Colonic bacteria express an ulcerative colitis pANCA-related protein epitope

Abstract

Bacteria are a suspected pathogenic factor in inflammatory bowel disease, but the identity of the relevant microbial species remains unresolved. The pANCA autoantibody is associated with most cases of ulcerative colitis (UC) and hence reflects an immune response associated with the disease process. This study addresses the hypothesis that pANCA identifies an antigen(s) expressed by bacteria resident in the human colonic mucosa. Libraries of colonic bacteria were generated using aerobic and anaerobic microbiologic culture conditions, and bacterial pools and clonal isolates were evaluated for cross-reactive antigens by immunoblot analysis using the pANCA monoclonal antibody Fab 5-3. Two major species of proteins immunoreactive to pANCA monoclonal antibodies were detected in bacteria from the anaerobic libraries. Colony isolates of the expressing bacteria were identified as Bacteroides caccae and Escherichia coli. Isolation and partial sequencing of the B. caccae antigen identified a 100-kDa protein without database homologous sequences. The E. coli protein was biochemically and genetically identified as the outer membrane porin OmpC. Enzyme-linked immunosorbent assay with human sera demonstrated elevated immunoglobulin G anti-OmpC in UC patients compared to healthy controls. These findings demonstrate that a pANCA monoclonal antibody detects a recurrent protein epitope expressed by colonic bacteria and implicates colonic bacterial proteins as a target of the disease-associated immune response.

FIG. 1

Immunoblot analysis of colonic bacterial cultures. (A) Mixed bacterial cultures were produced from colonic endoscopic biopsies of three patients under various medium and oxygen availabilities. Equivalent amounts (10 μg/lane) of mixed bacterial cultures were separated on a 10% polyacrylamide gel, transferred to nitrocellulose membranes, and probed with recombinant Fab monoclonal antibodies: Fab 5-3 pANCA (left panel), Fab 5-2 pANCA (middle panel), and P313 anti-tetanus toxoid (right panel). Medium A, sheep blood agar (aerobic) or BBA (anaerobic); medium B, Trypticase soy broth (aerobic) or TGL (anaerobic). Arrows indicate proteins specifically detected by Fab 5-3. (B) Equivalent amounts (10 μg/lane) of anaerobic colonic isolates were separated on a 10% polyacrylamide gel, transferred to nitrocellulose membranes, and probed with 5-3 pANCA monoclonal Fab. Isolates originated from the following anaerobic cultures: p1Bc5 and p1Bc9 were from the patient 1 specimen after BBA selection; p2c2 and p2c5 were from the patient 2 specimen after BBA selection; p2Lc2, p2Lc3, and p2Lc5 were from the patient 2 specimen after LKV selection. Two major immunoreactive protein species were identified: ∼35-kDa protein expressed by E. coli isolates and ∼100-kDa protein doublet expressed by B. caccae isolates. A ∼48-kDa species variably accompanied the ∼100-kDa B. caccae protein.

FIG. 2

Expression of Fab 5-3 immunoreactive protein in Bacteroides strains. Equivalent amounts (10 μg/lane) of Bacteroides clinical isolates were separated on a 13% polyacrylamide gel, transferred to nitrocellulose membranes, and probed with 5-3 pANCA monoclonal Fab. p2Lc3 is included as a positive control for the reactive ∼100-kDa protein.

FIG. 3

Identification of the E. coli immunoreactive protein. (A) The 19-amino-acid NH₂-terminal peptide sequence obtained from the E. coli ∼35-kDa immunoreactive protein was aligned with the peptide sequences of the E. coli OmpC and OmpF genes. The peptide initiates at position 22, corresponding to the beginning of the mature peptide (the first 21 amino acids are the leader peptide of the precursor protein). Sequences for the human histone H1.5 and Mycobacterium tuberculosis HupB gene products are also aligned, using the CLUSTAL W multiple sequence alignment program. (B) Equivalent number of cells (∼10⁷ per lane) of E. coli XL1 (wild type) and OmpC⁻, OmpF⁻, and OmpC⁻OmpF⁻ mutants were separated on a 12% polyacrylamide gel, transferred to nitrocellulose membranes, and probed with Fab 5-3 or P313 monoclonal antibody.

FIG. 4

Human seroreactivity to E. coli porin. ELISA wells were coated with OmpC-enriched porin (purified from OmpF⁻ E. coli) and reacted with serum samples at 1:200 (qualitatively similar results were obtained with 1:100 to 1:1,000 dilutions). Wells were developed with phosphatase–anti-IgG and chromogenic substrate, and absorbances were tabulated for 10 healthy control and 30 UC patient samples. Bars indicate arithmetic means for each group, and shaded areas indicate ± 1 SD. P values were calculated between the two groups by Student's t test. OD405, optical density at 405 nm.