Principal Supervisor: Professor Julian Ketley, Department of Genetics and Genome Biology
Co-supervisor: Dr Julie Morrissey
PhD project title: Iron acquisition from host lactoferrin by the bacterial pathogen Campylobacter jejuni: what is the role of extracellular glyceraldehyde 3-phosphate dehydrogenase?
University of Registration: University of Leicester
Campylobacter jejuniis a major cause of bacterial food-borne disease and is a 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 forms a major 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 suggested 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:
- Mutational analysis to verify that GAPDH, encoded by the gapAgene, is essential for iron acquisition. As we have evidence that GAPDH is essential, conditional knock-in mutants will be designed to complement the functional requirement for GAPDH so that the gapAgene 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 unable to interact with lactoferrin. Site directed mutants can also be tested in C. jejuniusing complementation and controllable promoters; one particular motif to target would be that responsible for extracellular fraction. Finally, using relevant assays we will determine the contribution of extracellular GAPDH in intestinal colonisation.
- 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 and lactoferrin, and GAPDH and CtuA, expressed GAPDH and targeted mutant GAPDH will be used in a variety of biophysical approaches. To interact with lactoferrin, GAPDH is surface associated; gene fusions to a new fluorescent tag we have established for C. jejuni and also antibodies will be used with microscopy to illustrate the surface localization and organization of GAPDH with respect to CtuA in situin 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. Use and further development of GAPDH assays and lactoferrin-binding assays.
- Imaging using antibody or fluorescently tagged proteins.
- Virulence assays using Waxworm system and, if acceptable to student, chicken intestinal colonisation experiments are also possible.