Signal transduction via the histidyl-aspartyl phosphorelay

The histidyl-aspartyl phosphorelay, formerly described as the two-component system, is the predominant mode of signal transduction in bacteria. Adaptation to environmental changes occurs through a sensor histidine protein kinase and a response regulator. The histidine protein kinase is usually a transmembrane receptor and the response regulator is a cytoplasmic protein. Together the histidyl-aspartyl phosphorelay proteins mediate reversible phosphorylation events that control downstream effectors. Following autophosphorylation at a conserved histidine residue, the histidine kinase serves as a phospho-donor for the response regulator. Once phosphorylated, the response regulator mediates changes in gene expression or cellular locomotion. The EnvZ-OmpR phosphorelay system in Escherichia coli , which monitors external osmolarity and responds by differentially modulating the expression of the OmpF and OmpC major outer membrane porins, will be described as a model system. While histidine kinases were thought to be present only in prokaryotes, they have recently been identified in eukaryotic systems. Here, we review the unique and conserved features of this growing family of signal transducers.     

A porin from Klebsiella pneumoniae: sequence homology, three-dimensional model, and complement binding.

A recombinant plasmid containing ompK36, the gene coding for the Klebsiella pneumoniae outer membrane protein OmpK36, was constructed by transposon mutagenesis and subcloning. Clones were identified in a cosmid library in Escherichia coli on the basis of their reaction with antiserum against the OmpK36 protein and by the presence in gel electrophoretic analysis of a band in E. coli outer membranes migrating with a mobility corresponding to 36 kDa. The ompK36-encoded protein exhibited characteristic properties of porins, such as heat modifiability and resistance to trypsin. The sequence of the gene revealed that OmpK36 is a close relative of the enterobacterial porin family, with a high degree of homology with E. coli OmpC, PhoE, and OmpF. On the basis of the structures of OmpF and PhoE porins, determined previously by X-ray analysis, it appears likely that the three-dimensional structure of OmpK36 also contains the motif of a 16-stranded beta-barrel, with long loops on one end and short turns on the other. Like the OmpC porin from E. coli, OmpK36 contains a long insertion in loop 4. The results of a binding study of complement component C1q to OmpK36 and the analysis of the OmpK36 model suggest that C1q binding sites are covered by the lipopolysaccharide core in the native porin.     

Salmonella porins induce a sustained, lifelong specific bactericidal antibody memory response

We examined the ability of porins from Salmonella enterica serovar typhi to induce a long-term antibody response in BALB/c mice. These porins triggered a strong lifelong production of immunoglobulin G (IgG) antibody in the absence of exogenous adjuvant. Analysis of the IgG subclasses produced during this antibody response revealed the presence of the subclasses IgG2b, IgG1, IgG2a and weak IgG3. Despite the high homology of porins, the long-lasting anti-S. typhi porin sera did not cross-react with S. typhimurium. Notably, the antiporin sera showed a sustained lifelong bactericidal-binding activity to the wild-type S. typhi strain, whereas porin-specific antibody titres measured by enzyme-linked immunosorbent assay (ELISA) decreased with time. Because our porin preparations contained the outer membrane proteins C and F (OmpC and OmpF), we evaluated the individual contribution of each porin to the long-lasting antibody response. OmpC and OmpF induced long-lasting antibody titres, measured by ELISA, which were sustained for 300 days. In contrast, although OmpC induced sustained high bactericidal antibody titres for 300 days, postimmunization, the bactericidal antibody titre induced by OmpF was not detected at day 180. These results indicate that OmpC is the main protein responsible for the antibody-mediated memory bactericidal response induced by porins. Taken together, our results show that porins are strong immunogens that confer lifelong specific bactericidal antibody responses in the absence of added adjuvant.     

