Immunological cross-reactivity between outer membrane pore proteins of Campylobacter jejuni and Escherichia coli

Immunocrossreactivity between the major outer membrane protein (MOMP) of Campylobacter jejuni 85H and the OmpC porin of Escherichia coli K-12 was observed. These results indicate that a common antigenic domain is conserved in both MOMP and OmpC. This antigenic region is detected only after a 96 degrees C treatment suggesting that it is buried in the native conformation of the respective porins. In addition, differences were observed between the major outer membrane proteins from various C. jejuni strains. About 60% of the C. jejuni pathogenic strains tested contained a protein exhibiting a similar electrophoretic profile to the 85H porin.     

Conformational analysis of the Campylobacter jejuni porin.

The major outer membrane protein (MOMP) of Campylobacter jejuni was purified to homogeneity by selective solubilization and fast protein liquid chromatography. The amino acid composition of the MOMP indicates the presence of cysteine residues. The amino-terminal sequence, determined over 31 residues, shows no significant homology with any other porin from gram-negative bacteria except in a discrete region. Immunocross-reactivity between Escherichia coli OmpC and the MOMP was analyzed, and a common antigenic site between these two porins was identified with an anti-peptide antibody. From circular dichroism and immunological investigations, the existence of a stable folded monomer, containing a high level of beta-sheet secondary structure, is evident. Conformational analyses show the presence of a native trimeric state generated by association of the three folded monomers; the stability of this trimer is reduced compared with that of E. coli porins. This study clearly reveals that the C. jejuni MOMP is related to the family of trimeric bacterial porins.     

Infection-induced antibodies against the major outer membrane protein of Campylobacter jejuni mainly recognize conformational epitopes

The major outer membrane protein (MOMP) of Campylobacter jejuni is an abundant surface protein with a pore-forming function and may be a potential candidate for vaccine development. Despite the fact that MOMP is immunogenic and the recombinant MOMP (rMOMP) can be readily produced in Escherichia coli, the nature of the antibody response to MOMP during in vivo infection is not well understood. In this study, various methods involving detergent replacement and liposome reconstitution were used to refold rMOMP, and antibody responses to MOMP elicited in Campylobacter-colonized chickens were evaluated using sera from chickens either naturally or experimentally infected by C. jejuni. The results demonstrated that proteoliposomes restored the reactivity of rMOMP to rabbit antibodies elicited by native MOMP, indicating the recovery of native MOMP conformation by this refolding method. Importantly, sera from naturally or experimentally infected chickens reacted weakly with denatured rMOMP, but strongly with rMOMP reconstituted in proteoliposome, suggesting that the chicken antibody response to MOMP is predominantly directed against conformational epitopes. These observations provide direct evidence for conformation-dependent humoral responses to MOMP induced by Campylobacter infection, demonstrate that C. jejuni MOMP is immunogenic in its natural host and suggest that proteoliposomes may be potentially used for the evaluation of rMOMP-based vaccines.     

High-level expression and epitope localization of the major outer membrane protein of Chlamydia trachomatis serovar L1

Fragments of the gene encoding the major outer membrane porin protein (MOMP) of Chlamydia trachomatis serovar L1 were ligated into the pUC plasmid vectors to give a series of overlapping recombinants expressing MOMP from the lac promoter. Induction of this promoter with IPTG leads to high-level expression of the recombinant porin protein. Electron microscopy shows the presence of insoluble inclusions within the Escherichia coli host cells. Probing the expressed MOMP fragments with a set of monoclonal antibodies permitted localization of the four binding sites (epitopes) of primary-sequence-dependent monoclonal antibodies that exhibit genus-, species-, subspecies- and type (serovar)-specific reactivities.     

Peptide translocation across MOMP,the major outer membrane channel from Campylobacter jejuni

Here we report on translocation of short poly-arginines across the MOMP porin, the major outer membrane protein in the cell wall of Campylobacter jejuni. MOMP was purified to homogeneity from a pathogenic strain of C. jejuni. Its reconstitution in lipid membranes and measuring the ion-current revealed two main distinct populations of protein channels which we interpreted as mono and trimers. Addition of poly-arginines causes concentration and voltage dependent ion-current fluctuations. Increasing the transmembrane potential decreases the residence time of the peptide inside the channel indicating successful translocation. We conclude that poly-arginines can cross the outer membrane of Campylobacter through the MOMP channel.     

