Global control of Campylobacter jejuni biology by protein lysine acetylation

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. C. jejuni has a small genome with only about 1650 genes and only a small number of proteins that are known to regulate gene expression. We hypothesise that in this bacterium, the control of the activity of proteins directly, through reversible modifications to their structure by additions of chemical groups, may assume greater importance than in bacteria with large genomes. In this project we wish to investigate one such modification; the addition of acetyl-groups (CH3-CO-) to proteins. We have discovered that over 70% of all of the proteins in C. jejuni are acetylated and that some of these modifications are reversible and controlled by a specific enzyme, a deacetylase. Moreover, we have found that important colonisation determinants such as the ability to swim and assimilate nitrogen for growth are impacted by acetylation. The project will investigate the consequences of acetylation at specific sites in selected proteins in detail and reveal how acetylation is controlled. The results should allow us to get a better knowledge of the way in which the bacterium uses acetylation in the colonisation process and to identify ways of disrupting acetylation, which could be exploited in future to limit its growth in chickens and to treat human infections.

Campylobacter jejuni is the commonest cause of food-borne gastroenteritis in the UK, mainly arising from under-cooked chicken. Research on the molecular basis of host colonisation is required to inform the rational design of strategies to control this zoonotic pathogen in the food-chain from farm to fork. It is not clear, however, how C. jejuni regulates colonisation critical protein function with a small genome encoding relatively few transcription factors. One possibility that has been overlooked is that post-translational modifications of specific amino-acid residues are crucial. Using antibody enrichment and high-resolution LC-MS, we have demonstrated for the first time that protein lysine acetylation is very widespread in the C. jejuni proteome. Some of these residues are specifically de-acetylated by Cj1050 (CobB), a lysine deacetylase we have characterised. From the wealth of data already obtained, we found that proteins involved in chemotaxis and motility and also nitrogen assimilation are impacted by acetylation as well as many other key enzymes and colonisation factors. The aim of this project is to fully understand the biological role of lysine acetylation in C. jejuni. We will achieve this by: (i) understanding the control of CobB and identifying proteins and small molecules it interacts with, (ii) investigating the balance of chemical versus enzymic acetylation and targeting several putative acetyltransferases for mutagenesis, biochemical characterisation and acetylome determination, (iii) determining the functional consequences of acetylation in controlling the activity of the major chemotaxis proteins and on N-assimilation (iv) probing the link between acetylation and phage replication, which may have implications for improving phage therapy by disrupting CobB activity (v) determination of the structure of CobB with and without effectors, to relate structure to function and to provide a basis for how inhibitors can be used to disrupt acetylation.

1. Who will benefit from the research?
The results from the proposed research project will contribute to knowledge-led approaches for improved food safety in the UK, thus addressing Policy priorities of the BBSRC, DEFRA and FSA with the aim of reducing infection of food animals with food borne pathogens. The work also has the longer term potential to impact on success of 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. The project matches the BBSRC strategic/scientific priorities of 'data driven biology', 'healthy and safe food' and 'Animal Health'.

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 uninvestigated mechanism for the control of Campylobacter metabolism and physiology that is directly relevant to avian colonization, as well as to infection of the human intestinal tract. Our research has identified a new specific protein control mechanism that, for example, could constitute a future new target for disruption by small molecule antagonists designed to weaken the ability of C. jejuni to colonise the avian or mammalian intestine. Moreover, if our hypothesis of a link between this control mechanism and phage replication is proven, the research will provide data that could inform future studies to improve the efficiency of phage therapy, which is one of the few realistic interventions known to reduce the caecal load of C. jejuni in chickens.