Abstract
Evidence from developing countries and volunteer studies indicates that immunity to Campylobacter jejuni and Campylobacter coli may be acquired, but the antigenic basis for this protection is poorly defined. We have purified to homogeneity four proteins with molecular weights of 28,000 (PEB1), 29,000 (PEB2), 30,000 (PEB3), and 31,000 (PEB4) from epidemic C. jejuni strain 81-176 using acid extraction and sequential ion-exchange, hydrophobic interaction, and gel filtration chromatography. The relative amino acid compositions of these four proteins are similar. NH2-terminal sequence analysis indicates that all four proteins are different, although the first 35 amino acids of PEB2 and PEB3 are 51.4% homologous. Isoelectric focusing showed that all four are basic proteins with pI of 8.5 for PEB1 protein and greater than 9.3 for the others. Use of the purified proteins as antigens in an IgG enzyme-linked immunosorbent assay (ELISA) found that seroconversion to the PEB1 or PEB3 proteins occurred in 15 of 19 patients with sporadic C. jejuni or C. coli infection. In comparison, only two, six, and 14 of these patients seroconverted to PEB2, PEB4, or the acid extract antigen. In an ELISA with whole bacterial cells as antigens, antiserum to the acid-extracted antigens showed broad recognition of C. jejuni, C. coli, C. fetus, C. lari, and Helicobacter pylori. Antiserum to PEB1 recognized all 35 C. jejuni and all 15 C. coli strains but none of the isolates of the other three bacterial species. The PEB1 and PEB3 proteins appear to be candidate antigens for both a Campylobacter vaccine and for serological assays for the pathogen.
Highlights
Nashville, Tennessee 37212, the §Medical and Research Seruices, Department of Veterans Affairs Medical Center, and the VDepartment of Medicine, Diuisionof Infectious Diseases, University of Colorado School of Medicine, Denuer, Colorado80220
The PEB2 ( M, = 29,000), PEB3 ( M,= 30,000),and PEB4 ( M,= 31,000) antigens were partially purified by cation-exchange separation on a Mono S column (Fig. 3)
The similarity inthe calculated PEBl from C. jejuni and C. coli are 70% identical (Fig. 7); the molecular weight of PEBl asdefined by SDS-PAGE and gel amino acid differences are consistent with antigenic differfiltration chromatography indicates that the native form of ences
Summary
Purification of C.jejuniAntigens-Whole bacterial cells and crude acid-extracted mixtures from four C.jejuni strains. The purification of the PEB antigens is summarized in Fig. 1.PEBl was purified to greater than 98% homogeneity by a single passage of the acid extract preparation on a phenyl-Superose column that fractionated material into three major protein peaks (Fig. 2). Of 19 patients with C. jejuni or C. coli diarrhea, seroconverted to the acid-extracted mixture, to PEBl or PEB3, two to PEB2, and six to PEB4. Rabbit antiserum to PEBl was used in Western blot to monitor the sensitivity of PEBl to the protease digestions This antiserum is monospecific and recognized a single antigen band migrating at M , = 28,000 in whole cell preparations, indicating that no degradation of the proteinoccurred during the extraction and purification processes. Acid-extract mixture recognized all 35 C. jejuni, all 15 C. coli, nine of 10 C. fetus, all five C. lari, and three of five H. pylori strains.
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