Function and maintenance of unidirectional actin-based motility in two bacterial pathogens

Listeria monocytogenes and Shigella flexneri are pathogens which hijack the actin-based motility system of their eukaryotic hosts in order to spread intracellularly without exposure to the extracellular environment. Each has been previously shown to require only one bacterial protein to do this—ActA or IcsA (VirG)—respectively. Here, we use video microscopy to describe the process of L. monocytogenes motility and spread in MDCK cells that constitutively express GFP-actin. We find that both motility and spread are affected by host cell monolayer age and subcellular location. We develop new methods for the visualization of protrusion formation into and uptake by recipient host cells. Importantly, we observe that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of “paracytophagy.” We conclude that intercellular spread is a sequence of contingent events with temporal and physical consistency revealing fundamental biochemical and structural involvement of both bacteria and their host.; Attempts to further characterize paracytophagy through the use of ActA-coated microspheres are thwarted by the inability of these particles to break symmetry and move in a unidirectional fashion, a property they have been previously shown to exhibit in cellular extracts. This underscores a critical difference between in vitro and in vivo actin-based motility. However, in characterizing the motility variability of such particles in vitro, we are able to partially describe the effects of altered viscosity, protein concentration, ActA surface density, and microsphere size.; We also quantitatively elucidate the mechanisms by which S. flexneri distributes IcsA on its surface in order to promote efficient actin-based motility. We show here that the focused polar gradient of IcsA is generated by its delivery exclusively to one pole followed by lateral diffusion through the outer membrane. The resulting gradient can be modified by altering the composition of the outer membrane either genetically or pharmacologically. The gradient can further be reshaped by the action of the protease IcsP (SopA), whose activity we show to be near-uniform on the bacterial surface. Further, we report polar delivery of IcsA in Escherichia coli and Yersinia pseudotuberculosis, suggesting that the mechanism for polar delivery of some outer membrane proteins is conserved across species, and that the virulence function of IcsA capitalizes on a more global mechanism for subcellular organization in bacteria.; Finally, we describe a heretofore undocumented feature of L. monocytogenes actin-based motility: longitudinal rotation of the bacterium as it moves. This rotation is unidirectional and periodic, with each bacterium exhibiting an individualized frequency of rotation. No existing biophysical models for force generation by actin polymerization predicts a source of torque. Current evidence supports the role of actin filaments in generating this force.