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.     

Size and Dynamics of the Vibrio cholerae Porins OmpU and OmpT Probed by Polymer Exclusion

The trimeric OmpU and OmpT porins form large, triple-barrel hydrophilic channels in the outer membrane of the pathogen Vibrio cholerae. They have distinct pore properties, such as conductance, block by deoxycholic acid, and sensitivity to acidic pH. Their three-dimensional structures are unknown, but they share significant sequence homologies. To gain insight into the molecular basis for the distinct functional properties of these two similar porins, we carried out polymer exclusion experiments using planar lipid bilayer and patch-clamp electrophysiology. By studying the partitioning of polyethylene glycols (PEGs) of different molecular weights into each porin, we determined an effective radius of 0.55 nm and 0.43 nm for OmpU and OmpT respectively, and found an increased OmpU effective radius at acidic pH. PEGs or high buffer ionic strength promotes the appearance of single step closures in OmpU similar to the acidic-pH induced closures we documented previously. In addition, these closing events can be triggered by nonpenetrating PEGs applied asymmetrically. We believe our results support a model whereby acidic pH, high ionic strength, or exposure to PEGs stabilizes a less conductive state that corresponds to the appearance of an additional resistive element on one side of the OmpU protein and common to the three monomers.     

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.     

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.     

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.     

Fosfomycin Permeation through the Outer Membrane Porin OmpF

Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.     

High-throughput sequencing reveals suppressors of Vibrio cholerae rpoE mutations: one fewer porin is enough

Analyses of suppressor mutations have been extremely valuable in understanding gene function. However, techniques for mapping suppressor mutations are not available for most bacterial species. Here, we used high-throughput sequencing technology to identify spontaneously arising suppressor mutations that enabled disruption of rpoE (which encodes σ^E) in Vibrio cholerae, the agent of cholera. The alternative sigma factor σ^E, which is activated by envelope stress, promotes expression of factors that help preserve and/or restore cell envelope integrity. In Escherichia coli, rpoE is an essential gene that can only be disrupted in the presence of additional suppressor mutations. Among a panel of independent V. cholerae rpoE mutants, more than 75% contain suppressor mutations that reduce production of OmpU, V. cholerae’s principal outer membrane porin. OmpU appears to be a key determinant of V. cholerae’s requirement for and production of σ^E. Such dependence upon a single factor contrasts markedly with regulation of σ^E in E. coli, in which numerous factors contribute to its activation and none is dominant. We also identified a suppressor mutation that differs from all previously described suppressors in that it elevates, rather than reduces, σ^E’s activity. Finally, analyses of a panel of rpoE mutants shed light on the mechanisms by which suppressor mutations may arise in V. cholerae.     

Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria

Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.     

Deoxycholic acid blocks vibrio cholerae OmpT but not OmpU porin

OmpT and OmpU are general diffusion porins of the human intestinal pathogen Vibrio cholerae. The sole presence of OmpT in the outer membrane sensitizes cells to the bile component deoxycholic acid, and the repression of OmpT in the intestine may play an important role in the adaptation of cells to the host environment. Here we report a novel important functional difference between the two porins, namely the sensitivity to deoxycholic acid. Single channel recordings show that submicellar concentrations of sodium deoxycholate induce time-resolved blocking events of OmpT but are devoid of any effect on OmpU. The effects are dose-, voltage-, and pH-dependent. They are elicited by deoxycholate applied to either side of the membrane, with some asymmetry in the sensitivity. The voltage dependence remains even when deoxycholate is applied symmetrically, indicating that it is intrinsic to the binding site. The pH dependence suggests that the active form is the neutral deoxycholic acid and not the negatively charged species. The results are interpreted as deoxycholic acid acting as an open-channel blocker, which may relate to deoxycholic acid permeation.     