Cloning and sequencing of Enterobacter aerogenes OmpC-type osmoporin linked to carbapenem resistance

Using outbreak-related strains of Enterobacter aerogenes, we cloned and sequenced ompK39, the structural gene coding for outer membrane protein OmpK39. Its lack of expression was closely associated with a phenotype exhibiting low-level carbapenem resistance. Detailed alignment of the predicted amino acid sequence revealed that OmpK39 is a member of the OmpC subclass of enterobacterial porins, with the highest degree of homology to Klebsiella pneumoniae OmpK36. Based on a computerized alignment including Escherichia coli PhoE and OmpF, the 3D structures of which are known from X-ray studies, OmpK39 can be assumed to form the typical beta-barrel structure which is common to all enterobacterial porins. Since no inhibitory DNA sequences could be detected in ompk39 in the resistant strains, porin deficiency leading to carbapenem resistance seems to involve alterations in key regulatory genes and/or the promotor sequence rather than a direct mutation in the structural gene.     

Antigenic determinants of the OmpC porin from Salmonella typhimurium.

The antigenic determinants of Salmonella typhimurium OmpC were investigated by the analysis of cyanogen bromide (CNBr)-generated porin peptides with antiporin monoclonal antibodies (MAbs). We identified six bands (f1 to f6) with estimated molecular masses of 35.5, 31.0, 25.0, 22.5, 13.8, and 10.0 kDa, respectively. In addition, two small fragments (f7 and f8; 3.0 to 6.0 kDa) were detected only infrequently. The OmpC monomer or its CNBr-generated peptides were electrophoretically transferred to a polyvinylidene difluoride membrane and then subjected to amino acid composition analysis and N-terminal sequencing. A comparison of the amino acid composition data with known compositions of Escherichia coli and Salmonella typhi OmpC showed some differences; however, the amino acid sequences of 71 residues identified in S. typhimurium showed 88 and 98% identity with OmpC from E. coli and S. typhi, respectively. The screening of CNBr peptides with the 12 anti-(S. typhimurium) OmpC MAbs by Western blot (immunoblot), in conjunction with the prediction of the OmpC folding pattern based on the known three-dimensional structure of E. coli OmpF, showed that four MAbs reacted with surface-exposed epitopes on loops L2, L8, and L4 to L7, four MAbs reacted with a region in the eyelet structure on loop L3, and four MAbs reacted with the buried epitopes on transmembrane beta strands. The MAbs reacting with surface-exposed loops showed no cross-reaction with E. coli OmpC, whose sequence has diverged extensively from that of S. typhi and (probably) S. typhimurium OmpC only in regions of the externally exposed loops. In contrast, MAbs reacting with transmembrane beta strands, whose sequence is strongly conserved, showed strong cross-reaction with E. coli OmpC. These results show that comparison with the E. coli OmpF structure predicts the folding pattern of S. typhimurium OmpC rather accurately and that evolutionary divergence in sequences is confined to the external loops. The possible roles of these surface-exposed and buried epitopes as potentially useful antigenic regions for diagnostic assays and vaccine development are discussed.     

Molecular characterization of amoxicillin-clavulanate resistance in a clinical isolate of Escherichia coli

The resistance phenotype of the clinical isolate of Escherichia coli 1941 was characterized by high-level resistance to penicillins and to combinations amoxicillin-ticarcillin/clavulanate and ampicillin/sulbactam. This resistance was carried by the conjugative plasmid pEC1941 that encoded a beta-lactamase activity. The purified enzyme focused at pI 5.4 and was strongly inhibited in vitro by clavulanic acid (IC50 = 0.09 microM). Nucleotide sequence analysis revealed identity between the plasmid borne blaTEM gene of E. coli 1941 and the blaTEM-1B gene, except for a single C-to-T substitution at position 32 in the promoter region leading to the overlapping promoters Pa and Pb. No alterations in the expression of outer membrane porins OmpC and OmpF have been detected. These findings show that the resistance of E. coli 1941 to the combinations of beta-lactams with beta-lactamase inhibitors is related to high-level production of TEM-1 enzyme expressed from the strong promoters Pa and Pb.     

Outer membrane and porin characteristics of Serratia marcescens grown in vitro and in rat intraperitoneal diffusion chambers.

