Analysis of the role of BimA phosphorylation on B. pseudomallei actin-based motility
Cotton, Sebastian William
Burkholderia pseudomallei is an intracellular pathogen that causes a severe disease known as melioidosis affecting both animals and humans in South East Asia and Northern Australia. B. pseudomallei displays actin-based motility in the cytoplasm of infected cells, although its mechanism is largely unknown. Located at the pole of the bacterium is BimA which is vital for motility, survival and virulence. BimA interacts with actin and initiates the formation of actin tails independently of the Arp 2/3 complex. Listeria monocytogenes actin-based motility has been shown to be affected by the host cell enzyme Casein Kinase II. This enzyme phosphorylates the bacterial factor ActA which is required for L. monocytogenes intracellular motility. My project aims to investigate the effect of phosphorylation of BimA on actin-based motility in B. pseudomallei. Online prediction tools were used to perform a bioinformatics analysis of BimA and identify potential phosphorylation sites. Actual phosphorylation sites were identified by mass spectrometry analysis of ectopically expressed BimA protein in HeLa cells and from in vitro kinase assays on recombinant GST-BimA protein. BimA phosphorylation mutant proteins were generated and expressed in a bimA mutant. Analysis of phosphorylation sites that are important to the function of BimA could lead to the development of novel anti-infectives.