Chitoporin from the Marine Bacterium Vibrio harveyi: PROBING THE ESSENTIAL ROLES OF TRP136 AT THE SURFACE OF THE CONSTRICTION ZONE*

VhChiP is a sugar-specific porin present in the outer membrane of the marine bacterium Vibrio harveyi. VhChiP is responsible for the uptake of chitin oligosaccharides, with particular selectivity for chitohexaose. In this study, we employed electrophysiological and biochemical approaches to demonstrate that Trp¹³⁶, located at the mouth of the VhChiP pore, plays an essential role in controlling the channel''s ion conductivity, chitin affinity, and permeability. Kinetic analysis of sugar translocation obtained from single channel recordings indicated that the Trp¹³⁶ mutations W136A, W136D, W136R, and W136F considerably reduce the binding affinity of the protein channel for its best substrate, chitohexaose. Liposome swelling assays confirmed that the Trp¹³⁶ mutations decreased the rate of bulk chitohexaose permeation through the VhChiP channel. Notably, all of the mutants show increases in the off-rate for chitohexaose of up to 20-fold compared with that of the native channel. Furthermore, the cation/anion permeability ratio P_c/P_a is decreased in the W136R mutant and increased in the W136D mutant. This demonstrates that the negatively charged surface at the interior of the protein lumen preferentially attracts cationic species, leading to the cation selectivity of this trimeric channel.     

Identification of the pore-forming region of the outer chloroplast envelope protein OEP16

The chloroplast outer envelope protein OEP16 forms a cation-selective high conductance channel with permeability to amines and amino acids. The region of OEP16 directly involved in channel formation has been identified by electrophysiological analysis of a selection of reconstituted OEP16 mutants. Because analysis of these mutants depended on the use of recombinant protein, we evaluated the electrophysiological properties of OEP16 isolated directly from pea chloroplasts and of the recombinant protein produced in Escherichia coli. The results show that the basic properties like conductance, selectivity, and open probability of the channel formed by native pea OEP16 are comparable with the channel activity formed by the recombinant source of the protein. Following electrophysiological analysis of OEP16 mutants we found that point mutations and insertion of additional amino acid residues in the region of the putative helix 1 (Glu(73) to Val(91)) did not change the properties of the OEP16 channel. The only exception was a Cys(71)-->Ser mutation, which led to a loss of the CuCl(2) sensitivity of the channel. Analysis of N- and C-terminal deletion mutants of OEP16 and mutants containing defined shuffled domains indicated that the minimal continuous region of OEP16, which is able to form a channel in liposomes, lies in the first half of the protein between amino acid residues 21 and 93.     

Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes

Purified porin OmpF from Escherichia coli outer membrane was chemically modified by acetylation and succinylation of amino groups and by amidation of the carboxyl groups. Native and chemically modified porins were incorporated into lipid bilayer membranes and the permeability properties of the pores were studied. Acetylation and succinylation of the porin trimers had almost no influence on the single channel conductance in the presence of small cations and anions and the cation selectivity remained essentially unchanged as compared with the native porin. Amidation had also only little influence on the single channel conductance and changed the pore conductance at maximum by less than 50%, whereas the cation selectivity of the porin is completely lost after amidation. The results suggest that the structure of the porin pore remains essentially unchanged after chemical modification of the pores and that their cation selectivity is caused by an excess of negatively charged groups inside the pore and/or on the surface of the protein. Furthermore, it seems very unlikely that the pore contains any positively charged group at neutral pH.     

Ion selectivity of the Kat1 K+ channel pore

Kat1 is a highly selective inward-rectifying K⁺ channel that opens for extended periods under conditions of extreme hyperpolarization. Over 200 point mutants in the pore region of the Kat1 K⁺ channel were generated and examined in the yeast Saccharomyces cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity. Substitutions at the tyrosine of the signature sequence G-Y-G resulted in the most significant alterations in ion selectivity, consistent with its role in the selectivity filter. However, greater than 80% of the mutations throughout the greater pore region also conferred a defect in selectivity demonstrating that the entire pore of Kat1 contributes to the ion selectivity of this channel. Surprisingly, we identified a novel class of mutant channel that conferred enhanced selectivity of K⁺ over Na⁺. Mutants of this class frequently displayed sensitivity to the competing ion Cs⁺. This finding has led us to speculate that the Kat1 channel pore has evolved to balance not only K⁺/Na⁺ selectivity, but selectivity over Cs⁺, and possibly a wide spectrum of potential competing ions.     

