Outer membrane protein PhoE as a carrier for the exposure of foreign antigenic determinants at the bacterial cell surface

PhoE protein is an abundant outer membrane protein of theEscherichia coli K-12 outer membrane. This protein can be used as an exposure system to produce foreign antigenic determinants and for their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature could be made in different cell surface-exposed regions of PhoE, without interfering with the assembly process of the mutant proteins into the outer membrane. Two antigenic determinants of the structural VP1 protein of foot-and-mouth disease virus were inserted in different combinations in four cell surface-exposed regions of PhoE. The epitopes were exposed at the bacterial cell surface and they keep their antigenic and immunogenic properties in this PhoE-associated conformation. Immunization of guinea pigs with one hybrid protein, containing a combination of the two epitopes inserted in the fourth exposed region, resulted in complete protection against challenge with the virus. A T-cell epitope of the 65 kDa heat shock protein ofMycobacterium tuberculosis was inserted in the fourth exposed region of PhoE and in vitro proliferation of two T-cell specific clones was demonstrated. Thus, the PhoE exposure system has been shown to be suitable for presentation of both B-cell and T-cell determinants to the immune system. Furthermore, good expression of the hybrid protein in attenuatedSalmonella strains, which can be used as live oral vaccines, was shown.Paper awarded the Kluyver Prize 1990 by the Netherlands Society of Microbiology to Dr. M.C. Agterberg     

Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein

To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane-spanning segments are essential for the assembly process.     

Topology of outer membrane pore protein PhoE of Escherichia coli. Identification of cell surface-exposed amino acids with the aid of monoclonal antibodies

Monoclonal antibodies which recognize the cell surface-exposed part of outer membrane protein PhoE of Escherichia coli were used to select for antigenic mutants producing an altered PhoE protein. The selection procedure was based on the antibody-dependent bactericidal action of the complement system. Using two distinct PhoE-specific monoclonal antibodies, seven independent mutants with an altered PhoE protein were isolated. Among these seven mutants, five distinct binding patterns were observed with a panel of 10 monoclonal antibodies. DNA sequence analysis revealed the following substitutions in the 330-residue-long PhoE protein: Arg-201----His (three isolates), Arg-201----Cys, Gly-238----Ser, Gly-275----Ser and Gly-275----Asp. It is argued that amino acid residues 201, 238, and 275 are most likely directly involved in antibody binding and, therefore, exposed at the cell surface. Together with Arg-158, which was previously shown to be cell surface exposed as it is changed in phage TC45-resistant phoE mutants, these four positions show a remarkably regular spacing, being approximately 40 residues apart. A model is suggested in which the PhoE polypeptide repeatedly traverses the outer membrane in an antiparallel beta-pleated sheet structure, exposing eight areas to the outside which are all separated by approximately 40 residues.     

Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein

Bacterial outer membrane proteins are supposed to span the membrane repeatedly, mostly in the form of amphipathic beta-sheets. The last ten C-terminal amino acid residues of PhoE protein are supposed to form such a membrane-spanning segment. Deletion of this segment completely prevents incorporation into the outer membrane. Comparison of the last ten amino acid residues of other outer membrane proteins from different Gram-negative bacteria revealed the presence of a potential amphipathic beta-sheet with hydrophobic residues at positions 1 (Phe), 3 (preferentially Tyr), 5, 7 and 9 from the C terminus, in the vast majority of these proteins. Since such sequences were not detected at the C termini of periplasmic proteins, it appears to be possible to discriminate between the majority of outer membrane proteins and periplasmic proteins on the basis of sequence data. The highly conserved phenylalanine at the C termini of outer membrane proteins suggests an important function for this amino acid in assembly into the outer membrane. Site-directed mutagenesis was applied to study the role of the C-terminal Phe in PhoE protein assembly. All mutant proteins were correctly incorporated into the outer membrane to some extent, but the efficiency of the process was severely affected. It appears that both the hydrophobicity and the aromatic nature of Phe are of importance.     

Immunological approach of assembly and topology of OmpF, an outer membrane protein of Escherichia coli.

