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Complete nucleotide sequence of the major outer membrane protein gen from Chlamydia trachomatis serovar L1
No full textThe complete nucleotide sequence encoding the major outer membrane protein (MOMP) of Chlamydia trachomatis serovar L1 has been determined. Comparison of the inferred amino acid sequence with that derived for the N-terminus of the MOMP from C. trachomatis serovar L2 reveals a conservation of amino acid residues and a putative 22 amino acid signal peptide at the N-terminus of the protein. A region displaying homology with a cloned fragment containing a species-specific domain of the MOMP from C. trachomatis L2 is also observed. The coding region of the C. trachomatis L1 MOMP is preceded by a probable Shine-Dalgarno ribosome binding site and followed by a typical ?-independent transcription termination site
Molecular cloning and sequence analysis of a developmentally regulated cysteine-rich outer membrane protein from Chlamydia trachomatis.
No full textTwo overlapping genomic fragments have been cloned from Chlamydia trachomatis serovar L1 DNA. Sequence determination of 2530 bp has revealed two open reading frames coding for 'cysteine-rich' (Cr) proteins. One of these proteins was confirmed, by analysis of the inferred amino acid sequence, as the 60-kDa Cr outer membrane protein associated with differentiation of reticulate bodies (RBs) into elementary bodies (EBs). The other smaller 15-kDa protein contained a high percentage of methionine and cysteine and may correspond to a reported smaller and co-ordinately synthesised Cr outer-membrane protein also associated with RB to EB differentiation. Sequencing showed three potential stem-loop structures within the 5', 3' and intergenic regions of the cloned fragment. Southern-blot analysis revealed that the cloned fragment is conserved in ten serovars of C. trachomatis and that a strongly cross-hybridising fragment is also present in Chlamydia psittaci
Chlamydia trachomatis major outer membrane protein epitopes expressed as fusions with LamB in an attenuated aroA strain of Salmonella typhimurium; their application as potential immunogens
No full textThe major outer-membrane protein (MOMP) of Chlamydia trachomatis is the focus of attention for chlamydial vaccine design, particularly those serovar- and subspecies-specific epitopes which provoke neutralizing immune responses. Selected surface-exposed B-cell epitopes of MOMP, incorporating B-subspecies specificities, were expressed as fusions with LamB, an inducible outer-membrane transport protein of Escherichia coli. These recombinant chlamydial-LamB proteins were correctly transported to the outer membrane of both E. coli and an aro A mutant of Salmonella typhimurium. The immunogenicity of the constructs was investigated in a mouse model of chlamydial salpingitis. After oral immunization, recombinant S. typhimurium were recovered from the livers of mice for up to two weeks, and a serum IgG response was induced both to the Salmonella and to the inserted chlamydial epitopes. By contrast, intravenous inoculation was ineffective. Although these LamB fusions proved only weakly immunogenic, this approach should be useful for investigating the ability of attenuated S. typhimurium vaccines incorporating chlamydial epitopes to stimulate protective mucosal immunity in the mouse model of chlamydial salpingitis
Sulfur-rich proteins of Chlamydia trachomatis: developmentally regulated transcription of polycistronic mRNA from tandem promoters
No full textRNA was extracted at various times from cells infected with Chlamydia trachomatis serovar L1. Northern-blot analysis showed that transcription of the CrP gene encoding the 60-kDa cysteine-rich outer membrane protein (CrP) produces a temporally controlled polycistronic mRNA. Primer extension analysis indicated the presence of tandem promoters separated by 66 nt with transcriptional start points (tsp) located 577 and 643 nt upstream from the start codon of the mature 60-kDa CrP. Nucleotide (nt) sequencing of this region revealed a small open reading frame (SORF) with coding potential for an 88-amino acid protein containing 13 cysteine residues. This SORF is transcribed as both a polycistronic 2300-nt mRNA together with the CrP gene, and as a separate 480-nt mRNA. Analysis of the upstream sequences, around the tsp for these mRNAs, revealed the presence of three inverted repeat structures that might act as binding domain(s) for a regulatory protein
Chlamydia trachomatis 60 kDa cysteine rich outer membrane protein: sequence homology between trachoma and LGV biovars
No full textAn 820 bp AccI-PstI fragment of the 60 kDa cysteine rich outer membrane protein (CrP) gene from C. trachomatis serovar L1 was used as a probe to locate the 60 kDa CrP gene of a recent serovar B trachoma isolate (Jali 20/OT). The probe hybridized to a single 1.8 kb SpeI fragment in Southern blot analyses of different restriction endonuclease digests of C. trachomatis serovar B DNA. This fragment was ligated into Lambda Zap II arms and Bluescript SK(-) recombinants, released by infection with the helper phage R408, were used as template for DNA sequence determination. Sequence analysis demonstrated a very high level of homology between the Jali 20/OT 60 kDa CrP and the previously published serovar L1 60 kDa CrP with only 8 out of 507 amino acid substitutions between the two proteins
Epitope mapping with solid-phase peptides: identification of type-, subspecies-, species- and genus-reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis
No full textThe major outer membrane protein (MOMP) of Chlamydia trachomatis carries serovar-, subspecies-, species- and genus immunodomains, antibodies to which may be protective. We have compared the inferred amino acid sequences for MOMP from different serovars of C. trachomatis and from Chlamydia psittaci to identify the likely locations of these sero-taxonomic epitopes. Overlapping peptides corresponding to each of these regions were synthesized on a solid phase and probed with monoclonal antibodies (MAbs) of appropriate specificities. We describe the primary structures of the binding sites of MAb to each of these four epitopes on C. trachomatis serovar L1 MOMP
High-level expression and epitope localization of the major outer membrane protein of Chlamydia trachomatis serovar L1
No full textFragments 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
Surface-exposed epitopes on the major outer-membrane protein of Chlamydia trachomatis defined with peptide antisera
No full textThe surface exposure of computer-predicted, linear B-cell epitopes on the major outer-membrane protein (MOMP) of Chlamydia trachomatis serovar B was assessed using antibodies raised against synthetic peptides in conjunction with immunogold transmission electron microscopy. Several of the chosen peptides elicited antibodies which reacted with both denatured and native MOMP. The majority of the exposed epitopes were found within the variable segments of MOMP. For each of the epitopes identified, the extent of their surface accessibility varied both among individual organisms and different developmental forms. Evidence for two distinct subspecies-specific epitopes within VS4 is presented
The major outer membrane protein of Chlamydia trachomatis: critical binding site and conformation determine the specificity of antibody binding to viable chlamydiae
No full textThe major outer membrane protein (MOMP) is the prime candidate for the development of a chlamydial vaccine. Antibodies to the subspecies-specific epitope neutralize chlamydial infection. Monoclonal antibodies (MAbs) to this epitope were prepared either by immunization with whole chlamydiae or with a 16 amino acid synthetic peptide. The critical binding site on the subspecies epitope for these MAbs was determined to single amino acid resolution using several hundred solid-phase peptides. A frame shift of just one amino acid in critical binding site completely prevented antibody binding to viable chlamydiae. A single MAb to whole organisms was capable of spanning both the surface-exposed, conformation-dependent, subspecies epitope and a buried, conformation-independent species epitope some 10 A distant. Immunization with peptide generated an MAb with reduced binding constraints which permitted the antibody to bind with broadened species-specificity at the subspecies binding site. The results show for the first time the importance of both critical binding site and conformation at the subspecies epitope. We suggest that the conformational flexibility of short, epitopic peptide vaccines may in some cases be advantageous, giving rise to extended specificity not attained with the natural protein
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