Disulfide bond tethering of extracellular loops does not affect the closure of OmpF porin at acidic pH

The permeability of the outer membrane of gram‐negative bacteria is essentially controlled by pore‐forming proteins of the porin family. The trimeric E. coli porin OmpF is assembled as a triple β‐barrel, where each monomer contains a central pore and extracellular loops. Electrophysiological analysis of the behavior of OmpF at acidic pH reveals that the protein undergoes a conformational change leading to the sequential step‐wise closure of the three monomers. A previous atomic force microscopy study suggested that the conformational change might be due to a bending of extracellular loops over the pore opening, and loop deletion experiments suggested that loops L1, L7, and L8 are involved. In order to test the hypothesis for loop movement, we engineered a series of double cysteine mutants in loops L1, L6, L7 and L8 in order to create disulfide bonds linking two loops to each other, or the two branches of a loop, or a loop to the β‐barrel. Five out of the six mutants showed the formation of the disulfide bond. However, none of these had an altered response to acidic pH relative to the wildtype channel. Although we cannot dismiss the possibility that the mobility restriction introduced by each disulfide bond was too localized to impact a more global conformational change of the three loops, the fact that all of the different types of disulfide bond tethering were similarly ineffective suggests that the extracellular loops L1, L7, and L8 may not undergo a major acidic‐pH induced conformational change leading to channel closure. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Discrimination of single‐porin Escherichia (E.) coli mutants by ATR and transmission mode FTIR spectroscopy

Vibrational spectroscopy has long been used in bacterial identification with different levels of taxonomic discrimination but its true potential for intra‐species differentiation remains poorly explored. Herein, both transmission Fourier‐transform infrared (FTIR) and attenuated total reflectance (ATR)‐FTIR spectroscopy are used to analyse E. coli strains that differ solely in their porin expression profile. In this previously unreported approach, the applicability of both FTIR‐spectroscopy techniques is compared with the same collection of unique strains. ATR‐FTIR spectroscopy proved to reliably distinguish between several E. coli porin mutants with an accuracy not replicated by FTIR in transmission mode (using previously optimized procedures). Further studies should allow the identification of the individual contribution of the single porin channel to the overall bacterial infrared spectrum and of molecular predictive patterns of porin alterations. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Permeability of the cell wall of Mycobacterium smegmatis

The cell wail of Mycobacterium smegmatis me²155 was shown to be an effective permeability barrier to hydrophilic compounds. Permeability coefficients to β-lactams ranged from 10 × 10 ⁻⁷ to 0.5 × 10 ⁻⁷ cm s⁻¹. Cell wall proteins were solubilized with EDTA and Genapol and were tested for channel-forming activity by reconstitution into lipid bilayers. Proteins were able to induce a voltage-gated cation-selective channel. The mycobacterial porin channel appeared to be water-filled since the single-channel conductance followed the mobility sequence of hydrated ions in the aqueous phase. On the basis of the Renkin equation and the single-channel conductance, the channel diameter was estimated to be around 3 nm. Model calculations showed that cation selectivity may be caused by four negative point-charges at the channel mouth. The permeability properties of the cell wall of intact cells were in good agreement with those of the reconstituted channel. Negatively charged cephalosporins, cefamandole and cephalothin, diffused at a 10- to 20-fold lower rate than the zwitterionic cephaloridine. The mycobacterial porin represents a major hydrophilic pathway of the cell wall of M. smegmatis.     

