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

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.

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.

1. Who will benefit from the research?
The results from the proposed research project may contribute to knowledge-led approaches for improved food safety in the UK, thus addressing Policy priorities of the BBSRC, and DEFRA, FSA and EU-driven aims of reducing infection of food animals with food borne pathogens. The work also has the longer term potential to impact on the poultry industry in the UK. It also therefore falls within the remit of the BBSRC Sustainable Agriculture Strategy and priority for research on Global Food Security. Most importantly, this proposal falls squarely into the research priorities of the UK Research and Innovation Strategy for Campylobacter, 2010-2015, jointly funded by the FSA, BBSRC, DEFRA, DARDNI and the Scottish Government, and matches the BBSRC scientific priority of 'Animal Health'. Policy priorities of 'economic and social impact' and 'welfare of managed animals' will also be addressed by this research.

2. How will they benefit?
The UK poultry industry faces numerous challenges in order to remain sustainable. These include the imminent move to more extensive rearing systems; the withdrawal of prophylactic and many therapeutic antibiotics, and other drugs such as anti-coccidials. These challenges will all have an impact on poultry health, but also have the potential to impact on human health. This proposal will shed light on a completely novel mechanism for defence against antimicrobial peptides that is directly relevant to avian colonization, as well as to infection of the human intestinal tract. Our research has identified a set of novel proteins that, for example, could constitute future new targets for small molecule antagonists designed to weaken the ability of C. jejuni to resist killing by host-derived antimicrobial peptides, and thus reduce levels of avian colonization. Alternatively, synthetic peptidomimetics may be designed that are not recognised by this resistance system, and so can be used directly as therapeutics. The proposed research has excellent complementarity with the recent BBSRC-LINK award to Prof Pete Kasier and the Co-PI Prof Mark Stevens (supported by Aviagen Ltd, the UK-based world's premier poultry breeding company), investigating quantitative trait loci associated with heritable resistance of chickens to C. jejuni (BBSRC-Aviagen BB/J006815/1). That project may uncover genes associated with innate immunity including those for antimicrobial peptide production and regulation, and will benefit from data derived from this proposal.

3. Engagement, collaboration and exploitation:
The impact of the proposed research will be conveyed to interested parties with the support of the Communications team at Sheffield/Roslin and Media Department of BBSRC. The Roslin Institute maintains well established strategies for communicating its research via several routes. These include (1) peer-reviewed scientific publications (2) presentations at scientific conferences (3) general presentations to the wider scientific community and the public via newsletters, press releases, and websites. We will also work in collaboration with the Communications teams and others to maximize utilization of outreach opportunities, via public showcases and participation in public events. Any commercially exploitable intellectual property that we gain will be protected in line with The University of Sheffield/BBSRC policies in this area.