Antigenic determinants of the OmpC porin from Salmonella typhimurium

Abstract

The antigenic determinants of Salmonella typhimurium OmpC were investigated by the analysis of cyanogen bromide (CNBr)-generated porin peptides with antiporin monoclonal antibodies (MAbs). We identified six bands (f1 to f6) with estimated molecular masses of 35.5, 31.0, 25.0, 22.5, 13.8, and 10.0 kDa, respectively. In addition, two small fragments (f7 and f8; 3.0 to 6.0 kDa) were detected only infrequently. The OmpC monomer or its CNBr-generated peptides were electrophoretically transferred to a polyvinylidene difluoride membrane and then subjected to amino acid composition analysis and N-terminal sequencing. A comparison of the amino acid composition data with known compositions of Escherichia coli and Salmonella typhi OmpC showed some differences; however, the amino acid sequences of 71 residues identified in S. typhimurium showed 88 and 98% identity with OmpC from E. coli and S. typhi, respectively. The screening of CNBr peptides with the 12 anti-(S. typhimurium) OmpC MAbs by Western blot (immunoblot), in conjunction with the prediction of the OmpC folding pattern based on the known three-dimensional structure of E. coli OmpF, showed that four MAbs reacted with surface-exposed epitopes on loops L2, L8, and L4 to L7, four MAbs reacted with a region in the eyelet structure on loop L3, and four MAbs reacted with the buried epitopes on transmembrane ␤ strands. The MAbs reacting with surface-exposed loops showed no cross-reaction with E. coli OmpC, whose sequence has diverged extensively from that of S. typhi and (probably) S. typhimurium OmpC only in regions of the externally exposed loops. In contrast, MAbs reacting with transmembrane ␤ strands, whose sequence is strongly conserved, showed strong cross-reaction with E. coli OmpC. These results show that comparison with the E. coli OmpF structure predicts the folding pattern of S. typhimurium OmpC rather accurately and that evolutionary divergence in sequences is confined to the external loops. The possible roles of these surface-exposed and buried epitopes as potentially useful antigenic regions for diagnostic assays and vaccine development are discussed.

Macrophages recognize and adhere to an OmpD-like protein of Salmonella typhimurium

“…This amino acid sequence is identical to the amino acid sequence for the OmpC of S. typhimurium LT2 SH 7457 [18] (Table 2).…”

“…Partial amino acid sequence data obtained from the N-terminus of the 44-kDa protein completely matched a 15-mer of the OmpD of S. typhimurium SH 7454 [27] and the OmpC of E. coli K-12 [28]. A similar but not identical sequence is associated with the 36-kDa OmpC from S. typhimurium LT2 [18] and 14028 (see Table 2). Based on these sequence data, the bacterial protein recovered in this study is OmpD-like; its physiological relationship with OmpD and other recognized porin proteins is, at present, unknown.…”

In Vitro Selection of RNA Aptamer Specific to Salmonella Typhimurium

“…Typhimurium, but not to E. coli O157:H7. E. coli and Salmonella OmpC proteins contain a different amino acid composition, and monoclonal antibodies reacting with surface-exposed loops of OmpC of Salmonella did not cross-react with that of E. coli [14]. This epitope region contains an amino acid sequence extensively diverged from that of E. coli.…”

“…Moreover, Sal. Typhimurium OmpC has a different amino acid sequence, compared with other Gramnegative bacteria including Escherichia coli (E. coli) [14]. Therefore, OmpC is a candidate target molecule for the specific detection of Sal.…”

The chaperone binding domain of SopE inhibits transport via flagellar and SPI‐1 TTSS in the absence of InvB

“…Samples of bacterial pellets (recovered from 0.2 ml of culture if not stated otherwise) and culture supernatants were separated on sodium dodecyl sulphate (SDS) or tricine gels and transferred onto a nitrocellulose membrane. Proteins were detected by Western blotting using a polyclonal anti‐SopE antiserum (Mirold et al ., 1999), a polyclonal anti‐SipC antiserum (Kaniga et al ., 1995), a polyclonal anti‐SptP antiserum (Ehrbar et al ., 2004), a polyclonal anti‐SopB antiserum (this study), a monoclonal anti‐M45 antibody (Obert et al ., 1994), a monoclonal anti‐OmpC antibody (Singh et al ., 1995), appropriate horseradish peroxidase (HRP)‐conjugated secondary antibodies and a chemoluminescent detection kit, as recommended by the manufacturer (Amersham Pharmacia). The blots were reprobed using the anti‐OmpC antibody to ensure that equal amounts were loaded.…”