Application of TraDIS to define the core essential genome of Campylobacter jejuni and Campylobacter coli

Abstract

Campylobacter species are the major cause of bacterial gastroenteritis. As there is no effective vaccine, combined with the rapid increase in antimicrobial resistant strains, there is a need to identify new targets for intervention. Essential genes are those that are necessary for growth and/or survival, making these attractive targets. In this study, comprehensive transposon mutant libraries were created in six C. jejuni strains, four C. coli strains and one C. lari and C. hyointestinalis strain, allowing for those genes that cannot tolerate a transposon insertion being called as essential. Comparison of essential gene lists using core genome analysis can highlight those genes which are common across multiple strains and/or species. Comparison of C. jejuni and C. coli, the two species that cause the most disease, identified 316 essential genes. Genes of interest highlighted members of the purine pathway being essential for C. jejuni whilst also finding that a functional potassium uptake system is essential. Protein–protein interaction networks using these essential gene lists also highlighted proteins in the purine pathway being major ‘hub’ proteins which have a large number of interactors across the network. When adding in two more species (C. lari and C. hyointestinalis) the essential gene list reduces to 261. Within these 261 essential genes, there are many genes that have been found to be essential in other bacteria. These include htrB and PEB4, which have previously been found as core virulence genes across Campylobacter species in other studies. There were 21 genes which have no known function with eight of these being associated with the membrane. These surface-associated essential genes may provide attractive targets. The essential gene lists presented will help to prioritise targets for the development of novel therapeutic and preventative interventions.

Identification of Campylobacter jejuni and Campylobacter coli genes contributing to oxidative stress response using TraDIS analysis

“…To compare the response to oxidative stress across the three strains, a pairwise BLASTP analysis was performed followed by the building of a network model [ 22 ]. Across all three genomes, 1391 genes were found to be core (present in all three strains tested here), with 140 genes being present only in the two C. coli strains (Fig.…”

“…Both C. coli strains had an ability to withstand higher H 2 O 2 concentrations, compared with C. jejuni 11168 which could not survive past 4mM of H 2 O 2 . These strains all came from different sources and belong to different sequence types [ 22 ]. The effect of these differences (possibly due to differences in genes present, and how the proteins interact under stress conditions) is likely to cause this variance in survival at different concentrations of H 2 O 2 .…”

“…Creation and analysis of the Tn-insertion mutant libraries within these stains have previously been documented [ 22 ] however are briefly detailed here. The C. jejuni 11168 Tn library consists of 79,301 unique Tn mutants, giving a rough density of a Tn insertion every 21 bp.…”

“…DNA was quantified using Qubit dsDNA BR assay kit (Invitrogen) and diluted to ~ 25ng/µl. Sequencing libraries were prepared according to Stoakes et al [ 22 ] and sequenced on an Illumina NextSeq 500 using a 500/550 high output V2 kit (75 cycles) (Illumina).…”

“…The molecular mechanisms underlying the survival strategies of Campylobacter spp, particularly C. jejuni and C. coli , under oxidative stress in the food chain are not fully understood. The generation of comprehensive Tn-insertion mutant libraries in both C. jejuni and C. coli [ 22 ] means that high throughput analysis of genes required for oxidative stress response can be completed.…”