g/kg: 1.08 +/- 0.44, P > 0.45 and 0.55 +/- 0.34, P > 0.16, respectively). By contrast, ghrelin-induced stimulation of food intake (g/kg: 3.96 +/- 0.56 g/kg vs. vehicle 0.82 +/- 0.59, P < 0.004) was not altered by amylin (1 and 5 microg/kg ip) (g/kg: 4.37 +/- 1.12, P > 0.69, and 3.01 +/- 0.78, respectively, P > 0.37). Ghrelin increased the number of FLI-positive neurons/section in the arcuate nucleus (ARC) compared with vehicle (median: 42 vs. 19, P < 0.008). Bombesin alone (4 and 8 microg/kg ip) did not induce FLI neurons in the paraventricular nucleus of the hypothalamus (PVN) and coadministered with ghrelin did not alter ghrelin-induced FLI in the ARC. However, bombesin (8 microg/kg) with ghrelin significantly increased neuronal activity in the PVN approximately threefold compared with vehicle and approximately 1.5-fold compared with the ghrelin group. Bombesin (8 microg/kg) with ghrelin injected intraperitoneally induced Fos expression in 22.4 +/- 0.8% of CRF-immunoreactive neurons in the PVN. These results suggest that peripheral bombesin, unlike amylin, inhibits peripheral ghrelin induced food intake and enhances activation of CRF neurons in the PVN.     

Coenzyme Q blocks biochemical but not receptor-mediated apoptosis by increasing mitochondrial antioxidant protection

Tau in Alzheimer neurofibrillary tangles has been shown to be hyperphosphorylated and CDK5, GSK3, MAP kinase and SAP kinases are the candidate kinases for the phosphorylation of tau. Recently, it was reported that the conversion of p35, the activator of CDK5, to p25 was upregulated in Alzheimer's disease (AD) brains, and that p35 is cleaved to yield p25 by calpain. Here we show that p35 is rapidly cleaved to p25 in rat and human brains within a short postmortem delay and that the conversion of p35 to p25 is partially dependent on calpain activity. Immunoblot analysis of brains prepared from patients with AD or age-matched control individuals with a short postmortem delay revealed no specific increase in the levels of p25 in AD brains, whereas the levels of active form of calpain were increased in AD brains compared to the those in controls. These observations suggest that the conversion of p35 to p25 is a postmortem degradation event and may not be upregulated in AD brains.