Re-annotation of the Campylobacter jejuni NCTC11168 genome and functional characterisation of selected genes involved in strain pathogenesis

Abstract

Campylobacter jejuni is the leading bacterial cause of foodborne human gastroenteritis worldwide. The first C. jejuni genome (strain NCTC11168) was sequenced in 2000. This original annotation was a milestone in Campylobacter research, but soon became outdated. A re-annotation and re-analysis of this genome sequence was performed resulting in updates to over 90% of coding sequences (CDSs) and modification of 18.2% of CDS product functions (Gundogdu et al., 2007). Following this re-annotation, 15 uncharacterised CDSs with revised functions relating to virulence, signal transduction or regulation of gene expression were selected for further investigation. Defined isogenic C. jejuni 11168H mutants were constructed and after preliminary analysis, the Cj1556 and Cj0248 mutants were selected for further characterisation. Cj1556 was originally annotated as a hypothetical protein and was updated to a MarR family transcriptional regulator. Further bioinformatic analysis indicated a putative role in regulating the oxidative stress response. A C. jejuni 11168H Cj1556 mutant exhibited increased sensitivity to oxidative and aerobic (O2) stress, decreased ability for intracellular survival in both Caco-2 intestinal epithelial cells (IECs) and J774A.1 mouse macrophages and a reduction in virulence in the Galleria mellonella infection model. Microarray analysis of gene expression changes in the Cj1556 mutant compared to the wild-type strain indicated negative autoregulation of Cj1556 expression and down-regulation of genes associated with oxidative and aerobic (O2) stress responses. Cj0248 was originally annotated as a hypothetical protein however the re-annotation identified a HD domain linked to a superfamily of metal-dependent phosphohydrolases with roles in signal transduction in bacteria. Previously a C. jejuni 81-176 Cj0248 mutant was shown to be deficient for motility and chick colonisation, however the exact function of Cj0248 was not investigated. The C. jejuni 11168H Cj0248 mutant also possessed a reduced motility phenotype and exhibited reduced interaction and invasion when co-cultured with Caco-2 IECs compared to the wild-type strain. However the Cj0248 mutant showed no difference in autoagglutination compared to the wild-type strain and TEM analysis indicated the mutant possessed intact flagella. Higher magnification TEM indicated the possibility of an altered flagella basal body region in the Cj0248 mutant. Secretion profile analysis identified no differences in the protein profile of the Cj0248 mutant compared to the wild-type strain. The exact function of Cj0248 remains unclear.