Campylobacter jejuni is the main cause of bacterial food-borne disease in the UK and is a global public health priority. Infection and transmission are usually linked with contaminated food or water, with chicken being a major source. Consumption of contaminated poultry meat forms an important source of clinical infection and therefore control of intestinal colonisation of chickens is an important strategy for the reduction in human infection. Despite the importance of this zoonotic pathogen, our understanding of the biology and molecular basis of C. jejuni virulence is comparatively limited. Furthermore, it is essential that the determinants involved in the colonization of the avian gut by campylobacters are better understood.
For the effective colonization of the intestine, including the chicken, C. jejuni, like other pathogens, needs to acquire iron, an essential co-factor in many physiological processes. In animals, iron is tightly complexed with proteins, such as haemoglobin, lactoferrin and transferrin, and is not readily available to microbes.
Campylobacters have several non-redundant iron acquisition systems that enable the bacterium to acquire iron from a variety of sources and several of these uptake systems are essential for the colonization of the intestine of poultry. Our previous work identified an outer membrane receptor protein, CtuA, and associated ABC transporter system that has a role in acquiring iron from lactoferrin and transferrin; this system is required for the colonisation of chickens. However, residual activity in a CtuA mutant indicated that other determinants are involved. Our recent findings with C. jejuni have shown that glyceraldehyde 3-phosphate dehydrogenase, GAPDH, along with the porin CtuA, plays a significant role in the acquisition of lactoferrin (and transferrin)-bound iron. GAPDH is a core metabolic enzyme in C. jejuni with unique properties and we have data to support that it is both essential and has a role in iron uptake. Our hypothesis is that extracellular GAPDH interacts with CtuA to remove iron from lactoferrin and enable its transport into the cell. The aims of the project are:
1. Mutational analysis to verify that GAPDH, encoded by the gapA gene, is essential for iron acquisition. As GAPDH is essential, conditional knock-in mutants will be designed to complement the functional requirement for GAPDH so that the gapA gene can be inactivated and a role in iron acquisition directly demonstrated by mutation. In addition, site directed mutagenesis will be undertaken to obtain mutant expressed protein able to act as a dehydrogenase for metabolism, but no longer able to interact with lactoferrin. Site directed mutants can also be tested in C. jejuni using complementation and controllable promoters; one particular motif to target would be that responsible for extracellular location. Finally, using relevant assays we will determine the contribution of extracellular GAPDH in intestinal colonisation.
2. Further characterize the role of GAPDH in the acquisition of iron from lactoferrin and directly show the location of the enzyme. Iron acquisition by GAPDH from lactoferrin appears to involve a direct contact. To understand the interaction between GAPDH, lactoferrin and CtuA, expressed GAPDH and targeted mutant GAPDH will be used in a variety of biophysical approaches, including ones to show intermolecular interactions like FRET. Interaction between GAPDH and lactoferrin will also be investigated by investigating the structure of interacting proteins by NMR and/or X-ray crystallography. To interact with lactoferrin, GAPDH is surface associated; gene fusions to an epitope tag will be used with electron microscopy to demonstrate the surface localization and organization of GAPDH with respect to CtuA in situ in isolated C. jejuni.
Techniques that will be undertaken during the project
Recombinant DNA techniques (cloning, PCR, RT-PCR, DNA sequencing etc)  and the construction of mutants . The latter will involve complex knock-in and knock-out genomic mutations, designed based on bioinformatic analysis  of certain metabolic pathways . Also, the production of site-directed mutant libraries and high-throughput screening .
Protein expression, purification and biophysical analysis techniques, possibly including X-ray crystallography or NMR . Use and further development of GAPDH assays and lactoferrin-binding assays .
Imaging (confocal and EM) using antibody or fluorescently tagged proteins .
Virulence assays using Waxworm system and, if acceptable to student, chicken intestinal colonisation experiments are also possible .
Available to UK/EU applicants only
Application information https://www2.le.ac.uk/research-degrees/doctoral-training-partnerships/bbsrc