The composition and antibiotic permeability barrier of the outer membrane of Serratia marcescens were assessed in cells grown in vivo and in vitro. Intraperitoneal diffusion chambers implanted in rats were used for the in vivo cultivation of bacteria. Outer membranes isolated from log-phase bacterial cells recovered from these chambers were compared with membranes isolated from cells grown in vitro. Analysis revealed that the suspected 41-kilodalton porin and the OmpA protein were recovered on sodium dodecyl sulfate-polyacrylamide gels in equal quantities. Several high-molecular-weight proteins, thought to be iron starvation induced, appeared in the diffusion chamber-grown cells. The outer membrane permeability barriers to cephaloridine were similar in in vivo- and in vitro-grown cells based on permeability coefficient calculations. The permeability coefficient of cephaloridine in S. marcescens cells (30.3 x 10(-5) to 38.9 x 10(-5) cm s-1) was greater than that obtained for an Escherichia coli strain expressing only porin OmpC but smaller than those obtained for the E. coli wild type and a strain expressing only porin OmpF. Functional characterization of the suspected porin was performed by using the planar lipid bilayer technology. The sodium dodecyl sulfate-0.4 M NaCl-soluble porin from both in vitro- and in vivo-grown cells showed an average single-channel conductance in 1 M KCl of 1.6. A partial amino acid sequence (19 residues) was obtained for the S. marcescens porin. The sequence showed a very high homology to the E. coli OmpC porin. These data identified the S. marcescens outer membrane 41-kilodalton protein as a porin by both functional and amino acid analyses. Also, the methodology used allowed for efficient growth and recovery of diffusion chamber-grown bacterial cells and permitted identification of specific in vivo-induced changes in bacterial cell membrane composition.     

Immunological analysis of porin polymorphism in Escherichia coli B and K-12

Two sets of monoclonal antibodies (MoF type I and MoF type II) directed against the OmpF protein were used to analyze the immunological reactivity of the major outer membrane porins of E. coli B and K-12. All these antibodies present a specificity to the native OmpF protein. In addition, among the type II antibodies, MoF 18, 19 and 20 could recognize an epitope present on both monomeric and trimeric forms of the porin as demonstrated by immunoblotting analyses. The use of two different screening methods led to the isolation of two different sets of MoF, one specific for a native conformation accessible only on E. coli B strain and the second directed against epitopes present on OmpF of the two strains, B and K-12. These various responses are discussed in relation to the lipopolysaccharide binding to OmpF and with respect to the screening test used.     

The OmpC protein of Yersinia enterocolitica: purification and properties

OmpC, one of the major outer membrane proteins of Yersinia enterocolitica, was isolated and purified to homogeneity. When solubilized at room temperature, this protein appeared on SDS polyacrylamide gel electrophoresis as an oligomer. After heating to the temperature of boiling water, the apparent molecular weight of the monomer was 36,000. The incorporation of purified OmpC into black lipid membranes resulted in an increase in membrane conductance demonstrating pore-forming activity. The reconstituted pores exhibited the characteristics of general diffusion pores. They showed cation selectivity and had a single channel conductance of 1.3 nS in 1.0 M KCl. Assuming a constant diameter of the pore, a length of 6 nm (the width of the outer membrane) and the same ion conductivity inside and outside the pore, the diameter of the pore protein was estimated as 1.0 nm. Polyclonal antibodies were raised against the native, pore-forming protein preparation. These antibodies did not recognize the denatured form of the protein, but cross-reacted with native OmpC and OmpF of Escherichia coli. The regulation of OmpC expression in Y. enterocolitica was dependent on the osmolarity of the medium in the same way as in E. coli.     

Immunological evidence for differences in the exposed regions of OmpF porins from Escherichia coli B and K-12

Nine monoclonal antibodies (MoF 0-8) directed against the native form (trimeric) of outer membrane protein OmpF of Escherichia coli B were obtained and characterized. All these antibodies bind to OmpF porin in intact E. coli B cells but not OmpF from E. coli K-12 cells which only differ at positions 66, 117 and 262 in the sequence. These antibodies exhibit a specificity to the native form, failing to recognize the denatured form in a liquid immunorecognition assay. Four tested antibodies are able to protect against colicin A, a bacteriotoxin using OmpF as receptor. One monoclonal antibody (MoF 0) is specific to the external topology of native porin in the outer membrane and three antibodies could recognize epitopes present in each conformation of subunits of trimer form. It is concluded that the region around the 66th and more probably around the 262nd amino acids are involved in cell-surface exposed epitopes. Moreover, these results support the assumption that the conformation of protruding regions of OmpF from E. coli B and K-12 are different.     

