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Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein.

ABSTRACT: To facilitate discrimination between the closely related enteropathogens Campylobacter jejuni and C. coli, unique differences in antigenic surface structure were examined. A genomic library of C. jejuni 81116 was constructed in plasmid pBluescriptIISK- and expressed in Escherichia coli K-12. Rabbit hyperimmune serum raised against C. jejuni ATCC 29428 recognized a clone expressing a C. jejuni 24-kDa membrane-associated protein. Antiserum raised against sonicated recombinant E. coli expressing the 24-kDa protein reacted with C. jejuni, whereas C. coli did not react specifically. Determination of the nucleotide sequence of the DNA insert of this recombinant plasmid revealed an open reading frame encoding 214 amino acids; the gene was designated mapA; and its gene product was designated MAPA. The 18 N-terminal amino acid residues constitute a signal sequence characteristic of prokaryotic membrane lipoproteins. In a dot blot hybridization assay with a mapA probe, 120 clinical isolates of C. jejuni were unequivocally discriminated from 126 other campylobacters, including 34 C. coli isolates. A PCR test based on the mapA sequence was developed for identification of C. jejuni. A PCR product was obtained with all of the clinical isolates of C. jejuni tested from human, dog, cat, bovine calf, and chicken sources. Recombinant MAPA with an added C-terminal six-histidine tail was affinity purified and used to immunize rabbits. The rabbit anti-MAPA serum specifically recognized the protein in whole cells of C. jejuni on Western blots (immunoblots). The MAPA protein was present in all of the C. jejuni strains tested and was absent in C. coli and related campylobacters.

SUBMITTER: Stucki U

PROVIDER: S-EPMC228055 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein.

Stucki U U Frey J J Nicolet J J Burnens A P AP

Journal of clinical microbiology 19950401 4

To facilitate discrimination between the closely related enteropathogens Campylobacter jejuni and C. coli, unique differences in antigenic surface structure were examined. A genomic library of C. jejuni 81116 was constructed in plasmid pBluescriptIISK- and expressed in Escherichia coli K-12. Rabbit hyperimmune serum raised against C. jejuni ATCC 29428 recognized a clone expressing a C. jejuni 24-kDa membrane-associated protein. Antiserum raised against sonicated recombinant E. coli expressing th ...[more]

PMID: 7790451

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Project description:We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.

Dataset Information

Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein.

Publications

Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein.

Similar Datasets

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