Various monoclonal antibodies (MoF) directed against cell-surface-exposed epitopes of OmpF, one major outer membrane pore protein of Escherichia coli B and K-12, have been used to study the assembly and the topology of the protein. This paper firstly describes the characterization of the OmpF epitopes recognized by the various monoclonal antibodies. A comparison between OmpC, OmpF and PhoE porins with respect to their primary amino acid sequence and their cell-surface exposed regions allows us to propose a rough model including 2 antigenic sites. The second part is focused on the assembly of the OmpF protein in the outer membrane. Various forms, precursor, unassembled monomer, metastable oligomer (pre-trimer) and trimer are detected with immunological probes directed against OmpF during a kinetic analysis of the process. The requirement for a concomitant lipid synthesis during the trimerization has been demonstrated by investigating the presence of a specific native epitope. The role of lipopolysaccharide during the stabilization of the conformation is discussed with regard to the various steps of assembly.     

Use of outer membrane protein PhoE as a carrier for the transport of a foreign antigenic determinant to the cell surface of Escherichia coli K-12

PhoE protein is an abundant transmembrane protein from the Escherichia coli K-12 outer membrane. A synthetic oligodeoxynucleotide corresponding to an antigenic determinant of the C-terminal part of the VP1 protein of foot-and-mouth disease virus was inserted into the phoE gene in an area corresponding to a cell surface-exposed region of the PhoE protein. The level of expression of the hybrid protein was normal and the protein was incorporated into the outer membrane. The VP1-epitope was exposed at the cell surface since intact cells were recognized by a monoclonal antibody which was raised against the virus.     

Expression of Escherichia coli PhoE protein in avirulent Salmonella typhimurium aroA and galE strains

Abstract Escherichia coli K-12 PhoE protein is found to be normally expressed and incorporated into the outer membrane of two avirulent Salmonella typhimurium strains, G30 and SH aroA . A hybrid protein which contains an insertion of an antigenic epitope of VP1 protein of foot-and-mouth disease virus into the PhoE protein, was also normally assembled into the Salmonella outer membranes. In the case of the G30 stain, which carries a galE mutation, the inserted epitope is accessible to antibodies in intact cells. In contrast, the epitope is less accessible in the case of the SH aroA strain, probably due to the shielding effect of the O-antigen in this strain.     

Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12

The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli K-12. A model, in which this protein crosses the membrane eight times in an antiparallel beta-sheet conformation and in which regions around amino acids 25, 70, 110 and 154 are exposed at the cell surface, had been proposed. Linkers were inserted into the ompA gene with the result that OmpA proteins, carrying non-OmpA sequences between residues 153 and 154 or 160 and 162, were synthesized. Intact cells possessing these proteins were treated with proteases. Insertion of 15 residues between residues 153 and 154 made the protein sensitive to proteinase K and the sizes of the two cleavage products were those expected following proteolysis at the area of the insertion. Addition of at least 17 residues between residues 160 and 162 left the protein completely refractory to protease action. Thus, the former area is cell surface exposed while the latter area appears not to be. The insertions did not cause a decrease in the concentration of the hybrid proteins as compared to that of the OmpA protein, and in neither case was synthesis of the protein deleterious to cell growth. It is suggested that this method may serve to carry peptides of practical interest to the cell surface and that it can be used to probe surface-located regions of other membrane proteins.     

Folding of a bacterial outer membrane protein during passage through the periplasm.

The transport of bacterial outer membrane proteins to their destination might be either a one-step process via the contact zones between the inner and outer membrane or a two-step process, implicating a periplasmic intermediate that inserts into the membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D-structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the outer membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the outer membrane.     

Amino terminus of outer membrane PhoE protein: localization by use of a bla-phoE hybrid gene

Expression of a recently constructed bla-phoE hybrid gene results in synthesis and incorporation into the outer membrane of PhoE protein containing an amino-terminal extension of 158 amino acid residues of beta-lactamase (Tommassen et al., EMBO J. 2:1275-1279, 1983). As the PhoE protein part of this hybrid protein is apparently normally incorporated into the outer membrane, the beta-lactamase part of the protein can be considered as a label of the amino terminus of PhoE protein. By using trypsin accessibility experiments, this beta-lactamase part was shown to be located at the periplasmic side of the membrane. Therefore, the amino terminus of PhoE protein most likely faces the periplasm.     

