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Mass spectrometric characterization of the Campylobacter jejuni adherence factor CadF reveals post‐translational processing that removes immunogenicity while retaining fibronectin binding
Author(s) -
Scott Nichollas E.,
Marzook N. Bishara,
Deutscher Ania,
Falconer Linda,
Crossett Ben,
Djordjevic Steven P.,
Cordwell Stuart J.
Publication year - 2009
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200900440
Subject(s) - campylobacter jejuni , biology , bacterial outer membrane , biochemistry , escherichia coli , genetics , gene , bacteria
Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn‐binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2‐DE and MS. Several spots were identified as products of the cadF gene. These included mass and p I variants of 34 and 30 kDa, as well as 24 kDa (CadF 24 ) and 22 kDa (CadF 22 ) mass variants. CadF variants were fully characterized by MALDI‐TOF MS and MALDI‐MS/MS. These data confirmed that CadF forms re‐folding variants resulting in spots with lower mass and varying p I that are identical at the amino acid sequence level and are not modified post‐translationally. CadF 22 and CadF 24 , however, were characterized as N ‐terminal, membrane‐associated polypeptides resulting from cleavage between serine 195 and leucine 196 , and glycine 201 and phenylalanine 202 , respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full‐length CadF (34 kDa), CadF 24 , and the deleted C ‐terminal OmpA domain (14 kDa; CadF 14 ) were created in Escherichia coli . Recombinant CadF variants were probed against patient sera and revealed that only full‐length CadF retained reactivity. Binding assays showed that CadF 24 retained Fn‐binding capability, while CadF 14 did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn‐binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni .