Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models

Abstract

Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. 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 chicken colonisation, survival in houseflies and under nutrient-rich and –poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.

Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection

“…Of the 98 mutations conferring increased in vitro fitness by DEseq2, validation experiments confirmed only 3 out of the 5 tested mutations (60%) ( Fig 2B ), revealing a slightly higher proportion of false-positive results with genes in this category. In general, false-positive results are not uncommon with Tn-seq screens [ 58 , 64 , 76 ] and may reflect a variety of reasons. The initial Tn-seq screen is a complex competitive environment involving thousands of mutants whereas validation studies tend to compare only a single mutant to the WT pathogen.…”

“…This result highlights that the 4 h in vitro library expansion step in our in vivo Tn-seq screen setting likely introduces some bias into our in vivo fitness datasets. Mutant pool expansion following in vivo Tn-seq screens, by mutant pool plating on agar [ 65 , 76 , 77 ] or culture in broth [ 64 , 78 ], is often required when library DNA extraction directly from tissue represents a limiting step to the Tn-seq procedure. This extra step is particularly important when using the Mme I-based Tn-seq approach [ 72 ], as it requires high quality DNA (no shearing).…”

Campylobacter jejuni transcriptome changes during loss of culturability in water

“…jejuni M1 adapts rapidly to introduction into a water environment, instigating gene expression changes that allow it to adapt to the stressful conditions, whilst maintaining viability. A recent complementary study by de Vries et al (2017) used Transposon Directed Insertion Sequencing (TRADIS) to identify genes essential for survival in vitro and in vivo [ 94 ]. Although comparing knockout studies to whole-transcriptome studies is difficult because gene knockouts will likely produce different transcriptional profiles due to deregulation or disruption of genetic pathways, in both studies a gene involved in the oxidative stress response: trxC , was identified as a key candidate gene for survival in model water systems.…”

CapC, a Novel Autotransporter and Virulence Factor of Campylobacter jejuni

“…To assess the role of CapC in C. jejuni, isogenic mutants of capC were constructed in C. jejuni 81116 and C. jejuni M1 by insertion of a chloramphenicol resistance cassette. We were unable to complement the capC mutants, as we were unable to clone the capC coding sequence into the pC46 or pSV009 complementation vector in the correct orientation, despite repeated attempts (36)(37)(38).…”