Haem scavenging by pathogenic Neisseriaceae bacteria through haemoglobin receptor HpuAB

The Neisseriaceae family of bacteria is home to the genus of Neisseria which contain two relevant pathogens in humans, Neisseria meningitidis and Neisseria gonorrhoeae. N. meningitidis is responsible for bacterial meningitis and a death toll of tens of thousands globally per annum. N. gonorrhoeae is responsible for the sexually transmitted infection gonorrhoea, classed by the World Health Organisation amongst the ‘HIGH’ priority bacterial pathogens, where new therapies are urgently needed to fight antibiotic resistance. Key to developing new therapies is the understanding and exploitation of bacterial nutrient acquisition systems within the host. Neisseria can utilise haemoglobin and the haemoglobin-haptoglobin complex as an additional iron source through the haemoglobin-binding TonB-dependent receptor HpuAB. The specifics of HpuAB functionality on a structural level is poorly understood. Significant progress was made by Wong et al. (2015) who described the structure and binding capabilities of HpuA, an extracellular lipoprotein that works in partnership with HpuB to facilitate the iron acquisition. For this system to be completely understood, similar structural and functional analysis must also be carried out for HpuB. In this project, HpuB was recombinantly produced in E. coli then extracted, solubilised, and reconstituted into a range of membrane memetics to facilitate downstream studies. Through pull down assays, recombinantly produced HpuB was shown to bind Hb and mutagenic analysis of HpuB’s flexible extracellular loops demonstrated that they are key to Hpub’s Hb binding capabilities. Electron microscopy (EM) was used for structural characterisation of HpuB in nanodiscs, peptidiscs and amphipols. Amphipol solubilised HpuB particles revealed interesting structural insights through 2D and 3D averaging and reconstruction. This sample was taken forward for cryo-EM screening. In this work, optimisation and development of methodologies to express, purify and reconstitute HpuB for downstream studies was undertaken resulting a pathway to cryoEM structure utilising amphipols as a workable membrane memetic.