Mass Spectrometric Characterization of the Surface-Associated 42 kDa Lipoprotein JlpA as a Glycosylated Antigen in Strains of Campylobacter jejuni

摘要

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in the developed world. Immunoproteornics highlighted a 42-45 kDa antigen that comigrated on two-dimensional (2-DE) gels with the C. jejuni major outer membrane protein (MOMP). Predictive analysis revealed two candidates for the identity of the antigen, the most likely of which was the surface-associated lipoprotein, JlpA. Recombinant JlpA (rJlpA) reacted with patient sera, confirming that JlpA is antigenic. Polyclonal antibodies raised against rJlpA reacted against 3 JlpA mass variants from multiple C. jejuni. These variants differed by approximately 1.5 kDa, suggesting the presence of the N-linked C. jejuni glycan on two sites. Soybean agglutinin affinity and 2-DE purified 2 JlpA glycoforms (43.5 and 45 kDa). Their identities were confirmed using mass spectrometry following trypsin digest. Glycopeptides within JlpA variants were identified by proteinase-K digestion, graphite micropurification and MS-MS. Sites of glycosylation were confirmed as asparagines 107 and 146, both of which are flanked by the N-linked sequon. Sequence analysis confirmed that the N-146 sequon is conserved in all C. jejuni genomes examined to date, while the N-107 sequon is absent in the reference strain NCTC 11168. Western blotting confirmed the presence of only a single JlpA glycoform in both virulent (0) and avirulent (GS) isolates of NCTC 11168. MS analysis showed that JlpA exists as 3 discrete forms, unmodified, glycosylated at N-146, and glycosylated at both N-146/107, suggesting glycan addition at N-146 is necessary for N-107 glycosylation. Glycine extracts and Western blotting revealed that doubly glycosylated JlpA was the predominant form on the C. jejuni JHH1 surface; however, glycosylation is not required for antigenicity. This is the first study to identify N-linked glycosylation of a surface-exposed C. jejuni virulence factor and to show strain variation in glycosylation sites.