Characterisation of FeoB, the iron transporting protein in Pseudomonas aeruginosa
The emergence of superbugs are becoming more virulent and resistant against antibiotics, increasing public health problem. The available antibacterial treatments are becoming less and less effective, making the discovery of new treatments and therapies more important than ever. An emerging field of fighting infection is the targeting of bacterial iron acquisition systems. Iron is vital for the survival of pathogens as well as being an important constituent of virulence and biofilm formation. Ferrous iron is acquired by the Feo transporter, composed of three proteins; FeoA, FeoB and FeoC. The former and latter are small soluble proteins that could potentially act as transport activator and transcriptional repressor respectively. FeoB contains a cytosolic N-terminal domain with GTPase activity and an integral membrane domain, putatively functioning as the Fe (II) permease domain. Despite the vital role of FeoB in the survival and virulence of pathogens and its unique property of being a GTPase directly tethered to a membrane domain, our knowledge about this transporter is still in infancy. This research focuses on characterising FeoB, in an attempt to elucidate the mechanisms governing the acquisition of ferrous iron. We have cloned, overexpressed and purified FeoA and FeoB, optimising the necessary conditions for improved expression and purification for biophysical and biochemical assays. The disputed oligomeric state of FeoB was investigated with Mass Spectrometry (MS), atomic force microscopy (AFM) and chemical cross-linking. Biophysical data indicated FeoB existing as a trimer, confirming previous observations with biochemical approaches. GTPase activity assays showed FeoB activated by FeoA and stimulated in the presence of Fe(II). We have confirmed the GTP binding site, by generating a D123N mutant of FeoB, where no GTPase activity was observed when mutation was introduced. Overall, the insight gained from this study might help devise new therapeutic strategies to combat infection by pathogenic bacteria such as P. aeruginosa.