The ClpB protein from Campylobacter jejuni: molecular characterization of the encoding gene and antigenicity of the recombinant protein

The ClpB heat-shock protein is necessary for the survival of Escherichia coli cells upon sudden increase of temperature. Using a PCR-based genomic walking method, the nucleotide sequence of a clpB homolog from Campylobacter jejuni was determined. The clpB gene encodes a protein of 857 amino acid (aa) residues, with a predicted molecular mass of 95.3kDa. Alignment of the deduced aa sequence with other known bacterial ClpB proteins revealed overall identity from 47% (E. coli) to 61% (Helicobacter pylori). Within the clpB promoter region, as indicated by primer extension analysis, we identified a sequence identical to the E. coli sigma70 consensus promoter. Northern blot analysis confirmed that clpB is heat-inducible in C. jejuni. The ClpB protein, fused to a 6xHis tag, was synthesized in E. coli and purified by metal-affinity and size exclusion chromatography. In ELISA studies, IgA levels reactive to recombinant ClpB were significantly higher in sera of patients with prior C. jejuni infections than in sera obtained from healthy control persons.     

Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin.

We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins.     

Structural and functional analyses of the major outer membrane protein of Chlamydia trachomatis

Chlamydia trachomatis is a major pathogen throughout the world, and preventive measures have focused on the production of a vaccine using the major outer membrane protein (MOMP). Here, in elementary bodies and in preparations of the outer membrane, we identified native trimers of the MOMP. The trimers were stable under reducing conditions, although disulfide bonds appear to be present between the monomers of a trimer and between trimers. Cross-linking of the outer membrane complex demonstrated that the MOMP is most likely not in a close spatial relationship with the 60- and 12-kDa cysteine-rich proteins. Extraction of the MOMP from Chlamydia isolates under nondenaturing conditions yielded the trimeric conformation of this protein as shown by cross-linking and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with different concentrations of acrylamide. Using circular dichroism spectroscopy, we determined that the trimers were formed mainly of beta-pleated sheet structures in detergent micelles. Using a liposomal swelling assay, the MOMP was found to have porin activity, and the size of the pore was estimated to be approximately 2 nm in diameter. The trimers were found to be stable in SDS at temperatures ranging from 4 to 37 degrees C and over a pH range of 5.0 to 8.0. In addition, the trimers of MOMP were found to be resistant to digestion with trypsin. In conclusion, these results show that the native conformation of the MOMP of C. trachomatis is a trimer with predominantly a beta-sheet structure and porin function.     

Proteomic analyses of a robust versus a poor chicken gastrointestinal colonizing isolate of Campylobacter jejuni

Campylobacter spp. are a significant contributor to the bacterial etiology of acute gastroenteritis in humans. Epidemiological evidence implicates poultry as a major source of the organism for human illness. However, the factors involved in colonization of poultry with Campylobacter spp. remain unclear. Determining colonization-associated factors at the proteome level should facilitate our understanding of Campylobacter spp. contamination of poultry. Therefore, proteomic analyses were utilized to identify expression differences between two Campylobacter jejuni isolates, a robust colonizer A74/C and a poor colonizing strain of the chicken gastrointestinal system designated NCTC 11168-PMSRU. Proteomic analyses by two-dimensional gel electrophoresis revealed the specific expression of an outer membrane-fibronectin binding protein, serine protease, and a putative aminopeptidase in the soluble portion of the robust colonizer A74C. Several proteins including a cysteine synthase and aconitate hydratase were detected specifically in the poor colonizer C. jejuni NCTC 11168-PMSRU isolate. Variation in the amino acid sequences resulting in different isoelectric points and relative mobility of the flagellin and C. jejuni major outer membrane (MOMP) protein were also detected between the two isolates. Western blotting of the bacterial proteins revealed the presence of two flagellin proteins in the poor colonizer versus one in the robust colonizing isolate, but no differences in MOMP. The results demonstrated that proteomics is useful for characterizing phenotypic variation among Campylobacter spp. isolates. Interestingly, different gene products potentially involved in robust colonization of chickens by Campylobacter spp. appear to conform to recently identified expression patterns in Biofilm or agar-adapted isolates.     