The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure–function relationships in the TraT protein

The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. The structure and function of the TraT protein determined by plasmid R6-5 was probed by genetic insertion of a foreign antigenic determinant, the C3 epitope of polio virus, at residues 61, 125, 180, 200 or 216 of the protein. The chimaeric proteins were transported to the outer membrane and, in three cases, immunoassays with an anti-C3 monoclonal antibody indicated that the C3 epitope was exposed on the cell surface. Three of the hybrids, with insertions at residues 125, 180 and 200, assembled into the trypsin-resistant oligomeric form characteristic of the wild-type protein, which suggested that these regions are not involved in TraT subunit:subunit interactions. Additionally, the hybrid protein carrying the C3 epitope at position 180 functioned in a genetic suppression assay and retained partial surface-exclusion activity. Thus, its localization, folding and organization does not appear to be grossly altered from that of the wild-type protein. Applications of the protein for the transport of foreign antigenic determinants to the cell surface are discussed.     

In vitro trimerization of outer membrane protein PhoE.

The folding of outer membrane protein PhoE of E coli into its native trimeric structure was studied in vitro by using monoclonal antibodies, which recognize cell-surface exposed, conformational epitopes of the protein. These antibodies were able to precipitate the in vitro synthesized PhoE protein, showing that the conformational epitopes are formed in vitro. From analysis by SDS--polyacrylamide gel electrophoresis, it appeared that the precipitated protein represents a folded monomer. The signal sequence interferes with the formation of the conformational epitopes. Outer membranes were required to induce the formation of the stable trimeric form of the protein. This trimerization was not accompanied by insertion into the outer membranes.     

Topology of PhoE porin: the 'eyelet' region

A model for the topology of the PhoE porin has been proposed according to which the polypeptide traverses the outer membrane sixteen times mostly as amphipathic β-sheets, thereby exposing eight loops at the cell surface. Until now, no evidence has been obtained for the surface exposure of the third loop. Recently, the structure of porin of Rhodobacter capsulatus has been determined. The proposed model of PhoE is very similar to the structure of the R capsulatus porin, which has an ‘eyelet’ region, extending into the interior of the pore. The proposed third external loop of PhoE might form a similar ‘eyelet’ region. To determine the location of the predicted third external loop of PhoE, multiple copies of an oligonucleotide linker encoding an antigenic determinant of VP1 protein of foot-and-mouth disease virus (FMDV) were inserted. All hybrid proteins were properly inserted in the outer membrane. The monoclonal antibody MA11, directed against the linear FMDV epitope, was able to bind only to intact cells expressing a hybrid PhoE protein with at least three copies of the FMDV epitope present. Antibiotic sensitivity tests and single-channel conductance measurements revealed that the insertions influenced the channel size. These results are consistent with a location of the third loop of PhoE within the pore channel.     

The assembly pathway of outer membrane protein PhoE of Escherichia coli.

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.     

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

Amino acid residue arginine-158 of the outer membrane protein PhoE of Escherichia coli K-12 has been shown to be cell-surface-exposed [Korteland et al. (1985) Eur. J. Biochem. 152, 691-697]. To study the effects of small insertions in this region of the protein on its biogenesis and characteristics, a unique restriction site was created by site-directed mutagenesis in a plasmid carrying the phoE gene and oligonucleotides of 12-74 bp were inserted. The insertions did not interfere with incorporation into the outer membrane since (a) several monoclonal antibodies, directed against the cell-surface-exposed part of PhoE protein, bound to whole cells producing the altered proteins and (b) the proteins formed functional pores for the uptake of beta-lactam antibiotics. The binding of one monoclonal antibody and of the PhoE-specific phages TC45 and TC45hrN3 was disturbed by the insertions, showing that this region of the protein is immunogenic and is involved in the binding of both of these phages. The functioning of the mutant pores was characterized both in vivo by studying the uptake of beta-lactam antibiotics and in vitro after the reconstitution of the proteins in black lipid films. The pore characteristics changed depending on the nature of the inserted amino acids. Addition of a negatively charged amino acid resulted in decreased anion-selectivity, whereas insertion of a positive charge and deletion of a negative charge had only a small influence.     

