Expression of Campylobacter hyoilei lipo-oligosaccharide (LOS) antigens in Escherichia coli

Abstract

Campylobacter spp. are well recognized as primary pathogens in animals and in people. To isolate and define the genetic regions encoding major surface antigens of Campylobacter hyoilei, genomic DNA of the type strain of the species, RMIT-32A, was cloned into a cosmid vector, plA2917, in Escherichia coli and the resulting genomic library was screened using antiserum raised to the parent C. hyoilei strain. Six cosmid clones were found to express a series of immunoreactive bands in the 15-25 kDa range. These bands were proteinase Kresistant and were found in the LPS fraction of the cells, suggesting that the recombinant cosmids expressed C. hyoirei lipo-oligosaccharide (LOS) antigen(s). The minimum DNA insert size required for expression of C. hyoilei LOS antigen@) in E. coli was 11.8 kb. This region was subcloned into the plasmid vector pBR322. The partial sequencing of the 11.8 kb region showed that it contains two ORFs, designated rfbf and rfbP, showing homology with the rfbF gene from Serratia marcescens and the rfbP gene from Salmonella typhimurium. Both genes are involved in LPS synthesis. The region also contained a sequence homologous to the rfaC gene of E. coli and Sal. typhimurium which is involved in core oligosaccharide synthesis.

Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis

“…The homology was over the last 124 amino acids in the rfpB gene product with 52% identity and 76% similarity. The GNMBB25f sequence was also homologous to a number of genes including Orf41x3 of Vibrio cholerae (Stroeher et al ., 1997), the rfbF gene of Campylobacter hyoilei (Korolik et al ., 1997), the cpsF gene of Proteus mirabilis (Gygi et al ., 1995), the Cap 1E of Streptococcus pneumoniae (Munoz et al ., 1997), the rfbF genes of Klebsiella pneumoniae (Clarke et al ., 1995; Kelly and Whitfield, 1996) and the rfbF gene of Serratia marcescens (Kelly and Whitfield, 1996). There was also similarity to an LPS glycosyltransferase icsA of Neisseria meningitidis (van der Ley et al ., 1996; also know as rfaK , Kahler et al ., 1996).…”

“…Clustal comparison of deduced amino acid sequences homologous to PglA. The sequences are: PglA Nm, Neisseria meningitidis strain MC58 (this work); RfpB Sd, rfpB gene of Shigella dysentariae serotype 1 (Gohmann et al ., 1994, also known as rfp Klena et al ., 1992); ORF41×3 Vc, Orf41x3 of Vibrio cholerae (Stroeher et al ., 1997); RfbF Ch, rfbF gene of Campylobacter hyoilei (Korolik et al ., 1997); CpsF Pm, the cpsF gene of Proteus mirabilis (Gygi et al ., 1995); Cps1 Sp, the Cap 1E of Streptococcus pneumoniae (Munoz et al ., 1997); RfbF KpO1, the rfbF genes of Klebsiella pneumoniae O1 (Clarke et al ., 1995); RfbF KpO8, the rfbF genes of Klebsiella pneumoniae O8 (Kelly and Whitfield, 1996); RfbF Sm and the rfbF gene of Serratia marcescens (Kelly and Whitfield, 1996).…”

The glycome

“…The possession of both glycosylation systems in C. jejuni is almost unprecedented among bacteria. 3) and initially the pgl genes were attributed a role in LOS biosynthesis (Section 2.1) [14,15]. The locus encoding this glycosylation system is adjacent to the genes responsible for LOS biosynthesis (Fig.…”

Pathogenesis of enteric Campylobacter infection

“…1998; Wood et al. 1999), as expression of these genes in E. coli led to the production of an O‐antigen recognized by antisera to C. jejuni LPS (Korolik et al. 1997).…”