A novel bacterial defence system against antimicrobial peptides: Implications for host colonisation in the foodborne pathogen Campylobacter jejuni

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

Some bacteria get into the food that we eat and cause food-poisoning. Some of these bacteria are quite common but are usually killed during cooking. When chicken meat is not cooked properly, one of these types of bacteria, called Campylobacter jejuni, is a particular problem. It normally lives harmlessly in the intestines of chickens but in humans causes severe diarrohea, which although not usually life-threatening, causes considerable impacts on time off work and in some cases can lead to serious complications. By understanding in detail the way in which this bacterium interacts with both its chicken and human hosts, we may be able to stop colonisation, prevent infection or design better treatments. We have discovered a previously unsuspected way in which this bacterium stops itself being killed by molecules normally produced by the host, called antimicrobial peptides. These molecules try to insert themselves into the membrane of the bacteria to create a pore, through which the bacterial contents leak out, causing death. But C. jejuni can sense the presence of these peptides, and in response deploys a protein at its surface which we have shown can bind them and stop them getting to the cell membrane, which therefore makes the bacteria highly resistant to their action. In this research, we wish to understand the details of how the bacteria sense and respond to these peptides, how the binding protein works and the role of some other proteins that are controlled by this system. The results should allow us to get a better knowledge of the way in which the bacterium uses this novel defence system in the colonisation process and to identify vulnerabilities, which could be exploited in future to limit its growth in chickens and to treat human infections.

Technical Summary

Research on the molecular basis of colonisation by Campylobacter jejuni of the chicken host is required to inform the rational design of strategies to eradicate this pathogen from poultry flocks. It is not clear, however, how C. jejuni evades avian innate immunity, particularly that mediated by cationic antimicrobial peptides (CAMPs). Many strains of C. jejuni are known to be naturally resistant to these molecules but the genetic and structural basis for this has not, up to now, been adequately explained. We have discovered an entirely novel, previously unrecognized, multi-component defence system in C. jejuni that can explain high-level CAMP resistance, which we believe is likely to be necessary for successful host colonisation. This system consists of a membrane bound sensor protein and a small DNA binding protein which control the expression of three genes encoding an inner membrane protein, an outer membrane anchored lipoprotein and a periplasmic protein, as well as certain dsb genes and genes involved in peptide transport and metabolism. We have solved the structure of the lipoprotein, shown that it binds the model CAMP polymyxin B and that a cognate null mutant is highly sensitive to polymyxin B killing. Our aim is to determine the function of each of the components of this complex system and their contribution to chicken colonisation. This will be achieved by investigating the mechanism of action of the lipoprotein and determining if it provides resistance to structurally diverse CAMPs; determining the roles of the uncharacterised genes by mutant studies; determining what the role of the Dsb system is in CAMP resistance; investigating the connection between peptide transport and CAMP transport and degradation; and determining the importance of this system in vivo by chicken colonisation studies with selected mutants. We believe this work could provide new avenues for intervention and will impact on future eradication programmes for C. jejuni in chickens.

Planned Impact

Please see Impact Summary submitted by lead applicant (joint ref. N1617101).

Publications

10 25 50
 
Description In this collaborative project with the University of Sheffied, we have tested the effect of mutations in a cluster of Campylobacter genes (cj0423-cj0425, which are putatively involved in resistance to innate defences) on colonisation of chickens. Across several studies in chickens infected experimentally with wild-type, mutant and repaired strains we have observed no significant role for the genes in colonisation of the avian host. We also re-derived a published Galleria mellonella (wax moth) replacement model in which some of the mutants exhibited defects in survival toward the 3Rs. Some delays were incurred as the parent strain originally used for construction of mutants proved to be a poor coloniser of chickens, necessitating that mutant strains were re-made in a different strain and re-tested in animal and replacement models. A draft manuscript reporting selected findings from the project is in preparation at the time of writing.
Exploitation Route Absence of a phenotype for mutants lacking Cj0423-0245) in chickens does not preclude a role in Campylobacter pathogenesis in humans or other hosts. Advances from laboratory-based research on the proteins under study will be reported by Prof. Kelly (BB/K005510/1).
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description Collaboration with Professor Dave Kelly, University of Sheffield 
Organisation University of Sheffield
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution This is a joint project led by Professor Dave Kelly at the University of Sheffield. Our component involved testing the ability of Campylobacter mutant strains lacking specific genes to colonise the intestines of chickens relative to the parent strain. Additionally, we established a Galleria mellonella 3R model to evaluate virulence of the strains.
Collaborator Contribution Provision of strains & expertise.
Impact Phenotypes for a series of mutant strains lacking specific genes, singly and in combinations, from chicken and Galleria models.
Start Year 2012
 
Description Feature for BBC2 Food Detectives programme 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Professor Stevens took part in a BBC2 Food Detectives programme which featured Campylobacter in chickens. He helped to design a survey of Campylobacter contamination in a sample of fresh chicken obtained from Scottish retailers and interpret the findings. This were close to Food Standards Agency findings, which were broadcast as part of an interview with Prof. Alice Roberts. The interview took place at The Roslin Institute and included a lay description of Campylobacter, the importance of chickens as a reservoir of infection and strategies by which consumers can protect themselves. It also highlighted BBSRC-funded work at Roslin to mitigate the problem based on genetic selection (this project) and vaccines (other BBSRC projects held by Prof. Stevens).
Year(s) Of Engagement Activity 2016
 
Description Public lecture on Campylobacter in chickens 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Public talk on 'Chickens and the enemy within' at The Roslin Institute Open Doors Day in 2012. This highlighted the problem of Campylobacter infections in people, the role of poultry as a reservoir, and research at Roslin to control the problem via genetic selection and vaccination in poultry and to understand the molecular basis of virulence of the organism.
Year(s) Of Engagement Activity 2012