A comparative study on the phoE genes of three enterobacterial species. Implications for structure-function relationships in a pore-forming protein of the outer membrane

The cloned phoE genes from Enterobacter cloacae and Klebsiella pneumoniae are normally expressed and regulated in Escherichia coli K-12, and their products are correctly assembled into the outer membrane. Differences between the three PhoE proteins were found with binding of two out of ten monoclonal antibodies directed against the cell-surface-exposed part and in pore characteristics, but not in phage receptor function. The DNA sequences of the E. cloacae and K. pneumoniae phoE genes were determined and used to predict the primary structures of the encoded proteins. In the upstream non-coding regions, which showed more variations among the three genes than the coding regions, conserved sequences were identified which might be involved in regulation of phoE gene expression. Comparison of the predicted PhoE primary structures revealed a high degree of homology, with 81% of the amino acid residues being identical in all three proteins. Four small variable regions were found where differences are the most pronounced, corresponding to regions which were previously predicted to be exposed at the cell surface. Implications of the sequence comparison for structure-function relationships in PhoE protein are discussed.     

Periplasmic accumulation of truncated forms of outer-membrane PhoE protein of Escherichia coli K-12

In order to localize the information within PhoE protein of Escherichia coli K-12 required for export of the protein to the outer membrane, we have generated deletions throughout the phoE gene. Immunocytochemical labelling on ultrathin cryosections revealed that the polypeptides encoded by the mutant alleles are transported to, and accumulate in, the periplasm. These results show that, except for the signal sequence, there is no specific sequence within the PhoE protein that is essential for transport through the cytoplasmic membrane. The overall structure of the protein, rather than a particular sequence of amino acids, seems to be important for assembly into the outer membrane.     

Lipopolysaccharides and divalent cations are involved in the formation of an assembly-competent intermediate of outer-membrane protein PhoE of E.coli.

To identify the requirements for the biogenesis of outer-membrane proteins in Gram-negative bacteria, the sorting and assembly of the trimeric, pore-forming protein PhoE was studied in vitro. Purified lipopolysaccharide (LPS) in combination with low amounts of Triton X-100 and divalent cations induced the formation of folded monomers. LPS of deep-rough strains was far less efficient in the formation of folded monomers than wild-type LPS was. These folded monomers could be converted into heat-stable trimers upon addition of outer membranes and higher amounts of Triton X-100. Trimerization could precede the insertion step. These in vitro data suggest that the assembly in vivo proceeds sequentially by (i) formation of a folded monomer by interaction with LPS; (ii) sorting of the folded monomers to assembly sites in the outer membrane; (iii) trimerization; and (iv) insertion.     

Analysis of structure-function relationships in Escherichia coli K12 outer membrane porins with the aid of ompC-phoE and phoE-ompC hybrid genes

Summary To study structure-function relationships in the outer membrane pore proteins OmpC and PhoE of Escherichia coli K12, we have constructed a series of phoE-ompC hybrid genes in which DNA encoding part of one protein is replaced by the homologous part of the other gene. The hybrid gene products were incorporated normally into the outer membrane, allowing their functional characterization. Combined with previous studies, the present results permit the identification of regions involved in determining functions and properties in which the native PhoE and OmpC proteins differ, such as pore characteristics, receptor activity for phages and binding of monoclonal antibodies. Most of these properties were found to be determined by multiple regions clearly separated in the primary structure. The combined phage and antibody binding data have demonstrated that at least five distinct regions in PhoE and OmpC are exposed at the cell surface. The locations of these regions are in agreement with a previously proposed model for porin topology.