A nitric oxide responsive regulatory network in Campylobacter jejuni: its role in intracellular survival and resistance to nitrosative stresses

The bacterium called Campylobacter jejuni (abbreviated to C. jejuni) is the most common cause of bacterial food poisoning in the UK. It causes a disease of the digestive system that results in painful and profuse diarrhoea. Whilst the disease is not generally fatal, it is highly unpleasant and causes much misery and economic loss. C. jejuni normally lives in the intestine of chickens where it does not cause disease. However, when chickens are slaughtered for food production the bacterium is transferred to the chicken meat, which, if not cooked properly, can cause food poisoning. As part of the body's first defence against infection, the immune system can generate a toxic gas called nitric oxide that, when dissolved in the fluid around the infecting bacteria, is able to destroy them. We have discovered that C. jejuni can resist nitric oxide. To do this the bacterium must first be able to detect the toxic gas and then produce proteins to neutralise the nitric oxide. We believe that we have found the detector for nitric oxide in C. jejuni and this project would use this information to find out how the bacterium resists this killing agent. Having characterised this system we would try to find out if the system is essential for infection. In future, the results could be used to develop novel ways of controlling this bacterium by blocking its ability to resist nitric oxide.

The response to NO is a key feature of bacterial pathogens because NO is a major bactericidal agent of the innate host immune system. Campylobacter jejuni is a prominent foodborne pathogen and will inevitably be exposed to NO during infection. As an outcome of grants D18368 & D18084, we have discovered a novel regulon (a central regulator, NssR, and four genes) that responds specifically to NO-stress. Through this work we have gained a position of international competitiveness and are now in a uniquely favourable position to fully characterise the response of C. jejuni to NO. We have begun to dissect some of the functions of the regulon but, to capitalise on our lead in this field, further work is required. Cgb (a globin) is involved in the detoxification of NO, but its mechanism is not yet known, and thus we propose to establish its activity. Whilst Ctb (a truncated globin) is induced by NO, it does not confer resistance to it. Instead, Ctb binds oxygen tightly and thus we seek to establish whether Ctb functions in the facilitation of O2 transfer to terminal oxidase(s), or if it facilitates O2 transfer to Cgb for NO detoxification. Our most recent studies have shown that a ctb, cgb double mutant, but not the corresponding single mutants, is hypersensitive to peroxynitrite (ONOO-) generators, and thus we will determine whether Ctb and Cgb act cooperatively to prevent ONOO- formation. The contribution of all members of the NssR regulon (and other independently NO-inducible proteins) to the defence of C. jejuni against NO will be established. Preliminary data suggest that the NssR-regulon plays a key role during the survival of C. jejuni in macrophages. Thus, the role of the regulon, and its key individual components, in intracellular environments where NO and ONOO- evolution is expected will be assessed. By studying the biochemical properties of purified NssR we also propose to confirm that NssR is the NO-sensor, and to determine the mechanism of NO sensing. (Joint with BB/E009476/1)