ena-STUDY-UNIVERSITY OF CAMBRDGE-08-01-2017-11:49:30:478-92
Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models
Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during in vitro growth, chicken colonisation, survival in houseflies and under nutrient-rich and –poor conditions at 4 degrees and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq), providing a measure for the relative abundance of each Tn mutant in the library. Tn-seq analysis after exposure to a challenge, compared with the control condition, provided a quantitative measure for the contribution of a gene to fitness in each of the models. For Tn-seq read-out, Tn mutant library DNA was fragmented by MmeI restriction digestion, adapters containing inline barcodes were ligated to the MmeI fragments, and amplified using the primers PBGSF29 and PBGSF30 with NEBNext high fidelity polymerase (New England Biolabs). Tn insertion sites were sequenced using single-end 40-50 bp sequencing on the Illumina HiSeq 2500 platform.
Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during in vitro growth, chicken colonisation, survival in houseflies and under nutrient-rich and –poor conditions at 4 degrees and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq), providing a measure for the relative abundance of each Tn mutant in the library. Tn-seq analysis after exposure to a challenge, compared with the control condition, provided a quantitative measure for the contribution of a gene to fitness in each of the models. For Tn-seq read-out, Tn mutant library DNA was fragmented by MmeI restriction digestion, adapters containing inline barcodes were ligated to the MmeI fragments, and amplified using the primers PBGSF29 and PBGSF30 with NEBNext high fidelity polymerase (New England Biolabs). Tn insertion sites were sequenced using single-end 40-50 bp sequencing on the Illumina HiSeq 2500 platform.