Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC

Abstract

The immunochemistry and structure of enteric bacterial porins are critical to the understanding of the immune response to bacterial infection. We raised 41 monoclonal antibodies (MAbs) to SalmoneUla typhimurium OmpD and OmpC porin trimers and monomers. Enzyme-linked immunosorbent assays, immunoprecipitations, and/or Western immunoblot techniques indicated that 39 in the panel are porin specific and one binds to the lipopolysaccharide; the specificity of the remaining MAb probably lies in the porin-lipopolysaccharide complex. Among the porin-specific MAbs, 10 bound cellsurface-exposed epitopes, one reacted with a periplasmic epitope, and the remaining 28 recognized determinants that are buried within the outer membrane bilayer. Many of the MAbs reacting with surface-exposed epitopes were highly specific, recognizing only the homologous porin trimers; this suggests that the cellsurface-exposed regions of porins tend to be quite different among S. typhimurium OmpF, OmpC, and OmpD porins. Immunological cross-reaction showed that S. typhimurium OmpD was very closely related to Escherichia coli NmpC and to the Lc porin of bacteriophage PA-2. Immunologically, E. coli OmpG and protein K also appear to belong to the family of closely related porins including E. coli OmpF, OmpC, PhoE, and NmpC and S. typhimurium OmpF, OmpC, and OmpD. It appears, however, that S. typhimurium "PhoE" is not closely related to this group. Finally, about one-third of the MAbs that presumably recognize buried epitopes reacted with porin domains that are widely conserved in 13 species of the family Enterobacteriaceae, but apparently not in the seven nonenterobacterial species tested. These data are evaluated in relation to host immune response to infection by gram-negative bacteria.

Immunochemical structure of the OmpD porin from Salmonella typhimurium

“…BALB/c mice were immunized with OmpD monomer and cell fusion was carried out as described previously (Singh e t al., 1992). Hybridomas were selected in hypoxanthine-aminopterin-thymidine medium (Kearney, 1984).…”

“…,1987). Recently, Hongo et al (1994) (Singh e t al., 1992). The porins in this family show roughly 65% identity and 80% similarity (Blasband et al, 1986; Mizuno e t a/., 1983; Puente e t al., 1989).…”

“…, 1991), but amphiphilic / 3 strands (7-14 residues each) in the porin barrel are structurally conserved (Gerbl-Rieger e t al., 1991 ;Jeanteur et al, 1994). Generally, porins contain five or more surface epitopes, 6-2s residues in length (Klebba et al, 1990;Puente e t al., 1989;Singh e t al., 1992Singh e t al., , 1995Singh e t al., , 1996Tommassen et al, 1993;van der Ley et al, 1986b), which are partially obscured by the lipopolysaccharide (LPS) core and completely blocked by O-antigen sugars (Bentley & Klebba, 1988 Ley e t al., 1986a). These proteins also contain conserved buried epitopes that are localized on the transmembranous p strands (Jeanteur etal., 1994;Klebba etal., 1990;Puente et al, 1989;Singh e t al., 1992Singh e t al., , 1995Singh e t al., , 1996.…”

“…Generally, porins contain five or more surface epitopes, 6-2s residues in length (Klebba et al, 1990;Puente e t al., 1989;Singh e t al., 1992Singh e t al., , 1995Singh e t al., , 1996Tommassen et al, 1993;van der Ley et al, 1986b), which are partially obscured by the lipopolysaccharide (LPS) core and completely blocked by O-antigen sugars (Bentley & Klebba, 1988 Ley e t al., 1986a). These proteins also contain conserved buried epitopes that are localized on the transmembranous p strands (Jeanteur etal., 1994;Klebba etal., 1990;Puente et al, 1989;Singh e t al., 1992Singh e t al., , 1995Singh e t al., , 1996. OmpD, a fourth general porin, is of special interest because it is regularly found in S. gpbimt/rizm but is absent from E. coli (Lee & Schnaitman, 1980;Lugtenberg & van Alphen, 1983;Nikaido & Vaara, 1985).…”

“…We also used a panel of 16 anti-OmpD mAbs to define several epitopes of S. gpbimtlrizlm OmpD. The CNBr-cleaved peptides containing internal Met residues and antiporin mAbs, as employed in this study, have been used previously to study the immunochemical structure of other bacterial porins (Haase et al, 1994;Klebba et al, 1990;Murphy & Bartos, 1988;Mutharia & Hancock, 1985;Rawling e t al., 1995;Singh et al, 1995). A folding pattern of OmpD is proposed; it predicts the same exposed loops, periplasmic turns and membrane-spanning strands of this protein as seen in the known threedimensional structure of E. coli OmpF (Cowan et al, 1992).…”