Characterization of a predominant immunogenic outer membrane protein of Riemerella anatipestifer

Abstract

The ompA gene, encoding the 42-kDa major antigenic outer membrane protein OmpA of Riemerella anatipestifer, the etiololgical agent of septicemia anserum exsudativa, was cloned and expressed in Escherichia coli. Recombinant OmpA displayed a molecular mass similar to that predicted from the nucleotide sequence of the ompA gene but lower than that observed in total cell lysates of R. anatipestifer. The ompA gene showed a conserved C-terminal region comprising the OmpA-like domain and a variable N-terminal region. This structure is similar to those of the analogous outer membrane proteins of several gram-negative bacteria. However, OmpA of R. anatipestifer contains six EF-hand calcium-binding domains and two PEST regions, which distinguish it from other outer membrane proteins. The occurrence of these motifs in OmpA suggests a possible role in virulence for this protein. The ompA gene is present in the R. anatipestifer type strain and in all serotype reference strains. However, it exhibits some minor genetic heterogeneity among different serotypes, which seems not to affect the strong antigenic characteristics of the protein. OmpA is a conserved and strong antigenic determinant of R. anatipestifer and hence is suggested to be a valuable protein for the serodetection of R. anatipestifer infections, independent of their serotype.

FIG. 1

Structure of the 2.2-kb insert in plasmid pJFFRaOmpA15. The upper part gives the localization of ompA and the partial open reading frame ORFX, encoding a protein with high similarity to the spore coat protein of B. subtilis. The promoter sequence upstream of ompA is indicated by a triangle. The inverted-repeat structure at the end of ompA, representing a potential transcription stop signal, is indicated by a hairpin. The different domains of OmpA are shown in the lower part. The inside-to-outside transmembrane helix-spanning domain (aa 5 to 22) is represented by a checkered box. The six EF-hand calcium-binding domains (aa 129 to 141) are shown by a vertically hatched box. The two PEST regions (aa 139 to 164 and 166 to 187) are represented by gray boxes, and the OmpA-like domain, consisting of a 45-aa stretch in the C-terminal half of the protein, is indicated by a black box.

FIG. 2

DNA sequence of the ompA gene and derived amino acid sequence. The first 1,320 bp of the DNA sequence of the insert in plasmid pJFFRaOmpA15 containing the entire ompA gene and its regulative sequences is shown. The nucleotides are numbered as deposited in the GenBank/EMBL database (accession number AF104936). The −10 and −35 promoter sequences and the ribosome binding site (RBS) are in italic. The start codon ATG is in boldface. The nucleotide sequences forming a stem-loop structure of a potential rho-independent transcription termination signal are indicated by two arrows above the DNA sequence. The amino acids forming an inside-to-outside transmembrane helix structure are underlined with a dashed line. The calcium-binding domains are shown in a gray box. The two PEST regions of OmpA are underlined, and the characteristic OmpA-like domain of 45 aa is underlined with a dotted line.

FIG. 3

Immunological reactions and Ca²⁺ binding of OmpA. (A) Immunoblot reacted with monospecific polyclonal anti-OmpA antibodies. (B) Immunoblot reacted with sera from convalescent ducks that were infected experimentally with R. anatipestifer serotype 15. (C) Autoradiography of the blot reacted with ⁴⁵Ca²⁺. Lanes 1, total cell lysate of R. anatipestifer strain CVL110/89 (serotype 15); lanes 2, purified recombinant His₆-OmpA1His₁₀ protein. As molecular mass standards (lanes st) we used broad-range prestained protein markers (New England Biolabs, Beverly, Mass.; no. 7708S) for the immunoblots (A and B) and Bio-Rad (Hercules, Calif.) prestained broad-range standards for the Ca²⁺ blot (C), which were shown to bind ⁴⁵Ca²⁺ (15). The arrows show the position of the OmpA triplet. Numbers on the left of each panel are molecular masses in kilodaltons.

FIG. 4

Expression of OmpA by the different R. anatipestifer serotypes. Immunoblots of total cell lysates of the R. anatipestifer type strain and the different serotype reference strains reacted with monospecific polyclonal anti-OmpA-His₆ mouse serum are shown. The numbers indicate the serotype (Table 1) a, strain CCUG 25055 890822; b, strain DRL 28020. C, purified recombinant His₆-OmpA1His₁₀ used as a control; st, prestained broad-range protein markers (New England Biolabs). Molecular masses of the protein markers are given in kilodaltons.