Faculty Bibliography

The protein PEB1 (28 kDa) is a common antigen and a major cell adherence molecule of Campylobacter jejuni and Campylobacter coli. We created a bank of chromosomal DNA fragments of C. jejuni strain 81-176 using lambda gt11. Screening this bank in Escherichia coli Y1090 cells with antibody raised against purified PEB1 enabled us to isolate and to purify a clone with a 2.6-kilobase insert expressing an immunoreactive protein of 28 kDa. DNA sequencing revealed that the insert contains three complete and two partial open reading frames (ORFs), designated 5' to 3' as ORFs A-E. The peb1A gene (ORF D) contains 780 bases encoding a 259-residue polypeptide having a calculated molecular mass of 28,181 Da. The peptide sequence starting at residue 27 matches that determined from aminoterminal sequencing of mature PEB1 from C. jejuni. The first 26 residues contain typical signal peptidase I and II cleavage sites. The deduced amino acid composition and pI of the recombinant mature protein are similar to those determined for purified PEB1. Gene bank searches indicated significant overall homology of peb1A and ORF C with operons for amino acid transport systems in other Gram-negative organisms. peb1A is homologous to the binding components of systems such as glnH (27.8%) and hisJ (28.9%), whereas ORF C has nearly 50% identity to glnQ and hisP. Thus, PEB1 could be involved both in binding to intestinal cells and in amino acid transport

Two immunogenic proteins of 27 (CBF1) and 29 (CBF2) kDa from enteropathogenic Campylobacter species appear to bind to mammalian cells. We purified these two proteins from a pathogenic and adherent Campylobacter jejuni strain to homogeneity by using acid extraction, preparative gel electrophoresis, and electroelution. Polyclonal rabbit antisera to these proteins were prepared. Immunologic studies indicate that CBF1 corresponds to the PEB1 and CBF2 corresponds to the PEB4 described by Pei et al. (Z. Pei, R. T. Ellison, and M. Blaser, J. Biol. Chem. 226:16363-16369, 1991). Immunogold labeling of a C. jejuni adherent strain with anti-CBF1, anti-CBF2, and anti-PEB1 suggested that CBF1 (PEB1) is surface exposed while CBF2 (PEB4) is not. Analysis of whole-cell extracts from 14 strains by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with 7 M urea and immunoblotting with antisera to CBF1 and CBF2 suggests that CBF proteins from adherent and nonadherent strains are different. Use of purified proteins in a microassay of adherence to cellular membranes indicated that CBF1 was much more adherent than CBF2. Adherence of C. jejuni to viable HeLa cells was markedly reduced with the antiserum to CBF1, whereas the CBF2 antiserum was a poor inhibitor. Purified CBF1 competitively inhibited adherence of whole bacteria to HeLa cells, whereas purified CBF2 was no better a competitor than bovine serum albumin. Adherence of CBF2 was markedly reduced in the presence of Tween 20 or SDS, whereas adherence of CBF1 was reduced only by SDS. We conclude that (i) CBF1 (PEB1) is surface exposed and may be the key protein for C. jejuni adhesion and (ii) CBF2 (PEB4) may be complexed with CBF1 and may passively coadhere with CBF1 under certain experimental conditions. Adherent and nonadherent strains contain different isotypes of these two proteins which could be useful markers of C. jejuni adhesion

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation

Wild-type Campylobacter fetus strains possess high-molecular-weight S-layer proteins (S+) and are highly resistant to serum-mediated killing and phagocytosis. Spontaneous mutant strains lacking these proteins (S-) are serum and phagocytosis sensitive and have reduced virulence in a mouse model. Intact S+ cells were treated with pronase, which made them S- although genotypically S+ and had essentially no effect on other cellular proteins or on viability. Treatment with pronase, but not buffer alone, rendered these cells serum and phagocytosis sensitive and reduced mouse virulence to the level observed for the S- mutant cells. In related studies, purified S-layer proteins diminished neutrophil chemoluminescent responses to a heterologous particulate antigen. Finally, passive administration of antiserum to the 97-kDa S-layer protein partially protected mice against lethal challenge with the S+ strain. These studies define the contribution of the S-layer proteins to C. fetus virulence