Effect of salicylate on expression of flagella by Escherichia coli and Proteus, Providencia, and Pseudomonas spp.

Osmotic stress, salicylate, and Mar (multiple antibiotic resistance) mutation are known to block the expression of the OmpF porin. Since these conditions have also been shown to inhibit the expression of P and CFA fimbriae in Escherichia coli, we speculated that they might affect the expression of flagella as well. Hyperosmotic conditions have been shown to block the synthesis of flagellin and expression of flagella in E. coli (C. Li, C. J. Louise, W. Shi, and J. Adler, J. Bacteriol. 175:2229-2235, 1993). In the current study, sodium salicylate was found to inhibit the motility of E. coli, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, and Providencia stuartii in a reversible, concentration-dependent manner. Swarming did not occur at 20 mM sodium salicylate. Salicylate also blocked the synthesis of flagellin in E. coli. Phenotypic Mar mutants of E. coli derived from motile strains were amotile. Flagella were markedly reduced as determined by scanning electron microscopy when P. mirabilis was grown in broth containing 20 mM salicylate. Salicylate had no apparent effect, however, on expression of a 40-kDa porin protein in P. mirabilis. This finding suggests that the noted effect of salicylate on Proteus spp. may be mediated through a mechanism other than porin production or that the Proteus porin may not be analogous to OmpF in E. coli. Salicylate decreased the motility of Pseudomonas cepacia but had no effect on Pseudomonas aeruginosa. The exact mechanism by which salicylate exerts its effect is not known, but it appears to be related to osmoregulation.     

Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic polypeptides

The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. Because Escherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on the assumption that differences in epitope specificity could influence protective or pathologic host reactions. Because of the high level of structural homology of OmpF to porins isolated from other enteric pathogens, the identification of T- and B-cell-stimulatory determinants of E. coli OmpF may have broader application.     

Immune recognition of porin and lipopolysaccharide epitopes of Salmonella typhimurium in mice

We investigated the antigenic specificity of the humoral immune response to infection by Salmonella typhimurium, by competitive inhibition enzyme-linked immunosorbent assay and Western immunoblots. A panel of eight murine monoclonal antibodies, raised to OmpC and OmpD porins and lipopolysaccharide (LPS)-O antigens, was used to define the specificity of the polyclonal immune response in mice. The monoclonal antibody panel recognized five distinct epitopes; these were localized to surface-exposed loops of OmpC and OmpD porin, to the "eye-let" forming loop L3 of OmpC/OmpD, and to LPS-O4 and O5 factors. The immune mouse serum raised to infections with S. typhimurium LT-2 strain WB600 (wild-type) competitively inhibited the binding of biotin-labelled monoclonal antibodies to the epitopes that they recognize, indicating that all five epitopes were targets of the host immune response to natural infection. However, only two epitopes, one within a surface-exposed loop of OmpC porin, and the other in the LPS-O4 factor, were immunodominant. Furthermore, the bacterial LPS core and O-antigen structure influenced the immune response to the porins. Surface epitopes of porins were dominant in the rough strain SH5014 (rfa), whereas the immune recognition of LPS epitopes was predominant in mice infected with the smooth, wild-type strain (WB600). Finally, the immune response to LPS epitopes O4 and O5 was more pronounced in mice immunized with heat-killed cells than those infected with live S. typhimurium.     

OmpC is involved in invasion of epithelial cells by Shigella flexneri.

