How does the bacterial transcription-coupling repair factor promote adaptive mutagenesis in Campylobacter jejuni?

Campylobacter jejuni is a bacterium that causes food poisoning. Many animals that are raised for food production are colonised by C. jejuni, although this does not usually make the animals ill. However, when humans are infected (for example by infected chicken meat that has not been properly cooked) they can suffer from diarrhoea, and sometimes more serious illnesses. It is estimated that in developed countries like the UK about 1 in every 100 people suffer Campylobacter-related illness each year. This represents a large cost to the community in terms of lost working days, as well as discomfort for the individuals concerned. Most people recover from C. jejuni, infections without medical intervention, but when treatment is considered necessary the patients are often prescribed a class of antibiotics called Fluoroquinolones. Fluoroquinolones kill cells by inhibiting specific enzymes within the cell, but if bacteria change the sequence of the DNA that codes for these enzymes they can become resistant to the antibiotic. The increasing prevalence of antibiotic resistant bacteria is a growing problem for both human and animal health. When stressed, bacteria can sometimes change their physiology so that their DNA mutates more quickly than it would do normally. Although most of the mutations that occur will be detrimental to the cell, some (such as those that confer antibiotic resistance) will be beneficial. Because bacteria reproduce very quickly if one or two individuals acquire mutations that allow them to survive in a new environment they will rapidly grow into a substantial population of adapted bacteria. It has recently been discovered that C. jejuni can rapidly mutate to become Fluoroquinolone-resistant because when it encounters the antibiotic it makes more of a protein called Mfd. The same mechanism for increasing mutation frequency also seems to be used when C. jejuni is exposed to other antibiotics, and it may represent a universal strategy for helping C. jejuni to adapt to extreme changes in its environment. It is important to understand how this adaptive strategy works, not only so that we can understand how C. jejuni reacts to antibiotic treatment, but also so that we can understand how it might adapt to changes that we make in the environment that food-producing animals are reared in, or mutate to cause more serious disease. The finding that the Mfd protein increases the mutation rate is paradoxical, because the protein is best known as a DNA repair protein that prevents mutations arising in bacterial genomes. For several years we have been studying the Mfd protein in a model organism called Escherichia coli. We have gained a good understanding of this multi-functional protein, and we have identified many altered forms of the protein, each of which is specifically defective in a single function. The central aim of the work in this proposal is apply this knowledge to understand how overproduction of Mfd increases the rate of mutation in C. jejuni. We will identify which functions of Mfd, and which of the proteins that it cooperates with, are needed for the process, and we hope that the understanding that we gain will help microbiologists to control the rate of mutation of C. jejuni by developing strategies that avoid or interfere with the mutation-generating pathway.

The food-borne pathogen C. jejuni has a high rate of mutagenesis to flouroquinolone (FQ) resistance, but has no conventional SOS response. Expression of the mfd gene, encoding the bacterial transcription-repair coupling factor, is elevated in C. jejuni exposed to the FQ antibiotic ciprofloxacin. The increase in Mfd production is causally linked to the high mutation frequency, as deletion of the mfd gene decreased the mutation frequency by 100-fold, and increasing the mfd gene number increased the mutation frequency 10-fold. The effect of Mfd on C. jejuni is not restricted to FQ-resistance, and may represent a general mechanism for inducing a stress-induced mutator phenotype that enables Campylobacter spp. to adapt to extreme changes in environment. The finding that a DNA repair protein promotes mutation appears paradoxical, but the multi-functional nature of the Mfd protein supports several alternative explanations for the effect. The aims of this project are to determine how Mfd promotes mutation in C. jejuni, and to begin to investigate how prevalent the pro-mutagenic role of Mfd is in other organisms. In previous work we have characterised various mutant forms of E. coli Mfd that are specifically defective in individual functions. We will analyse the ability of the equivalent mutant C. jejuni Mfd proteins to promote mutagenesis, and will determine the requirement for the Uvr repair proteins that Mfd cooperates with. We will also determine whether Mfd causes strand-specific DNA repair in C. jejuni. Together, the results of these experiments will clarify the mechanism by which Mfd promotes mutagenesis in C. jejuni, and will pave the way for future work aimed at subverting the process. Finally, we will determine whether the stress-induced upregulation of mfd is observed in wild-type veterinary isolates of C. jejuni, and we will investigate whether upregulation of mfd may circumvent attempts to moderate the mutagenesis rate of E. coli by disrupting the SOS response.

The aim of this project is to define the mechanism by which C. jejuni, an organism responsible for frequent gastrointestinal disease in humans, induces a mutator phenotype that promotes adaptation to antibacterial treatment and other changes to its environment. Infections by Campylobacter spp. are estimated to affect around 1% of the UK population each year, and the majority of infections within the UK arise from the consumption of foodstuffs contaminated during the slaughter and processing of colonised animals. The resulting loss of working days has an economic cost to both businesses and government. The BBSRC have identified 'research aimed at combating infectious diseases that reduce the health and welfare of animals farmed for food production in the UK', including 'food-borne or other zoonotic diseases with implications for public health that are carried by farmed animals but do not necessarily have a significant impact on animal welfare' as a strategic priority, because these threaten to undermine the sustainability of the UK livestock and poultry industries. The work in this project will address this prioritised issue by providing the information needed to understand a fundamental aspect of the way that the organism responds and adapts to treatment with antibacterial agents, or changes in the livestock environment intended to reduce persistence of the organism (such as changes to the litter conditions in poultry houses). We anticipate that the principal route by which our findings will have impact on applications relating to animal husbandry and human health is by underpinning the work of industrial and academic groups that are developing anti-microbial treatments, or procedures to control the incidence of C. jejuni colonisation within food-production facilities. The outcomes of such translational/applied research, built upon the basic research described in this proposal, are expected to have beneficial impacts for the health and wellbeing of the general population. We shall ensure that these groups are able to exploit our work by publishing the results in peer-reviewed scientific journals, presenting them at appropriate scientific meetings and in review articles, and making materials available after publication. In addition, we will work with the Research and Enterprise Development unit at the University of Bristol to identify findings that have clear potential for exploitation and to identify suitable academic or industrial partners with which to develop them. In addition, this project will contribute to the provision of a scientifically literate workforce by supporting the training of a postdoctoral research assistant, and the training of undergraduate students at the University of Bristol. Finally, the project will promote the engagement of the public with scientific issues via outreach and media activities conducted by the staff engaged on the project.