Genome-Wide Fitness Analyses of the Foodborne PathogenCampylobacter jejuniinIn VitroandIn VivoModels

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 nutrientrich 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.Infection by Campylobacter is the most common cause of foodborne bacterial diarrhoeal disease worldwide, responsible for ~96 million foodborne illnesses and ~21,000 foodborne deaths in 2010 1 . While most cases are self-limiting, for some, campylobacteriosis is a particularly serious infection, and it is also associated with severe post-infection complications, including irritable bowel and Guillian-Barré syndromes. Consumption of undercooked poultry, unpasteurised dairy products and contaminated water represent the most common sources of

Analysis of Campylobacter jejuni infection in the gnotobiotic piglet and genome-wide identification of bacterial factors required for infection

“…Previous Tn mutant screens in chicken colonisation models showed extensive random dropout of colonisation-proficient Tn mutants and extensive between-experiment variation15222324, for example Johnson et al ., reported a 51% loss of the Tn mutants present in the input pool after recovery from colonised 1-day-old chickens15. Unpredictable variations in population structure were even observed when birds were inoculated with just two competing wild-type isogenic-tagged strains (WITS) that have indistinguishable phenotypes in pure culture25.…”

“…To identify genes implicated in infection of piglets, the developed model was used to screen a mariner Tn mutant library in C. jejuni strain M1cam2226. Consisting of 9,951 unique Tn insertion mutants.…”

“…For reference, a total of 512 genes in the C. jejuni M1cam genome are indispensable for in vitro growth (for more details see Methods and ref. 22), hence these were eliminated from downstream analyses. A total of 8,340 (5,484 ± 3,332) unique Tn mutants were detected in the output pools recovered from the piglets (isolator 1 and 2), representing 96% recovery of the Tn mutant library complexity from infected piglets.…”

“…Thus, it appears that the molecular mechanisms involved in C. jejuni intestinal infection of gnotobiotic piglets, overlap largely with those implicated in colonization of natural reservoir host organisms such as chickens but also with those required for invasion of human gut epithelial tissue culture cells. Our recent work revealed that 65 out of 172 genes required for colonisation of commercial broiler chickens and 34 out of 57 genes required for invasion of Caco-2 cells and were also found to play a role during the infection of gnotobiotic piglets22. The large overlap suggests that this model presents an opportunity to inform us about the mechanisms of disease in humans, however a detailed molecular and immunological characterisation of the model system would be required, for example through monitoring the transcriptional response of C. jejuni and the host during infection.…”

“…The mutagenized DNA was repaired by T4 DNA polymerase and E. coli DNA ligase and introduced into C. jejuni M1cam through an adapted biphasic natural transformation method4647. Tn mutants that were obtained by selective plating were pooled to generate a large Tn mutant library of 9,951 unique Tn insertion mutants22.…”