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.     

Isolation and phenotyping of Vibrio cholerae strains with the enhanced production of main protective antigens

Two V. cholerae strains of classical biovar, 2414 (serovar Ogawa) and 2415 (serovar Inaba), with of increased production of main protective antigens--cholera toxin, toxin-coregulated pili of adhesion (TCP), outer membrane protein OmpU, as well as phospholipases and proteases, have been detected among natural and recombinant strains under study. A simultaneous increase in the production of the above-mentioned main immunogenicity factors in strains 2414 and 2415 is seemingly linked with the presence of recombinant plasmid pCT105 with cloned genes of cholera toxin in these microbial cells. As the result of plasmid-chromosomal relationships, this plasmid probably ensures the effective expression of global regulating gene toxR. The strains capable of the hyperproduction of cholera toxin, TCP and protein OmpU may be used for the manufacture of more effective chemical vaccine (choleragen-toxoid).     

A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation

ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.     

Porins of Vibrio cholerae: purification and characterization of OmpU.

Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity. The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded. The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units. Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit. Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein. The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium. The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU. Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated E. coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes. OmpU is also not antigenically related to OmpF. Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V. cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium. OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol. 170:2575-2583, 1988).     

Phenotypical analysis of heterogenic Vibrio cholerae strain Dacca 35 Ogawa

The comparative analysis of the production of the main pathogenicity factors by toxigenic and non-toxigenic clones of V. cholerae natural classical strain Dacca 35 Ogawa has been carried out. The data obtained in this analysis indicate that the appearance of turbid colonies, not synthesing cholera toxin, is linked with the production of an exopolysaccharide layer on the outer surface of the cells, which determines their morphology. The suggestion has been made that the regulatory gene toxR controls the expression of not only cholera toxin, protein OmpU, but also exopolysaccharide.     

Modulation of Vibrio cholerae porin function by acidic pH

The outer membrane of Gram-negative bacteria contains porins, large pore-forming proteins which allow the traffic of hydrophilic compounds between the external medium and the periplasm. The oral mode of infection of Vibrio cholerae, the agent of cholera, implies that the bacteria must adapt to severe changes in the environment, such as acidic pH and the presence of bile. Because of their localization and the regulation of their expression in response to these external factors, the OmpU and OmpT porins of V. cholerae are thought to be involved in the adaptation of the bacteria to the host environment. Using patch clamp and planar lipid bilayer electrophysiology, we assessed the effect of pH on the channel properties of OmpU and OmpT. OmpT does not show any major modification in its activity between pH 4 and pH 7.2. In the case of OmpU, the effect of acidic pH is manifested by promoting single-step closures, whose duration, frequency and current size increase as pH is lowered, thereby producing a pH-dependent decrease in the channel open probability. Surprisingly, the increase in current size of this single-step closure is not coupled with an increase of the total current through the porin, indicating that the trimeric conductance remains unchanged. This observation suggests that coordinated events take place at the level of the trimer, and various explanations for this peculiar effect of acidic pH on porin gating and conductance are provided.     

Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions

The current model for autodisplay suggests a mechanism that allows a passenger protein to be translocated across the outer membrane by coordinate action of a C-terminal beta-barrel and its preceding linking region. The passenger protein, linker, and beta-barrel are together termed the autotransporter, while the linker and beta-barrel are here referred to as the translocation unit (TU). We characterized the minimal TU necessary for autodisplay with the adhesin-involved-in-diffuse-adherence (AIDA-I) autotransporter. The assumed beta-barrel structure at the C terminus of the AIDA-I autotransporter was studied by constructing a set of seven AIDA-I-cholera toxin B subunit fusion proteins containing various portions of AIDA-I. Surface exposure of the cholera toxin B moiety was assessed by dot blot experiments and trypsin accessibility of the chimeric proteins expressed in Escherichia coli JK321 or UT5600. Export of cholera toxin B strictly depended on a complete predicted beta-barrel region. The absolute necessity for export of a linking region and its influence on expression as an integral part of the TU was also demonstrated. The different electrophoretic mobilities of native and denatured chimeras indicated that the proposed beta-barrel resides within the C-terminal 312 amino acids of AIDA-I. Together these data provide evidence for the predicted beta-barrel structure and support our formerly proposed model of membrane topology of the AIDA-I autotransporter.     

Strains of Vibrio cholerae serogroups O1 and O139 that produce the basic protective antigens

To find out stable and effective producers of major protective antigens intended for use as components of cholera chemical vaccine against V. cholerae strains of serogroups O and O139, the comparative analysis of the production of cholera toxin, toxin-coregulated pili (TCP), antigens O1 and O139, polysaccharide capsule and outer membrane protein OmpU in different V. cholerae strains groups O1 and O139 has been made. V. cholerae strain KM68, serogroup O1, has been found capable of the production of antigen O1, serovar Ogawa, protein OmpU at a sufficiently high level and the hyperproduction of cholera toxin and TCP, and thus suitable for use in the manufacture of cholera bivalent vaccine as the source of these antigens. Specially selected alysogenic noncapsular strain KM137 of serogroup O139, characterized by a high and stable level of the biosynthesis of this somatic antigen when grown in both laboratory and production conditions, may serve as the produces of antigen O139.     

Comparison of Escherichia coli K-12 outer membrane protease OmpT and Salmonella typhimurium E protein.

The predicted amino acid sequence of OmpT, an Escherichia coli outer membrane protease, was found to be highly homologous to that predicted for the pgtE gene product of Salmonella typhimurium. In this paper, it is shown that pgtE codes for a protein functionally homologous to OmpT as judged by its ability to proteolyze T7 RNA polymerase and to localize in the outer membrane of E. coli.