The major outer membrane protein of Haemophilus ducreyi consists of two OmpA homologs.

The major outer membrane protein (MOMP) of Haemophilus ducreyi is an OmpA homolog that migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels as three species with apparent molecular weights ranging from 37,000 to 43,000. Monoclonal antibodies directed against this macromolecule were used to identify recombinant clones containing fragments of the gene encoding this protein. Nucleotide sequence analysis of these fragments confirmed that the MOMP encoded by the intact gene (momp) was a member of the OmpA family of outer membrane proteins. Construction of an isogenic H. ducreyi mutant unable to express the MOMP led to the discovery of a second outer membrane protein which migrated at the same rate on SDS-PAGE gels as the MOMP. N-terminal amino acid sequence analysis of this second protein revealed that its N terminus was nearly identical to that of the MOMP and also had homology with members of the OmpA family. Nucleotide sequence analysis of the region downstream from the momp gene revealed the presence of a partial open reading frame encoding a predicted OmpA-like protein. A modification of anchored PCR technology was used to obtain the nucleotide sequence of this downstream gene which was shown to encode a second OmpA homolog (OmpA2). The N-terminal amino acid sequence of OmpA2 was identical to that of the OmpA-like protein detected in the momp mutant. The H. ducreyi MOMP and OmpA2 proteins, which comigrated on SDS-PAGE gels and which were encoded by the tandem arranged momp and ompA2 genes, were 72% identical.     

四种类型启动子控制下空肠弯曲杆菌血红蛋白对大肠杆菌生长速度的影响

细菌血红蛋白可增加细胞在低氧条件下的氧气利用率,提高细胞的增长速率。为提高E. coli在现有发酵水平下的生物量,获得新型的基因工程底盘细胞,基于空肠弯曲杆菌(Campylobacter jejuni)血红蛋白基因,比较分析了组成型和诱导型启动子对血红蛋白在E. coli基因工程菌中的表达状况及对E. coli生长的影响。首先合成了空肠弯曲杆菌血红蛋白基因chb,并让chb基因在诱导型启动子(P_T7和P_vgh)和组成型启动子(P2和P_spoI-Ⅱ)控制下表达;随后,测定了由四种类型启动子控制的血红蛋白工程菌在摇瓶和蓝盖瓶中的生长状况。结果表明四种重组菌可以表达出有活性的血红蛋白,并可显著提高大肠杆菌的生长;在发酵罐中的试验也表明四种工程菌同样对细胞生长具有出促进作用;进一步比较分析了菌体密度、细胞鲜重和CO差式光谱值间的相互关系,讨论了四种类型启动子控制的基因工程菌在诱导表达和生长调控等方面的特点,并为在微氧和好氧等不同发酵条件下选用基因工程底盘细胞提供了参考。     

Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3' end in mRNA stability

The major outer membrane protein (MOMP)-encoding gene (omp1) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant omp1 is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous omp1 promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3' end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed omp1 gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.     

Comparison of methods used to separate the inner and outer membranes of cell envelopes of Campylobacter spp

The outer membrane of Campylobacter coli, C. jejuni and C. fetus cell envelopes appeared as three fractions after sucrose gradient centrifugation. Each outer membrane fraction was contaminated with succinate dehydrogenase activity from the cytoplasmic membrane fraction. Similarly the inner membrane fraction was contaminated with 2-ketodeoxyoctonate and outer membrane proteins including the porin(s). The separation of these two membranes was not facilitated by variations in lysozyme treatment, cell age, presence or absence of flagella, or longer lipopolysaccharide chain length. Sodium lauroyl sarcosinate extraction resulted in an outer membrane fraction which contained some inner membrane contamination and produced multiple bands upon sucrose gradient centrifugation. Triton X-100 extraction removed the inner membrane from the outer membrane and Triton X-100/EDTA treatment extracted lipopolysaccharide-rich regions of the outer membrane which contained almost exclusively the Campylobacter porin(s). These data indicated that the inner and outer membranes of the Campylobacter cell envelope were very difficult to separate, possibly because of extensive fusions between these two membranes.     