Campylobacter fetus strains may be of serotype A or B, a property associated with lipopolysaccharide (LPS) structure. Wild-type C. fetus strains contain surface array proteins (S-layer proteins) that may be extracted in water and that are critical for virulence. To explore the relationship of S-layer proteins to other surface components, we reattached S-layer proteins onto S- template cells generated by spontaneous mutation or by serial extractions of S+ cells with water. Reattachment occurred in the presence of divalent (Ba2+, Ca2+, Co2+, and Mg2+) but not monovalent (H+, NH4+, Na+, K+) or trivalent (Fe3+) cations. The 98-, 125-, 127-, and 149-kDa S-layer proteins isolated from strains containing type A LPS (type A S-layer protein) all reattached to S- template cells containing type A LPS (type A cells) but not to type B cells. The 98-kDa type B S-layer protein reattached to SAP- type B cells but not to type A cells. Recombinant 98-kDa type A S-layer protein and its truncated amino-terminal 65- and 50-kDa segments expressed in Escherichia coli retained the full and specific determinants for attachment. S-layer protein and purified homologous but not heterologous LPS in the presence of calcium produced insoluble complexes. By quantitative enzyme-linked immunosorbent assay, the S-layer protein copy number per C. fetus cell was determined to be approximately 10(5). In conclusion, C. fetus cells are encapsulated by a large number of S-layer protein molecules which may be specifically attached through the N-terminal half of the molecule to LPS in the presence of divalent cations

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

We developed a mouse model to compare the virulence of Campylobacter fetus strains with (S-plus) and without (S-minus) surface array protein (S-protein) capsules. In adult HA/ICR mice pretreated with ferric chloride, the LD50 for S-plus strain 84-32 was 43.3 times lower than its spontaneous S-minus mutant 84-54. Seven strains of inbred mice were no more susceptible than the outbred strain. In contrast to the findings with Salmonella typhimurium by others, 3 X 10(7) CFU of strain 84-32 caused 90% mortality in C3H/HeN (LPSn) mice and 40% mortality in C3H/HeJ (LPSd) mice. High-grade bacteremia in HA/ICR mice occurred after oral challenge with S-plus C. fetus strains and continued for at least 2 d, but was not present in any mice challenged with S-minus strains. Bacteremia at 30 min after challenge was 51.6-fold lower in mice pretreated with 10 microliters of rabbit antiserum to purified S-protein than after pretreatment with normal rabbit serum. Challenge of mice with a mixture of S-minus strain 84-54 and free S-proteins at a concentration 31.1-fold higher than found in wild-type strain 84-32 caused 30% mortality, compared with 0% with strain 84-54 or S-protein alone. These findings in a mouse model point toward the central role of the S-protein in the pathogenesis of C. fetus infection. The S-protein is not toxic per se, but enhances virulence when present on the bacterial cell surface as a capsule

A variety of Gram-negative and Gram-positive bacteria possess crystalline surface layers, although little is known of their function. We previously have shown that the high molecular weight surface-array proteins of Campylobacter fetus are important in both the pathogenicity and antigenicity of this organism. For biochemical and immunological characterization, we purified high molecular weight (100,000, 127,000, 149,000) surface-array proteins from three C. fetus strains using sequential gel filtration and ion exchange high performance liquid chromatography. These proteins are acidic with pI values between 4.12 and 4.25 and contain large proportions of acidic amino acids (19.7%-22.0%) in addition to hydrophobic amino acids (37.3%-38.5%). They share a novel amino-terminal sequence through at least 19 residues. Carbohydrate analysis using periodic acid-Schiff staining and treatment with trifluoromethanesulfonic acid shows no evidence of glycosylation. Antiserum to a purified Mr = 100,000 protein from C. fetus 82-40 LP cross-reacts with three other purified C. fetus surface-array proteins by enzyme-linked immunosorbent assay with titers greater than 12,800. We conclude that: 1) there is a family of surface-array proteins of C. fetus with common structural and antigenic characteristics; 2) that these molecules have similar biochemical characteristics to surface-array proteins described for other bacteria; but however, 3) by amino-terminal sequence analysis these are unique