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Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis.

ABSTRACT: During studies in which we serotyped large numbers of Chlamydia trachomatis clinical isolates by using monoclonal antibodies, three novel serological variants (D-, D*, and I-) were identified. To determine the molecular basis for the altered monoclonal antibody reactions of these strains and other previously identified variants (Da, Ia, and L2a), we determined the nucleotide sequences of the variable domains in their major outer membrane protein genes. Da, D-, and D* variants differed by a single nucleotide and also an amino acid in the carboxy terminus of variable domain IV (VDIV) from the D serovar. The L2a variant also differed from L2 by a single amino acid but in VDII. Ia variants differed in VDI, III, and IV and I- variants differed in all four VDs from the I serovar. These studies demonstrate the potential for using major outer membrane protein VD sequencing as a highly sensitive typing method and further identify immunologically reactive major outer membrane protein epitopes.

SUBMITTER: Lampe MF

PROVIDER: S-EPMC302707 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis.

Lampe M F MF Suchland R J RJ Stamm W E WE

Infection and immunity 19930101 1

During studies in which we serotyped large numbers of Chlamydia trachomatis clinical isolates by using monoclonal antibodies, three novel serological variants (D-, D*, and I-) were identified. To determine the molecular basis for the altered monoclonal antibody reactions of these strains and other previously identified variants (Da, Ia, and L2a), we determined the nucleotide sequences of the variable domains in their major outer membrane protein genes. Da, D-, and D* variants differed by a singl ...[more]

PMID: 8418043

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Project description:BackgroundChlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes.ResultsC. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) beta-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands.ConclusionsC. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with beta-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide modified antigens for immunological studies and vaccination, including live subunit vaccines, and might be useful to co-express MOMP with other chlamydial membrane proteins.

Project description:OBJECTIVES--Whereas serovars A, B, Ba and C of Chlamydia trachomatis are usually associated with trachoma, two of these serovars (Ba and C) are occasionally observed in urogenital infections. Variation in the gene encoding the major outer membrane protein (MOMP) was explored to distinguish urogenital from trachoma specimens of the same serovar. METHODS--A large portion of the MOMP gene was amplified by nested PCR directly from clinical samples from trachoma or urogenital infection and the serovar of the infecting C trachomatis was determined by restriction fragment length polymorphism (RFLP). Amplified DNA from trachoma serovars B, Ba and C and from urogenital serovars Ba, C, D and E was sequenced by the dideoxy chain termination method. RESULTS--While almost identical in variable segment (VS)I, three urogenital Ba samples differed from all trachoma B and Ba samples at eight nucleotides including two sites which changed amino acids in the constant region upstream of VSI. An identical sequence in this region was observed for the reference urogenital D serovar. Variation in this same region upstream of VSI also distinguished 40% of serovar D samples from prototype D including three that were sequenced. Two urogenital C differed from trachoma C samples at four sites that changed the MOMP amino acid sequence including two changes in the constant region between VSII and III and single changes in VSII and III. On the basis of these sequence determinations, RFLP was predicted which allowed extension of these observations to 20 other urogenital Ba, 12 trachoma B or Ba, seven variant D, 12 D, four urogenital C and three trachoma C samples without further sequencing. CONCLUSION--Urogenital Ba and C samples have VSI or II and III sequences identical or very similar to trachoma strains of the same serovar, but resemble more closely other serovars in the constant regions. Urogenital serovar D samples can also be divided into two genotypes on the basis of sequence differences in the constant region preceding VSI.

Dataset Information

Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis.

Publications

Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis.

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