Identification of positively charged residues of FomA porin of Fusobacterium nucleatum which are important for pore function.

FomA porin is the major outer-membrane protein of Fusobacterium nucleatum. It exhibits the functional properties of a general diffusion porin, but has no sequence similarity to other porins. According to the proposed topology model, each monomer of this trimeric protein is a beta-barrel consisting of 16 transmembrane segments with eight surface-exposed loops. Several conserved charged residues are proposed to extend from the beta-barrel wall into the aqueous channel lumen, and may contribute to a transverse electric field similar to that at the pore constriction of porins with known structure. The goal of our study was to identify particular basic residues contributing to such an electric field in FomA. Several arginines and lysines were replaced by negatively charged glutamates or uncharged alanines. The mutated FomA porins were expressed in Escherichia coli, and the effects on pore function were studied in vivo, by assaying the uptake rate of beta-lactam antibiotics, and in vitro after reconstitution of the purified proteins in lipid bilayer membranes. Some of the point mutations had a significant impact on the channel properties. The substitution R92A produced a 130% increased permeability of the zwitterionic beta-lactam cephaloridine, and the cation selectivity of R92E increased by 70%. The effects of the R90E substitution on channel properties were similar. Most of the point mutations had a minor effect on the voltage gating of the FomA channel, resulting in an increased sensitivity, except for K78E, which showed a decreased sensitivity. The latter mutation had no effect on cation selectivity, but the K78A substitution improved the uptake rate of cephaloridine. The results presented here indicate that arginines 90 and 92 are probably part of the constriction zone of the FomA porin, and lysine 78 and arginines 115 and 117 are probably in close proximity to this region as well.     

Brownian dynamics simulation of ion flow through porin channels

Bacterial porins, which allow the passage of solutes across the outer bacterial membrane, are structurally well characterized. They therefore lend themselves to detailed studies of the determinants of ion flow through transmembraneous channels. In a comparative study, we have performed Brownian dynamics simulations to obtain statistically significant transfer efficiencies for cations and anions through matrix porin OmpF, osmoporin OmpK36, phosphoporin PhoE and two OmpF charge mutants.The simulations show that the electrostatic potential at the highly charged channel constriction serves to enhance ion permeability of either cations or anions, dependent on the type of porin. At the same time translocation of counterions is not severely impeded. At the constriction, cations and anions follow distinct trajectories, due to the segregation of basic and acidic protein residues.Simulated ion selectivity and relative conductance agree well with experimental values, and are dependent crucially on the charge constellation at the pore constriction. The experimentally observed decrease in ion selectivity and single channel conductance with increasing ionic strength is well reproduced and can be attributed to electrostatic shielding of the pore lining.     

Role of charged residues at the OmpF porin channel constriction probed by mutagenesis and simulation

The channel constriction of OmpF porin, a pore protein in the bacterial outer membrane, is highly charged due to the presence of three arginines (R42, R82, and R132) and two acidic residues (D113 and E117). The influence of these charges on ion conductance, ion selectivity, and voltage gating has been studied with mutants D113N/E117Q, R42A/R82A/R132A/D113N/E117Q, and V18K/G131K, which were designed to remove or add protein charge at the channel constriction. The crystal structures revealed no or only local changes compared to wild-type OmpF, thus allowing a comparative study. The single-channel conductance of the isosteric D113N/E117Q variant was found to be 2-fold reduced, and that of the pentuple mutant was 70% of the wild-type value, despite a considerably larger pore cross section. Ion selectivity was drastically altered by the mutations with cation/anion permeability ratios ranging from 1 to 12. Ion flow through these and eight other mutants, which have been characterized previously, was simulated by Brownian dynamics based on the detailed crystal structures. The calculated ion selectivity and relative channel conductance values agree well with the experimental data. This demonstrates that ion translocation through porin is mainly governed by pore geometry and charge, the two factors that are properly represented in the simulations.