Bacterial Manipulation of Actin Dynamics via p21 Activated Kinases and Cofilin

The actin cytoskeleton is a complex and dynamic network of protein filaments involved in a great number of cellular processes essential for cellular homeostasis and survival. The dynamic nature of the cytoskeleton requires careful control of actin stabilisation and severing. Cofilin is an important promoter of actin turnover, promoting the severing and disassembly of actin filaments, resulting in increased availability of actin and driving assembly of new actin structures . A major reported negative regulator of cofilin is p21 activated kinase 1 (PAK1). These dynamic structures and the importance of actin turnover result in targets for manipulation by bacterial pathogens, such as Salmonella Typhimurium’s ability to enter non-phagocytic cells, and Shigella flexneri to assemble actin ‘comet tails’ for intracellular propulsion and bacterial dissemination. This work sought to investigate the role that cofilin and PAK1, as well as its upstream and downstream regulators has in actin remodelling, and how this may be usurped by bacterial enteric pathogens, with a particular focus on Shigella flexneri comet tail formation. Shigella LPS O-antigen null mutants have been previously shown to have poor comet tail formation, however the mechanism behind this defect remains controversial. Previous studies have focused on the relationship between LPS length and the functionality of IcsA, the protein primarily responsible for intracellular motility. Here we demonstrate that LPS length does not affect IcsA’s ability to bind the vital actin related proteins necessary to produce actin comet tails, leading to investigation of the wider actin dynamics and the role they play in Shigella infection post-invasion. This work establishes key roles for both PAK1 and cofilin in bacterial manipulation of the actin cytoskeleton, demonstrating that the activation of both cofilin and PAK is required for Salmonella invasion and Shigella comet tail invasion. I provide evidence that the artificial activation of PAK and cofilin via ATP and calcium signalling results in the restoration of several attenuated bacterial mutant strains ability to manipulate the actin cytoskeleton. I provide evidence that Salmonella is able to limit its invasion, by the inhibition of cofilin post-invasion via the bacterial effector SopB, preventing the overloading of host cells and prolonging infection. I propose that Shigella activates cofilin post-invasion in response to caspase-1 activation by the bacterial effector protein IpaB, maintaining the dynamic cytoskeleton required for comet tail formation. This work also provides a potential mechanism for the failure of R-LPS Shigella mutants to form actin comet tails by indicating that the loss of O-antigen results in the secretion impairment of IpaB, resulting in its association to the bacterial membrane. Together these finding outline a novel function for IpaB’s activation of caspase-1 in the promotion of comet tail formation and provides further insight into the relationship between inflammation and actin reorganisation