Phylogenetic relationships of Campylobacter jejuni based on porA sequences

Campylobacter porins are the dominant major outer membrane protein (MOMP) of these bacteria. They are composed of hypervariable, surface-exposed, peptide loops and membrane-embedded, conserved peptide regions. Porins are functionally important and may also be useful for molecular subtyping methods but have not yet been well characterized. We therefore sequenced the porA gene from 39 Campylobacter isolates, including multilocus sequence type (MLST) reference strains, isolates from patients with the Guillain-Barré syndrome, other clinical isolates, and serotyping reference strains. These were compared with additional sequences available from GenBank. Three distinct porA lineages were observed after phylogenetic analysis. Both Campylobacter coli and Campylobacter jejuni were found with group 3 porA sequences, and this was the only group showing any evidence of recombination among porA genes. There was no recombination between porA genes from C. jejuni groups 1 and 2, suggesting there may be functional constraints on changes at this locus. Most of the amino acid differences among the three groups were present in surface-exposed loops, and dissimilar substitutions were found when groups 1 and 2 MOMP were compared. Different MOMP sequence groups may have different biological or antigenic properties, which in turn may be associated with survival in different environments, host adaptation, or virulence.     

MOMP (major outer membrane protein) of Campylobacter jejuni; a versatile pore-forming protein

The great majority of trimeric porins of Gram-negative bacteria cannot be dissociated into monomers without disrupting their folded conformation. The porin of Campylobacter jejuni, however, displays two folded structures, a classical oligomer and a monomer resistant to detergent denaturation. We probed the transition of trimer to monomer using light scattering experiments and examined the secondary structures of these two molecular states by infra-red spectroscopy. The channel-forming properties of both trimer and monomer were studied after incorporation into artificial lipid bilayers. In these conditions, the trimer induced ion channels with a conductance value of 1200 pS in 1 M NaCl. The pores showed marked cationic selectivity and sensitivity to low voltage. Analysis of the isolated monomer showed nearly the same single-channel conductance and the same selectivity and sensitivity to voltage. These results indicate that the folded monomer form of C. jejuni MOMP displays essentially the same pore-forming properties as the native trimer.     

EnvZ functions through OmpR to control porin gene expression in Escherichia coli K-12.

The regulatory proteins OmpR and EnvZ are both required to activate expression of the genes for the major outer membrane porin proteins, OmpF and OmpC, of Escherichia coli K-12. Here we show that OmpR, under certain conditions, could activate porin expression in the complete absence of EnvZ. In addition, the pleiotropic phenotypes conferred by a particular envZ mutation (envZ473) required the presence of functional OmpR protein. These results lead us to conclude that EnvZ and OmpR act in sequential fashion to activate porin gene expression; i.e., EnvZ modifies or in some way directs OmpR, which in turn acts at the appropriate porin gene promoter.     

Solute transport proteins and the outer membrane protein NmpC contribute to heat resistance of Escherichia coli AW1.7

This study aimed to elucidate determinants of heat resistance in Escherichia coli by comparing the composition of membrane lipids, as well as gene expression, in heat-resistant E. coli AW1.7 and heat-sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids than that of E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression in these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR. DnaK and 30S and 50S ribosomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in that of E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50- to 1,000-fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7, but the heat resistance of this strain is dependent on additional factors, which likely include the composition of membrane lipids, as well as solute transport proteins.     

Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis and capable of eliciting protective antibodies in infected hosts, and therefore has potential as a candidate vaccine to prevent infection with this significant human pathogen. The recombinant MOMP clone, L2rMOMP, contained the entire MOMP gene including the encoded leader sequence. Large quantities of chlamydial MOMP were expressed, some of which was processed and translocated to the E. coli surface. Surface localization of the MOMP was demonstrated by the binding of anti-MOMP monoclonal antibodies to the surface of the induced clone, and was visualized by fluorescence and electron microscopy. The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including the contribution of the MOMP variable segments to the topographical interactions which determine the antigenic structure responsible for human immune response.