The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C trachomatis serovar L2 Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

Identification of the immunogenic proteins that induce Chlamydia trachomatis (CT)-specific T cell responses is crucial to the development of protective vaccines and understanding the mechanisms of chlamydia-induced pathology. To characterize the targets of the human T cell response we have used chlamydia-reactive human T cell clones as cellular probes to screen a CT genomic library expressed in Escherichia coli using peripheral blood mononuclear cells to present antigens. The library was screened with three chlamydia-reactive T cell clones of unknown specificity and three novel stimulatory chlamydia antigens were identified. These E. coli recombinants were shown to express the chlamydia proteins, enolase, pmpD and CT579. Enolase and pmpD proteins were purified and shown to induce the proliferation of synovial fluid mononuclear cells isolated from the knee joints of patients suffering from chlamydia-associated reactive arthritis. We suggest that these stimulatory antigens are common targets of the T cell response in this group of patients. A greater understanding of T cell-mediated immunity in uncomplicated CT infection, and in patients with CT-induced chronic inflammatory disease (trachoma, salpingitis, arthritis) may identify the principal immune responses associated with immunopathology.

Identification of Chlamydia trachomatis antigens recognized by human CD4+ T lymphocytes by screening an expression library

DNA obtained from the Sheila Smith strain of Rickettsia rickettsii was digested to completion with the restriction endonucleases BamHI and SalI and ligated with the plasmid vector pUC19. The ligation mixture was used to transform Escherichia coli. A total of 465 bacterial clones were screened for antigen production with hyperimmune rabbit serum. One of the reactive clones, containing a recombinant plasmid designated pSS124, was solubilized and subjected to immunoblot analysis and revealed expression of a 17-kilodalton protein reactive with anti-R. rickettsii serum that comigrated with an antigen from R. rickettsii. A 1.6-kilobase PstI-BamHI fragment from pSS124 was subcloned and continued to direct synthesis of the 17-kilodalton antigen. The nucleotide sequence was determined for this 1.6-kilobase subclone, which encompassed the gene encoding the polypeptide as well as flanking regions containing potential regulatory sequences. The open reading frame consisted of 477 nucleotides that specified a 159-amino-acid protein with a calculated molecular weight of 16,840. The deduced amino acid sequence contained a hydrophobic sequence near the amino terminus that resembled signal peptides described for E. coli. The carboxy terminus was hydrophilic in nature and probably contained the exposed epitopes.

Sequence analysis of the 17-kilodalton-antigen gene from Rickettsia rickettsii.

The 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 ϱ-independent transcription termination site.

Complete nucleotide sequence of the major outer membrane protein gene from Chlamydia trachomatis serovar L1

Summary 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 colL 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, L2rM0MP, 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 rapidfy lethal effect on the L2rM0MP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli done 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.

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

A gene library of Chlamydia trachomatis (serovar L1) DNA has been prepared in the phage vector lambda 1059. From this bank, 20 recombinant phage-expressing components which reacted with serum from a patient with a C. trachomatis (L1) infection were chosen. Selective expression and radiolabeling of phage polypeptides in irradiated Escherichia coli demonstrated that one of these clones encoded a polypeptide doublet with an apparent molecular weight similar to that of the C. trachomatis (L1) major outer membrane protein. Both species of this cloned doublet (40 and 41 kilodaltons) could be immunoprecipitated by serum from a patient with a C. trachomatis (L1) infection but not by normal human serum. Components of this apparent molecular weight were not precipitated from irradiated E. coli infected with vector phage lambda 1059 by either of these sera. Comparison of the Staphylococcus aureus-V8 protease peptide maps of these two cloned polypeptides and chlamydial major outer membrane protein extracted from elementary bodies showed all three polypeptides to produce peptide fragments of 15.5, 13.8, and 11.5 kilodaltons. Due to the identical apparent molecular weights of the fragments produced from the 40- and 41-kilodalton cloned polypeptides, these were concluded to be different conformational forms of the same molecular species. These cloned components were indistinguishable from C. trachomatis (L1) major outer membrane protein.

https://iai.asm.org/content/iai/45/3/637.full.pdf

T M Cunningham

Papers

Molecular cloning of the major outer membrane protein of Chlamydia trachomatis.