Dissecting the role of the Campylobacter haem uptake system in host colonisation and disease

Iron is a nutrient that bacteria need for many essential processes in the cell. As part of the response to infection by bacteria, animals restrict the amount of iron available. In mammals lactoferrin (in mucosal secretions) and transferrin (in serum) tightly bind free iron to restrict its availability. Unfortunately, successful disease-causing bacteria have found ways to grab iron back from the host. The bacterial mechanisms involved in acquiring iron in the host usually consist of a specific pore on the outer surface of the bacterial cell and an associated transport system that makes the iron available inside the cell. The most important food-associated bacterium, which infects humans, is Campylobacter and as such is responsible for much misery and economic loss in the UK. Campylobacters normally live in the intestine of many animals, notably chickens, where they do not always cause disease. However, when humans eat food contaminated with campylobacters, a highly unpleasant cramping diarrhoea can follow. Food producers strive to minimise the risks of food poisoning for consumers, but if Campylobacter could be eliminated from the intestines of farm animals, particularly chickens, it would have a significant impact on human health by preventing many thousands of cases of food borne disease each year. If control is to be achieved it is important that we better understand how Campylobacter colonises the intestine. Several components of campylobacters that are essential for growth in the intestine are involved in acquiring iron within the host. In our preliminary work, we have identified and begun to characterise a system in campylobacters that can grab iron directly from haem to support bacterial growth. Haem is a prosthetic group, or non-amino acid component of a protein that is important in the protein's biological activity, that contains iron. The Campylobacter Haem Uptake system, or Chu, includes a surface pore (ChuA) that imports haem into the cell and an enzyme (ChuZ) that removes iron from haem. The Chu system is highly conserved in isolates of Campylobacter. In contrast, other iron uptake pores, namely CfrA and Cj0178, are thought to be essential in some strains but are not present in others. Our first task is to determine which components play a role in acquiring iron in those strains where CfrA and Cj0178 are absent; these components include the Chu and Cj0444 pores. Our second task is to further investigate how the ChuA pore removes the haem group from proteins and to carry out a more detailed characterisation of how ChuZ works, including working out the structure of the protein. With a better understanding of Campylobacter iron acquisition systems we will be able to determine if they would be an attractive target for intervention on the farm in order to block growth in the animal gut and reduce food contamination.

Iron acquisition systems are topical and widely studied because during infection pathogens are likely to be in a state of near continual iron deficiency in the face of fierce competition from the normal microbiota and iron limitation by the host. At present four ferric iron uptake systems have been identified in Campylobacter jejuni; these comprise the CfrA/Ceu (enterobactin uptake), Chu (haem uptake), Cj0178 (iron from transferrins) and p19 (rhodotorulic acid uptake) systems. In those strains, including NCTC11168, that possess all four systems, CfrA and Cj0178 have been shown to be required for intestinal colonisation. In another group of strains, cfrA and cj0178 are absent and, in addition to the highly conserved Chu and p19 systems, an uncharacterised receptor gene, cj0444 is present. Our hypothesis is that the importance and role of each iron uptake system in campylobacters is complex and depends upon genomic context. We predict that in the absence of CfrA and Cj0178, the Chu system and/or Cj0444 have a role in iron acquisition in the intestinal niche. Our investigation on the roles of iron uptake systems in infection primarily focuses on strain 81-176 with comparisons to NCTC11168. In this context, 81-176 does not contain the cfrA or cj0178 genes required for intestinal colonisation by NCTC11168. Mutants in the relevant iron uptake systems will be assessed in iron uptake assays and then the biological role determined in the chicken colonisation and pig loop models. Intestinal colonisation will be determined in both models, spread to extra-intestinal sites will be assessed in chickens and pathological changes measured in pig loops. Further investigation of the Chu system will be undertaken, including the nature of the interaction of ChuA with haemoproteins, the contribution of the Chu system to haem metabolism, and the structure of the novel haem oxygenase, ChuZ. Finally, we propose to characterise the specificity of the Cj0444 iron uptake receptor.