Osmoregulation of the Shigella flexneri ompC gene and the role of OmpC in Shigella virulence have been investigated. OmpC was highly expressed when bacteria were grown in medium of either low or high osmolarity. This constitutive expression is in contrast with the regulation observed in Escherichia coli, in which the expression of OmpC is repressed at low osmolarity and induced at high osmolarity. In addition, the Shigella ompC gene was barely expressed by a delta ompB (delta ompR and delta envZ) mutant. We described in a previous report that such a mutant was severely impaired in virulence both in vitro and in vivo. Starting from this observation, and in order to assess which gene(s) regulated by ompR and envZ are involved in virulence, we constructed an S. flexneri delta ompC mutant. Three S. flexneri mutants, ompF'-lacZ, delta ompC, and delta ompB, were compared for virulence. The ompF'lacZ mutant behaved like the S. flexneri serotype 5 wild-type strain M90T in all in vitro and in vivo virulence tests. On the contrary, the delta ompB and delta ompC strains were considerably impaired in their virulence phenotypes. The ability of these two mutants to spread from cell to cell and to kill epithelial cells was severely affected. Consequently delta ompC, as previously described for delta ompB, was unable to elicit a positive Sereny test. The delta ompB mutant was restored to virulence by introducing a recombinant multicopy plasmid carrying the cloned E. coli ompC gene, indicating that a functional OmpC protein was necessary and sufficient to restore virulence to this mutant of S. flexneri.     

Structural and functional characterization of the OmpF and OmpC porins of the Escherichia coli outer membrane: studies involving chimeric proteins

The roles of submolecular regions of OmpF and OmpC, major outer membrane proteins of Escherichia coli, as concerns their biogenesis, structure and function were studied using a large number of chimeric genes constructed from the ompF and ompC genes through single or double homologous in vivo recombination. When recombination between the two genes took place at certain regions of their central regions, no chimeric protein was detected, irrespective of whether the amino-terminal and carboxy-terminal regions were derived from OmpF or OmpC. Biochemical studies revealed that these proteins were synthesized and exported across the cytoplasmic membrane normally, but that they were not properly assembled into the outer membrane and hence were degraded rapidly. Characterization of these chimeric proteins, in which recombination between OmpF and OmpC took place once or twice, suggested that the central region of each of these proteins plays an important role in the respective assembly, whereas the roles of the amino-terminal and carboxy-terminal regions may be marginal. Functional characterization of these chimeric proteins revealed the regions important for the receptor functions of OmpF and OmpC for phages TuIa and TuIb, respectively.     

Two Salmonella OmpC K(b)-restricted epitopes for CD8+-T-cell recognition

We report the identification of two peptides from Salmonella OmpC porin that can bind to major histocompatibility complex class I K(b) molecules and are targets of cytotoxic T lymphocytes from Salmonella-infected mice. These peptides are conserved in gram-negative bacterial porins and are the first Salmonella porin-specific epitopes described for possible CD8(+)-T-cell elimination of infected cells.     

Role of surface-exposed loops of Haemophilus influenzae protein P2 in the mitogen-activated protein kinase cascade

The outer membrane of gram-negative bacteria contains several proteins, and some of these proteins, the porins, have numerous biological functions in the interaction with the host; porins are involved in the activation of signal transduction pathways and, in particular, in the activation of the Raf/MEK1-MEK2/mitogen-activated protein kinase (MAPK) cascade. The P2 porin is the most abundant outer membrane protein of Haemophilus influenzae type b. A three-dimensional structural model for P2 was constructed based on the crystal structures of Klebsiella pneumoniae OmpK36 and Escherichia coli PhoE and OmpF. The protein was readily assembled into the beta-barrel fold characteristic of porins, despite the low sequence identity with the template proteins. The model provides information on the structural features of P2 and insights relevant for prediction of domains corresponding to surface-exposed loops, which could be involved in the activation of signal transduction pathways. To identify the role of surface-exposed loops, a set of synthetic peptides were synthesized according to the proposed model and were assayed for MEK1-MEK2/MAPK pathway activation. Our results show that synthetic peptides corresponding to surface loops of protein P2 are able to activate the MEK1-MEK2/MAPK pathways like the entire protein, while peptides modeled on internal beta strands are unable to induce significant phosphorylation of the MEK1-MEK2/MAPK pathways. In particular, the peptides corresponding to loops L5 (Lys206 to Gly219), L6B (Ser239 to Lys253), and L7 (Thr280 to Lys287) activate, as the whole protein, essentially JNK and p38.