Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface

Abstract

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and SalmoneUla typhimurium strains. Western immunoblots showed complete immunological crossreactivity between E. cofi B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coi B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli 08:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S.typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact 0 antigen, as in E. coli 08:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an 0 antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfia E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.The outer membrane of gram-negative bacteria is an asymmetric bilayer composed of lipopolysaccharide (LPS), phospholipids, and proteins (for reviews, see references 16 and 26). It plays a dominant role in resistance to host factors and antibiotics (6, 36), while it provides uptake channels for nutrients and ions (25). LPS is confined to the outer leaflet of the outer membrane, comprising 45% of its surface area (10, 16). It consists of a hydrophobic lipid A region embedded in the bilayer and two peripheral structures: the oligosaccharide core and the 0-antigenic side chain. These last two saccharide domains give the cell surface its hydrophilic character. Lipid A is similar in composition among the enteric bacteria, but the core oligosaccharide shows variation from strain to strain; six different structures have been characterized (7). In studies with rfa mutants, which lack the O antigen or have a shortened core, the role of LPS as a permeability barrier has been demonstrated (5, 35).Porins are trimeric proteins which span the outer membrane bilayer and serve as water-filled channels for the passive diffusion of hydrophilic molecules of less than 650 daltons (25,26). The Escherichia coli K-12 porins OmpF, OmpC, and PhoE share extensive regions of sequence homology and some limited local homology with OmpA (23...

Evolution of the ferric enterobactin receptor in gram-negative bacteria

“…(5-cm) strip containing proteins in the molecular mass range of 50 to 150 kDa was cut from the paper and incubated overnight in diluted monoclonal ascitic fluid at ambient temperature. Western blots were developed with goat anti-mouse immunoglobulinalkaline phosphatase and nitroblue tetrazolium-bromochloroindoyl phosphate (5). The reaction was stopped after 30 min by washing the strips in distilled water.…”

“…The incubation of OM or cell envelopes with lysozyme (1 mg/ml) for 30 min at 0°C and the addition 0.03 M EDTA and 0.2 M NaCl to the SDS-PAGE sample buffer improved electrophoretic separation. For silver-stained gels, 5 ,ug of each sample was boiled for 5 min and spun in the microcentrifuge for 1 min, and the supernatant was loaded into the slab.…”

Immunochemical structure of the OmpD porin from Salmonella typhimurium

“…, 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.…”

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

“…The primary structure of porins is quite hydrophilic overall but contains short stretches (10 to 14 residues) that cross the OM bilayer as amphiphilic 13-strands (24,59,60). Porins have numerous (five or more) surface epitopes, 6 to 25 residues in length (24), that are at least partially obscured by the lipopolysaccharide (LPS) core and completely blocked by 0-antigen sugars (2,32). The primary structure of porins varies significantly among different gram-negative bacteria (16), but it is likely that their predominant structural motif of amphiphilic ,B-strands forming a barrel is rather conserved (16,22,60).…”